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TW202511481A - Novel esterases and uses thereof - Google Patents

Novel esterases and uses thereof Download PDF

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TW202511481A
TW202511481A TW113117991A TW113117991A TW202511481A TW 202511481 A TW202511481 A TW 202511481A TW 113117991 A TW113117991 A TW 113117991A TW 113117991 A TW113117991 A TW 113117991A TW 202511481 A TW202511481 A TW 202511481A
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esterase
seq
combination
amino acid
substitution
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伊莎貝爾 安德烈
格雷戈里 阿爾諾
尼可拉斯 夏保特
馬克 蓋魯
艾倫 瑪蒂
亞歷山卓 托信
文森特 圖爾尼爾
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法商卡爾畢歐斯公司
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
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    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01074—Cutinase (3.1.1.74)
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    • C08J2367/00—Characterised by the use of polyesters obtained by reactions forming a carboxylic ester link in the main chain; Derivatives of such polymers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W30/00—Technologies for solid waste management
    • Y02W30/50—Reuse, recycling or recovery technologies
    • Y02W30/62—Plastics recycling; Rubber recycling

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Abstract

The present invention relates to novel esterases, more particularly to esterase variants having improved activity and/or improved thermostability under basic conditions compared to the parent esterase of SEQ ID NO:1 or SEQ ID NO:2 and the uses thereof for degrading polyester containing material, such as plastic products. The esterases of the invention are particularly suited to degrade polyethylene terephthalate, and material containing polyethylene terephthalate.

Description

新穎之酯酶及其用途Novel esterase and its use

本發明係關於新穎之酯酶，更尤其係關於與親本酯酶相比在包含於6與10之間的pH下，較佳在包含於7與9之間的pH下具有改良之降解活性及/或改良之熱穩定性的酯酶。本發明亦係關於該等新穎之酯酶用於使含聚酯材料(諸如塑膠產品)降解之用途。本發明之酯酶尤其適合於使聚對苯二甲酸伸乙酯及含有聚對苯二甲酸伸乙酯之材料降解。The present invention relates to novel esterases, more particularly to esterases having improved degradation activity and/or improved thermostability at a pH comprised between 6 and 10, preferably at a pH comprised between 7 and 9, compared to a parent esterase. The present invention also relates to the use of these novel esterases for the degradation of polyester-containing materials such as plastic products. The esterases of the present invention are particularly suitable for the degradation of polyethylene terephthalate and materials containing polyethylene terephthalate.

酯酶能夠催化包括聚酯在內的各種聚合物之水解。在此上下文中，酯酶已展現在許多工業應用中具有有前景的效果，該等工業應用包括用於洗碗及洗衣應用之清潔劑、用於處理生物質及食物之降解酶、環境污染物之去毒的生物催化劑，或用於處理紡織工業中之聚酯織品。酯酶作為用於使聚對苯二甲酸伸乙酯(PET)水解之降解酶的用途尤其受到關注。實際上，PET用於許多技術領域中，諸如用於製作衣服、地毯，或以熱固性樹脂之形式用於製造封裝或汽車塑膠等，使得填埋場中之PET積聚成為逐漸增加的生態問題。Esterases are able to catalyze the hydrolysis of various polymers, including polyesters. In this context, esterases have shown promising results in many industrial applications, including detergents for dishwashing and laundry applications, degradative enzymes for the treatment of biomass and food, biocatalysts for the detoxification of environmental pollutants, or for the treatment of polyester fabrics in the textile industry. The use of esterases as degradative enzymes for the hydrolysis of polyethylene terephthalate (PET) has received particular attention. In fact, PET is used in many technical fields, such as for the production of clothes, carpets, or in the form of thermosetting resins for the manufacture of packaging or automotive plastics, making the accumulation of PET in landfills an increasing ecological problem.

聚酯且尤其為PET之酶促降解被視為用於減少塑膠及織物廢料積聚的受關註解決方案。實際上，酶可加快含聚酯材料，且更尤其為塑膠及織物產品之水解，甚至高達單體水準。此外，水解產物(亦即，單體及寡聚物)可再循環作為用於合成新聚合物之材料。The enzymatic degradation of polyesters, and in particular PET, is considered an attractive solution for reducing the accumulation of plastic and textile waste. In fact, enzymes can accelerate the hydrolysis of polyester-containing materials, and more particularly plastic and textile products, even up to the monomer level. In addition, the hydrolysis products (i.e., monomers and oligomers) can be recycled as materials for the synthesis of new polymers.

在此上下文中，已鑑定出若干酯酶為用於聚酯之候選降解酶，且已研發出此類酯酶之一些變異體。在酯酶當中，角質酶(亦被稱作角皮質水解酶(EC 3.1.1.74))尤其受到關注。已自各種真菌(P.E. Kolattukudy，「Lipases」，B. Borg- strόm及H.L. Brockman編，Elsevier 1984，471-504)、細菌及植物花粉中鑑定出角質酶。最近，宏基因體學方法已鑑定出其他酯酶。舉例而言，宏基因體衍生之角質酶(如Sulaiman等人, Appl Environ Microbiol. 2012年3月中所描述，且在SwissProt中參考G9BY57)及其變異體(如對於WO2018011284、WO2018011281、WO2020021116、WO2020021117、WO2020021118中之實例所描述)展現使聚酯，且更尤其為聚對苯二甲酸伸乙酯降解之能力。In this context, several esterases have been identified as candidate degrading enzymes for polyesters, and some variants of these esterases have been developed. Among the esterases, cutinases, also called cutin hydrolases (EC 3.1.1.74), are of particular interest. Cutinases have been identified from various fungi (P.E. Kolattukudy, "Lipases", edited by B. Borgstrόm and H.L. Brockman, Elsevier 1984, 471-504), bacteria and plant pollen. Recently, metagenomic approaches have identified other esterases. For example, metagenomic-derived cutinases (as described in Sulaiman et al., Appl Environ Microbiol. Mar 2012, and referenced in SwissProt as G9BY57) and variants thereof (as described for the examples in WO2018011284, WO2018011281, WO2020021116, WO2020021117, WO2020021118) exhibit the ability to degrade polyesters, and more particularly polyethylene terephthalate.

然而，仍需要與已知酯酶相比具有改良之活性及/或改良之熱穩定性的酯酶，以尤其在6與10之間的pH下提供更高效且從而更具有競爭性的聚酯降解過程。However, there is still a need for esterases with improved activity and/or improved thermostability compared to known esterases, in order to provide a more efficient and thus more competitive polyester degradation process, in particular at a pH between 6 and 10.

本發明提供新的酯酶變異體，其與具有SEQ ID NO: 1中所示之胺基酸序列的親本酯酶相比，在6與10之間的pH下展現增加之聚酯降解活性及/或增加之熱穩定性。The present invention provides novel esterase variants which exhibit increased polyester degradation activity and/or increased thermostability at a pH between 6 and 10 compared to a parent esterase having the amino acid sequence shown in SEQ ID NO: 1.

本發明之酯酶尤其適用於在鹼性條件下使塑膠產品，更尤其為含有PET之塑膠產品降解的過程。The esterase of the present invention is particularly suitable for the process of degrading plastic products, more particularly plastic products containing PET, under alkaline conditions.

在此方面，因此本發明之目標為提供一種酯酶，其：(i)與SEQ ID NO: 1中所示之全長胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%一致性；(ii)與SEQ ID NO: 1相比，具有至少一種選自以下之胺基酸取代：H183Q/L/A/Y/N/D/E/W、R30G、A64V、A68N、T109S、L124G、S145G、L152M、E158A、K159R、S193L/Q、W228R/S、V242E、G7A、N9T、A24Q、Y26H、T27A、G39S、G46V、T50M、A64T、R73H、L74V、Y106F、R108L、V167L、I169M、P179N、I185V、F187L/Q、P192S、P213A、L227N、Q237L、L239M、R251S、S66T、F90C/V、P151V、A246R、A215E/D、D230E、L15K/R、N143D、V200I及N253D，或至少一種選自以下之取代的組合：N204G + M208L + N211E、F90E + N204G + N211E、F90N + N204G + N211E、F90Q + N204G + N211E、F90R + N204G + N211E、F90W + N204G + N211E、F187I + N204G + N211E、S98R + E173Q、M208Q + N211M及M208N + N211M，其中參考SEQ ID NO: 1中所示之胺基酸序列對位置進行編號；(iii)具有聚酯降解活性；及(iv)與SEQ ID NO: 1之酯酶相比，在包含於6與10之間的pH下展現增加之熱穩定性及/或增加之聚酯降解活性。In this regard, it is therefore an object of the present invention to provide an esterase which: (i) has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the full-length amino acid sequence shown in SEQ ID NO: 1; (ii) has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the full-length amino acid sequence shown in SEQ ID NO: 1, having at least one amino acid substitution selected from the group consisting of H183Q/L/A/Y/N/D/E/W, R30G, A64V, A68N, T109S, L124G, S145G, L152M, E158A, K159R, S193L/Q, W228R/S, V242E, G7A, N9T, A24Q, Y26H, T27A, G39S, G46V, T50M, A64T, R73H, L74 V, Y106F, R108L, V167L, I169M, P179N, I185V, F187L/Q, P192S, P213A, L227N, Q237L, L239M, R251S, S66T, F90C/V, P151V, A246R, A215E/D, D230E, L15K/R, N143D, V200I, and N253D, or a combination of at least one substitution selected from the following: N204G + M208L + N211E, F90E + N204G + N211E, F90N + N204G + N211E, F90Q + N204G + N211E, F90R + N204G + N211E, F90W + N204G + N211E, F187I + N204G + N211E, S98R + E173Q, M208Q + N211M and M208N + N211M, wherein the positions are numbered with reference to the amino acid sequence shown in SEQ ID NO: 1; (iii) have polyester degradation activity; and (iv) exhibit increased thermostability and/or increased polyester degradation activity at a pH comprised between 6 and 10 compared to the esterase of SEQ ID NO: 1.

較佳地，酯酶包含選自以下之至少一種胺基酸取代或至少一種取代之組合：H183Q/L/A/Y/N/D/E/W、R30G、A64V、A68N、T109S、L124G、S145G、L152M、E158A、K159R、S193L/Q、W228R/S、V242E、G7A、N9T、A24Q、Y26H、T27A、G39S、G46V、T50M、A64T、R73H、L74V、Y106F、R108L、V167L、I169M、P179N、I185V、F187L/Q、P192S、P213A、L227N、Q237L、L239M、R251S、S66T、F90C/V、P151V、A246R、A215E/D、D230E、L15K/R、N253D、N204G + M208L + N211E、F90E + N204G + N211E、F90N + N204G + N211E、F90Q + N204G + N211E、F90R + N204G + N211E、F90W + N204G + N211E、F187I + N204G + N211E、S98R + E173Q及M208N + N211M。Preferably, the esterase comprises at least one amino acid substitution or a combination of at least one substitution selected from the group consisting of H183Q/L/A/Y/N/D/E/W, R30G, A64V, A68N, T109S, L124G, S145G, L152M, E158A, K159R, S193L/Q, W228R/S, V242E, G7A, N9T, A24Q, Y26H, T27A, G39S, G46 V, T50M, A64T, R73H, L74V, Y106F, R108L, V167L, I169M, P179N, I185V, F187L/Q, P192S, P213A, L 227N, Q237L, L239M, R251S, S66T, F90C/V, P151V, A246R, A215E/D, D230E, L15K/R, N253D, N204G + M208L + N211E, F90E + N204G + N211E, F90N + N204G + N211E, F90Q + N204G + N211E, F90R + N204G + N211E, F90W + N204G + N211E, F187I + N204G + N211E, S98R + E173Q and M208N + N211M.

在特定實施例中，酯酶包含至少一種選自N204G + H183Q + F187I、M208N + N211M或H183Q + F187I + N204G + N211M + S193E之取代的組合。In particular embodiments, the esterase comprises at least one combination of substitutions selected from N204G + H183Q + F187I, M208N + N211M, or H183Q + F187I + N204G + N211M + S193E.

本發明之另一目標為提供一種酯酶變異體，其：(i)與SEQ ID NO: 1中所示之全長胺基酸序列具有至少97%、98%或99%一致性；(ii)與SEQ ID NO: 1之胺基酸序列相比，具有至少一種選自以下之胺基酸取代：H183Q/L/A/Y/N/D/E/W、R30G、A64V、A68N、T109S、L124G、S145G、L152M、K159R、S193L/Q、W228R/S、V242E、G7A、N9T、A24Q、Y26H、T27A、G39S、G46V、T50M、A64T、R73H、L74V、Y106F、R108L、V167L、I169M、P179N、I185V、F187L/Q、P192S、L227N、Q237L、L239M、R251S、N9E、V28I、A62T、F90Y/R/W/L、V115I、S181K、H183S、F187I、M208T、A246K、S66N、N243Y、S66T、F90C/V、P151V、A246R、A215E/D、D230E、L15K/R、T16K/R、Y4K/R、V219E/D、N143D、N253D、E182D、M208G、T16E、Q142E、Q237E、M208K、N105D、N85D、M208A、M208D，或至少一種選自以下之取代的組合：N204G + M208L + N211E、F90E + N204G + N211E、F90N + N204G + N211E、F90Q + N204G + N211E、F90R + N204G + N211E、F90W + N204G + N211E、F187I + N204G + N211E、S98R + E173Q、M208Q + N211M及M208N + N211M，其中參考SEQ ID NO: 1中所示之胺基酸序列對位置進行編號；(iii)與SEQ ID NO: 1之親本酯酶一樣至少具有胺基酸C240、C275、I170、G92、P213、E182、L13及E158；(iv)具有聚酯降解活性；及(v)與SEQ ID NO: 1之酯酶相比，在包含於6與10之間的pH下展現增加之熱穩定性及/或增加之聚酯降解活性。Another object of the present invention is to provide an esterase variant which: (i) has at least 97%, 98% or 99% identity to the full-length amino acid sequence shown in SEQ ID NO: 1; (ii) has at least 97%, 98% or 99% identity to the full-length amino acid sequence shown in SEQ ID NO: 1, has at least one amino acid substitution selected from the following: H183Q/L/A/Y/N/D/E/W, R30G, A64V, A68N, T109S, L124G, S145G, L152M, K159R, S193L/Q, W228R/S, V242E, G7A, N9T, A24Q, Y26H, T27A, G39S, G46V, T50M, A64T, R73H, L74V, Y106F, R108L, V167L, I169M, P179N, I185V, F187L/Q, P192S, L227N, Q237L, L239M, R251S, N9E, V28I, A62T, F90Y/R/W/L, V115I, S181K, H183S, F187I, M208T, A246K, S66N, N243Y, S66T, F90C/V, P151V, A246R, A215E/D, D230E, L15K/R, T16K/R, Y4K/R, V219E/D, N143D, N253D, E182D, M208G, T16E, Q142E, Q237E, M208K, N105D, N85D, M208A, M208D, or a combination of at least one substitution selected from the following: N204G + M208L + N211E, F90E + N204G + N211E, F90N + N204G + N211E, F90Q + N204G + N211E, F90R + N204G + N211E, F90W + N204G + N211E, F187I + N204G + N211E, S98R + E173Q, M208Q + N211M and M208N + N211M, wherein the positions are numbered with reference to the amino acid sequence shown in SEQ ID NO: 1; (iii) with SEQ ID NO: (iv) having polyester degradation activity; and (v) exhibiting increased thermostability and/or increased polyester degradation activity at a pH comprised between 6 and 10 compared to the esterase of SEQ ID NO: 1.

本發明之另一目標為提供一種酯酶變異體，其：(i)與SEQ ID NO: 2中所示之全長胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%一致性；(ii)與SEQ ID NO: 2之胺基酸序列相比，包含至少一種選自以下之胺基酸取代：H183Q/L/A/Y/N/D/E/W、R30G、A64V、A68N、T109S、L124G、T145G、L152M、E158A、K159R、S193L/Q、W228R/S、V242E、G7A、N9T、A24Q、Y26H、S27A、G39S、G46V、T50M、A64T、R73H、L74V、Y106F、R108L、Q167L、I169M、P179N、I185V、F187L/Q、P192S、P213A、L227N、Q237L、L239M、R251S、S66T、L90C/V、F92K、P151V、A246R、A215E/D、D230E、L15K/R、I143D、N253D及V200I，或至少一種選自以下之取代的組合：N204G + M208L + N211E、L90E + N204G + N211E、L90N + N204G + N211E、L90Q + N204G + N211E、L90R + N204G + N211E、L90W + N204G + N211E、F187I + N204G + N211E、S98R + E173Q、M208A + N211M、M208G + N211M、M208K + N211M、M208N + N211M、M208P + N211M、M208Q + N211M、M208H + N211M及M208T + N211M，其中參考SEQ ID NO: 2中所示之胺基酸序列對位置進行編號；(iii)具有聚酯降解活性；及(iv)與SEQ ID NO: 2之酯酶相比，在包含於6與10之間的pH下展現增加之熱穩定性及/或增加之聚酯降解活性。Another object of the present invention is to provide an esterase variant which: (i) has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the full-length amino acid sequence shown in SEQ ID NO: 2; (ii) has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the full-length amino acid sequence shown in SEQ ID NO: 2, comprising at least one amino acid substitution selected from the following: H183Q/L/A/Y/N/D/E/W, R30G, A64V, A68N, T109S, L124G, T145G, L152M, E158A, K159R, S193L/Q, W228R/S, V242E, G7A, N9T, A24Q, Y26H, S27A, G39S, G46V, T50M, A64T, R73H, L74 V, Y106F, R108L, Q167L, I169M, P179N, I185V, F187L/Q, P192S, P213A, L227N, Q237L, L239M, R251S, S66T, L90C/V, F92K, P151V, A246R, A215E/D, D230E, L15K/R, I143D, N253D, and V200I, or a substituted combination of at least one selected from the following: N204G + M208A + N211M, M208G + N211M, M208K + N211M, M208N + N211M, M208P + N211M, M208Q + N211M, M208H + N211M and M208T + N211M, with reference to SEQ ID NO: (iii) having polyester degradation activity; and (iv) exhibiting increased thermostability and/or increased polyester degradation activity at a pH comprised between 6 and 10 compared to the esterase of SEQ ID NO: 2.

本發明之另一目標為提供一種酯酶變異體，其：(i)與SEQ ID NO: 2中所示之全長胺基酸序列具有至少97%、98%或99%一致性；(ii)與SEQ ID NO: 2之胺基酸序列相比，具有至少一種選自L90W、M208A、M208N、M208S、N122D、Q237E、Q258E及S212T之胺基酸取代，或至少M208I + N211M之取代的組合，其中參考SEQ ID NO: 2中所示之胺基酸序列對位置進行編號；(iii)與SEQ ID NO: 2之親本酯酶一樣至少具有胺基酸C240、C275、I170、F92、P213、E182、L13及E158；(iv)具有聚酯降解活性；及(v)與SEQ ID NO: 2之酯酶相比，在包含於6與10之間的pH下展現增加之熱穩定性及/或增加之聚酯降解活性。Another object of the present invention is to provide an esterase variant which: (i) has at least 97%, 98% or 99% identity to the full-length amino acid sequence shown in SEQ ID NO: 2; (ii) has at least one amino acid substitution selected from L90W, M208A, M208N, M208S, N122D, Q237E, Q258E and S212T, or a combination of at least M208I + N211M substitutions compared to the amino acid sequence of SEQ ID NO: 2, wherein the positions are numbered with reference to the amino acid sequence shown in SEQ ID NO: 2; (iii) has at least amino acids C240, C275, I170, F92, P213, E182, L13 and E158 as the parent esterase of SEQ ID NO: 2; (iv) has polyester degradation activity; and (v) has at least one amino acid substitution selected from L90W, M208A, M208N, M208S, N122D, Q237E, Q258E and S212T, or a combination of at least M208I + N211M substitutions compared to the amino acid sequence of SEQ ID NO: 2, wherein the positions are numbered with reference to the amino acid sequence shown in SEQ ID NO: 2; 2, exhibiting increased thermostability and/or increased polyester degradation activity at a pH comprised between 6 and 10.

本發明之另一目標為提供一種編碼本發明之酯酶的核酸。本發明亦係關於一種包含該核酸之表現卡匣或表現載體，以及一種包含該核酸、表現卡匣或載體之宿主細胞。Another object of the present invention is to provide a nucleic acid encoding the esterase of the present invention. The present invention also relates to an expression cassette or expression vector comprising the nucleic acid, and a host cell comprising the nucleic acid, expression cassette or vector.

本發明亦提供一種組合物，其包含本發明之酯酶、本發明之宿主細胞或其萃取物。The present invention also provides a composition comprising the esterase of the present invention, the host cell of the present invention or an extract thereof.

本發明之另一目標為提供一種產生本發明之酯酶的方法，其包含： (a) 在適合於表現編碼酯酶之核酸的條件下培養根據本發明之宿主細胞；及視情況 (b) 自細胞培養物回收該酯酶。 Another object of the present invention is to provide a method for producing the esterase of the present invention, comprising: (a) culturing a host cell according to the present invention under conditions suitable for expressing a nucleic acid encoding the esterase; and optionally (b) recovering the esterase from the cell culture.

本發明之另一目標為提供一種使聚酯或含聚酯材料之聚酯降解的方法，其包含 (a) 使該聚酯與根據本發明之酯酶或根據本發明之宿主細胞或根據本發明之組合物接觸；及視情況 (b) 回收單體及/或寡聚物。 Another object of the present invention is to provide a method for degrading polyester or polyester-containing material, which comprises (a) contacting the polyester with an esterase according to the present invention or a host cell according to the present invention or a composition according to the present invention; and, as the case may be, (b) recovering monomers and/or oligomers.

有利的是，至少步驟a)係在鹼性條件下，尤其在6與10之間的pH下、較佳在7與9之間的pH下進行。Advantageously, at least step a) is carried out under alkaline conditions, in particular at a pH between 6 and 10, preferably at a pH between 7 and 9.

特定言之，本發明提供一種使PET降解之方法，其包含使PET與本發明之至少一種酯酶接觸，及視情況回收PET之單體及/或寡聚物。有利的是，至少使PET與本發明之該酯酶接觸之步驟係在鹼性條件下，尤其在6與10之間的pH下、較佳在7與9之間的pH下進行。In particular, the present invention provides a method for degrading PET, comprising contacting PET with at least one esterase of the present invention, and optionally recovering monomers and/or oligomers of PET. Advantageously, at least the step of contacting PET with the esterase of the present invention is carried out under alkaline conditions, in particular at a pH between 6 and 10, preferably at a pH between 7 and 9.

本發明亦係關於本發明之酯酶用於使PET或含有PET之塑膠產品降解的用途。有利的是，該用途係在鹼性條件下，尤其在6與10之間的pH下、較佳在7與9之間的pH下進行。The present invention also relates to the use of the esterase according to the present invention for degrading PET or a plastic product containing PET. Advantageously, the use is carried out under alkaline conditions, in particular at a pH between 6 and 10, preferably at a pH between 7 and 9.

本發明亦係關於一種含聚酯材料，其中包括本發明之酯酶或宿主細胞或組合物。The present invention also relates to a polyester-containing material, which comprises the esterase or host cell or composition of the present invention.

本發明亦係關於一種清潔劑組合物，其包含根據本發明之酯酶或宿主細胞或包含本發明之酯酶的組合物。The present invention also relates to a cleaning agent composition comprising an esterase according to the present invention or a host cell or a composition comprising an esterase according to the present invention.

定義藉由參考以下定義將最佳理解本揭示。 Definitions The present disclosure will be best understood by reference to the following definitions.

本文中，術語「 肽」、「 多肽」、「 蛋白質」、「 酶」係指由肽鍵連接之胺基酸鏈，與形成該鏈之胺基酸的數目無關。胺基酸在本文中根據以下命名法藉由其一個字母或三個字母之代碼表示：A：丙胺酸(Ala)；C：半胱胺酸(Cys)；D：天冬胺酸(Asp)；E：麩胺酸(Glu)；F：苯丙胺酸(Phe)；G：甘胺酸(Gly)；H：組胺酸(His)；I：異白胺酸(Ile)；K：離胺酸(Lys)；L：白胺酸(Leu)；M：甲硫胺酸(Met)；N：天冬醯胺(Asn)；P：脯胺酸(Pro)；Q：麩醯胺酸(Gln)；R：精胺酸(Arg)；S：絲胺酸(Ser)；T：蘇胺酸(Thr)；V：纈胺酸(Val)；W：色胺酸(Trp)；及Y：酪胺酸(Tyr)。 As used herein, the terms " peptide ", " polypeptide ", " protein ", and " enzyme " refer to a chain of amino acids linked by peptide bonds, regardless of the number of amino acids forming the chain. Amino acids are referred to herein by their one-letter or three-letter codes according to the following nomenclature: A: alanine (Ala); C: cysteine (Cys); D: aspartic acid (Asp); E: glutamine (Glu); F: phenylalanine (Phe); G: glycine (Gly); H: histidine (His); I: isoleucine (Ile); K: lysine ( Lys); L: leucine (Leu); M: methionine (Met); N: asparagine (Asn); P: proline (Pro); Q: glutamine (Gln); R: arginine (Arg); S: serine (Ser); T: threonine (Thr); V: valine (Val); W: tryptophan (Trp); and Y: tyrosine (Tyr).

術語「 酯酶」係指屬於根據酶命名法(Enzyme Nomenclature)分類為EC 3.1.1的一類水解酶的酶，其催化酯水解成酸及醇。術語「 角質酶」或「 角皮質水解酶」係指根據酶命名法分類為EC 3.1.1.74的酯酶，其能夠催化由角皮質及水產生角皮質單體的化學反應。 The term " esterase " refers to an enzyme belonging to the class of hydrolases classified as EC 3.1.1 according to Enzyme Nomenclature, which catalyzes the hydrolysis of esters into acids and alcohols. The term " cutinase " or " keratin hydrolase " refers to an esterase classified as EC 3.1.1.74 according to Enzyme Nomenclature, which is capable of catalyzing the chemical reaction that produces keratin monomers from keratin and water.

術語「 親本蛋白質」係指具有SEQ ID NO: 1或SEQ ID NO: 2中所示之胺基酸序列的酯酶。 The term " parent protein " refers to an esterase having the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

術語「 突變」及「 變異體」係指衍生自SEQ ID NO: 1或SEQ ID NO: 2且與SEQ ID NO: 1或SEQ ID NO: 2相比在一或多個(例如若干個)位置包含至少一個修飾或改變，亦即取代、插入及/或缺失，且具有聚酯降解活性的多肽。變異體可藉由此項技術中熟知之各種技術獲得。特定言之，用於改變編碼親本蛋白質之DNA序列的技術之實例包括但不限於定點突變誘發、隨機突變誘發及合成性寡核苷酸構築。由此，本文中關於特定位置所使用之術語「 修飾」及「 改變」意謂此特定位置中之胺基酸與親本蛋白質中此特定位置中之胺基酸相比已經修飾。 The terms " mutation " and " variant " refer to a polypeptide derived from SEQ ID NO: 1 or SEQ ID NO: 2 and comprising at least one modification or change, i.e., substitution, insertion and/or deletion, at one or more (e.g., several) positions compared to SEQ ID NO: 1 or SEQ ID NO: 2, and having polyester degradation activity. Variants can be obtained by various techniques well known in the art. Specifically, examples of techniques for altering the DNA sequence encoding the parent protein include, but are not limited to, site-directed mutagenesis induction, random mutagenesis induction, and synthetic oligonucleotide construction. Thus, the terms " modification " and " change " used herein with respect to a specific position mean that the amino acid in this specific position has been modified compared to the amino acid in this specific position in the parent protein.

「 取代」意謂胺基酸殘基由另一胺基酸殘基置換。較佳地，術語「 取代」係指藉由選自以下之另一胺基酸殘基來置換胺基酸殘基：天然產生之20種標準胺基酸殘基；天然產生之稀有胺基酸殘基(例如，羥基脯胺酸、羥基離胺酸、別羥基離胺酸、6-N-甲基離胺酸、N-乙基甘胺酸、N-甲基甘胺酸、N-乙基天冬醯胺、別異白胺酸、N-甲基異白胺酸、N-甲基纈胺酸、焦麩胺酸、胺基丁酸、鳥胺酸、正白胺酸、正纈胺酸)；及通常以合成方式製備的非天然產生之胺基酸殘基(例如，環己基-丙胺酸)。較佳地，術語「 取代」係指藉由選自天然產生之20種標準胺基酸殘基(G、P、A、V、L、I、M、C、F、Y、W、H、K、R、Q、N、E、D、S及T)之另一胺基酸殘基來置換胺基酸殘基。符號「+」指示取代之組合。在本文件中，以下術語用於表示取代：L82A表示親本序列之位置82處的胺基酸殘基(白胺酸，L)經丙胺酸(A)取代。A121V/I/M表示親本序列之位置121處的胺基酸殘基(丙胺酸，A)經以下胺基酸中之一者取代：纈胺酸(V)、異白胺酸(I)或甲硫胺酸(M)。取代可為保守性或非保守性取代。保守性取代之實例在以下之群內：鹼性胺基酸(精胺酸、離胺酸及組胺酸)、酸性胺基酸(麩胺酸及天冬胺酸)、極性胺基酸(麩醯胺酸、天冬醯胺及蘇胺酸)、疏水性胺基酸(甲硫胺酸、白胺酸、異白胺酸、半胱胺酸及纈胺酸)、芳族胺基酸(苯丙胺酸、色胺酸及酪胺酸)，及較小胺基酸(甘胺酸、丙胺酸及絲胺酸)。 " Substitution " means that an amino acid residue is replaced by another amino acid residue. Preferably, the term " substitution " refers to replacing an amino acid residue by another amino acid residue selected from the following: the 20 standard amino acid residues occurring naturally; rare amino acid residues occurring naturally (e.g., hydroxyproline, hydroxylysine, allohydroxylysine, 6-N-methyllysine, N-ethylglycine, N-methylglycine, N-ethylaspartate, alloisoleucine, N-methylisoleucine, N-methylvaline, pyroglutamine, aminobutyric acid, ornithine, norleucine, norvaline); and non-naturally occurring amino acid residues that are usually prepared synthetically (e.g., cyclohexyl-alanine). Preferably, the term " substitution " refers to replacing an amino acid residue by another amino acid residue selected from the 20 standard amino acid residues (G, P, A, V, L, I, M, C, F, Y, W, H, K, R, Q, N, E, D, S and T) produced naturally. The symbol "+" indicates a substituted combination. In this document, the following terms are used to represent substitutions: L82A means that the amino acid residue at position 82 of the parent sequence (leucine, L) is replaced by alanine (A). A121V/I/M means that the amino acid residue at position 121 of the parent sequence (alanine, A) is replaced by one of the following amino acids: valine (V), isoleucine (I) or methionine (M). Substitutions can be conservative or non-conservative substitutions. Examples of conservative substitutions are within the following groups: basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamine and aspartic acid), polar amino acids (glutamine, asparagine, and threonine), hydrophobic amino acids (methionine, leucine, isoleucine, cysteine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and smaller amino acids (glycine, alanine, and serine).

除非另外規定，否則本申請案中所揭示之胺基酸位置係參考SEQ ID NO: 1中所示之胺基酸序列進行編號。Unless otherwise specified, amino acid positions disclosed in this application are numbered with reference to the amino acid sequence shown in SEQ ID NO: 1.

依本文所用，術語「 序列一致性」或「 一致性」係指兩個多肽序列之間的匹配物(相同胺基酸殘基)之數目(或表現為百分比%之分數)。序列一致性係藉由在比對以使重疊及一致性達到最大，同時使序列間隙減至最小時比較序列來確定。特定言之，視兩個序列之長度而定，可使用多種數學全域或局域比對演算法中之任一者來確定序列一致性。具有相似長度之序列較佳使用在整個長度內最佳比對序列之全域比對演算法(例如，Needleman及Wunsch演算法；Needleman及Wunsch，1970)來進行比對，而具有實質上不同長度之序列較佳使用局域比對演算法(例如，Smith及Waterman演算法(Smith及Waterman，1981)或Altschul演算法(Altschul等人，1997；Altschul等人，2005))來進行比對。用於確定胺基酸序列一致性百分比之目的之比對可以此項技術中之技能內的各種方式達成，例如使用可在諸如http://blast.ncbi.nlm.nih.gov/或http://www.ebi.ac.uk/Tools/emboss/)之網際網路網站上獲得的公開可用的電腦軟體。熟習此項技術者可確定用於量測比對之適當參數，包括用於達成所比較序列之全長內之最大比對所需的任何演算法。出於本文之目的，胺基酸序列一致性%值係指使用成對序列比對程式EMBOSS Needle產生的值，該程式使用尼德曼-翁施算法(Needleman-Wunsch algorithm)產生兩個序列之最佳全域比對，其中所有搜尋參數設定成預設值，亦即計分矩陣=BLOSUM62，Gap開頭=11，Gap延長=1。 As used herein, the term " sequence identity " or " identity " refers to the number (or fraction expressed as a percentage %) of matches (identical amino acid residues) between two polypeptide sequences. Sequence identity is determined by comparing the sequences while aligning them to maximize overlap and identity while minimizing sequence gaps. In particular, sequence identity can be determined using any of a variety of mathematical global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar length are preferably aligned using a global alignment algorithm (e.g., the Needleman and Wunsch algorithm; Needleman and Wunsch, 1970) that optimally aligns sequences over their entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g., the Smith and Waterman algorithm (Smith and Waterman, 1981) or the Altschul algorithm (Altschul et al., 1997; Altschul et al., 2005)). Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways within the skill in the art, for example, using publicly available computer software available on Internet websites such as http://blast.ncbi.nlm.nih.gov/ or http://www.ebi.ac.uk/Tools/emboss/). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximal alignment over the full length of the sequences being compared. For the purposes of this article, % amino acid sequence identity values refer to values generated using the pairwise sequence alignment program EMBOSS Needle, which uses the Needleman-Wunsch algorithm to produce the best global alignment of two sequences, with all search parameters set to the default values, i.e., Scoring Matrix = BLOSUM62, Gap Start = 11, Gap Extension = 1.

「 聚合物」係指結構係由藉由共價化學鍵連接之多個單體(重複單元)構成的化合物或化合物混合物。在本發明之上下文內，術語聚合物包括天然或合成聚合物，其由單一類型之重複單元(亦即，均聚物)或由不同重複單元之混合物(亦即，共聚物或雜聚物)構成。根據本發明，「 寡聚物」係指含有2至約20個單體之分子。 " Polymer " refers to a compound or mixture of compounds whose structure is composed of multiple monomers (repeating units) connected by covalent chemical bonds. In the context of the present invention, the term polymer includes natural or synthetic polymers that are composed of a single type of repeating unit (i.e., homopolymer) or a mixture of different repeating units (i.e., copolymer or heteropolymer). According to the present invention, " oligomer " refers to a molecule containing 2 to about 20 monomers.

在本發明之上下文中，「 含聚酯材料」或「 含聚酯產品」係指包含呈結晶形式、半結晶形式或完全非晶形式之至少一種聚酯的產品，諸如塑膠產品。在特定實施例中，含聚酯材料係指由含有至少一種聚酯且可能含有其他物質或諸如塑化劑、礦物質或有機填充劑之添加劑的至少一種塑膠材料製成的任何物品，該至少一種塑膠材料諸如為塑膠片材、管、棒、型材、成型件、薄膜、大塊體、纖維等。在另一特定實施例中，含聚酯材料係指適合於製造塑膠產品之呈熔融或固體狀態的塑膠化合物或塑膠調配物。在另一特定實施例中，含聚酯材料係指包含至少一種聚酯之織物、織品或纖維。在另一特定實施例中，含聚酯材料係指包含至少一種聚酯之塑膠廢料或纖維廢料。特定言之，塑膠物品係製品，諸如硬質或軟質包裝(瓶、盤、杯等)、農業膜、袋及大袋、一次性物品或類似物品、地毯廢料、織品、織物等。塑膠物品可含有其他物質或添加劑，諸如塑化劑、礦物質、有機填充劑或染料。在本發明之上下文中，塑膠物品可包含半結晶及/或非晶形聚合物及/或添加劑之混合物。 In the context of the present invention, " polyester-containing material " or " polyester-containing product " refers to a product, such as a plastic product, comprising at least one polyester in crystalline, semi-crystalline or completely amorphous form. In a specific embodiment, the polyester-containing material refers to any article made of at least one plastic material containing at least one polyester and possibly containing other substances or additives such as plasticizers, minerals or organic fillers, such as plastic sheets, tubes, rods, profiles, molded parts, films, bulks, fibers, etc. In another specific embodiment, the polyester-containing material refers to a plastic compound or plastic formulation in a molten or solid state suitable for making plastic products. In another specific embodiment, polyester-containing material refers to fabrics, textiles or fibers containing at least one polyester. In another specific embodiment, polyester-containing material refers to plastic waste or fiber waste containing at least one polyester. In particular, plastic articles are articles such as hard or soft packaging (bottles, trays, cups, etc.), agricultural films, bags and sacks, disposable items or similar items, carpet waste, textiles, fabrics, etc. Plastic articles may contain other substances or additives, such as plasticizers, minerals, organic fillers or dyes. In the context of the present invention, plastic articles may include mixtures of semi-crystalline and/or amorphous polymers and/or additives.

在本說明書中，術語「 聚酯」涵蓋但不限於聚對苯二甲酸伸乙酯(PET)、聚對苯二甲酸伸丙酯(PTT)、聚對苯二甲酸伸丁酯(PBT)、聚異山梨醇對苯二甲酸伸乙酯(PEIT)、聚乳酸(PLA)、聚羥基烷酸酯(PHA)、聚丁二酸伸丁酯(PBS)、聚丁二酸己二酸伸丁酯(PBSA)、聚己二酸對苯二甲酸伸丁酯(PBAT)、聚呋喃酸伸乙酯(PEF)、聚己內酯(PCL)、聚(己二酸伸乙酯) (PEA)、聚萘二甲酸伸乙酯(PEN)及此等聚合物之摻合物/混合物。聚酯亦可涵蓋「聚烯烴類」聚酯，較佳為「聚乙烯類」聚酯，其對應於已引入酯片段(通常藉由長鏈α,ω-雙官能單體之聚縮合來達成)之聚烯烴(較佳聚乙烯)，如Lebarbé等人Green Chemistry Issue 4 2014中所定義。 In this specification, the term " polyester " includes but is not limited to polyethylene terephthalate (PET), polypropylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), polycaprolactone (PCL), poly(ethylene adipate) (PEA), polyethylene naphthalate (PEN) and blends/mixtures of these polymers. Polyesters may also encompass "polyolefin-based" polyesters, preferably "polyethylene-based" polyesters, which correspond to polyolefins (preferably polyethylene) into which ester segments have been introduced (usually obtained by polymerization of long-chain α,ω-difunctional monomers), as defined in Lebarbé et al., Green Chemistry Issue 4 2014.

新的酯酶本發明提供在鹼性條件下，尤其在包含於6與10之間的pH下與親本酯酶相比具有改良之活性及/或改良之熱穩定性的新穎之酯酶。更特定言之，本案發明人已設計出尤其適合於在工業過程中在鹼性條件下使用的新穎之酶。本發明之酯酶尤其適合於使聚酯，更尤其為PET，包括含有PET之材料，且尤其為含有PET之塑膠產品降解。在特定實施例中，酯酶在鹼性條件下展現與親本酯酶相比增加之活性及增加之熱穩定性兩者。 The present invention provides novel esterases having improved activity and/or improved thermostability compared to a parent esterase under alkaline conditions, particularly at a pH comprised between 6 and 10. More particularly, the inventors have designed novel enzymes that are particularly suitable for use under alkaline conditions in industrial processes. The esterases of the present invention are particularly suitable for degrading polyesters, more particularly PET, including materials containing PET, and particularly plastic products containing PET. In particular embodiments, the esterases exhibit both increased activity and increased thermostability compared to a parent esterase under alkaline conditions.

根據本發明，「鹼性條件」係指在包含於6與10之間的pH下的條件(例如培養基、溶液等)。特定言之，「鹼性條件」係指進行聚酯之降解步驟的條件，亦即使酯酶在pH介於6與10之間的培養基中與聚酯接觸。According to the present invention, "alkaline conditions" refer to conditions (such as culture medium, solution, etc.) at a pH comprised between 6 and 10. Specifically, "alkaline conditions" refer to conditions for carrying out the polyester degradation step, that is, esterase is contacted with polyester in a culture medium with a pH between 6 and 10.

本發明之酯酶在鹼性條件下展現與親本酯酶相比增加之活性及/或增加之熱穩定性。特定言之，當在6與10之間的pH下進行時，本發明之酯酶展現與親本酯酶相比增加之活性及/或增加之熱穩定性。The esterase of the present invention exhibits increased activity and/or increased thermal stability compared to the parent esterase under alkaline conditions. In particular, when carried out at a pH between 6 and 10, the esterase of the present invention exhibits increased activity and/or increased thermal stability compared to the parent esterase.

根據本發明，可在6與10之間的特定pH下及/或6與10之間的pH範圍內觀測到增加之活性及/或增加之熱穩定性。特定言之，可至少在pH 6、pH 6.5、pH 7、pH 7.5、pH 8、pH 8.5、pH 9、pH 9.5及/或pH 10下觀測到增加之活性及/或增加之熱穩定性。亦可在pH 6至10之整個範圍內、在pH 6至9之整個範圍內、在pH 6.5至9之整個範圍內、在pH 7至9之整個範圍內、在pH 7.5至8.5之整個範圍內、在pH 7至8.5之整個範圍內、在pH 7.5至9之整個範圍內觀測到增加之活性及/或增加之熱穩定性。According to the present invention, increased activity and/or increased thermostability may be observed at a specific pH between 6 and 10 and/or within a pH range between 6 and 10. In particular, increased activity and/or increased thermostability may be observed at least at pH 6, pH 6.5, pH 7, pH 7.5, pH 8, pH 8.5, pH 9, pH 9.5 and/or pH 10. Increased activity and/or increased thermostability may also be observed over the entire range of pH 6 to 10, over the entire range of pH 6 to 9, over the entire range of pH 6.5 to 9, over the entire range of pH 7 to 9, over the entire range of pH 7.5 to 8.5, over the entire range of pH 7 to 8.5, over the entire range of pH 7.5 to 9.

因此，本發明之目標為提供酯酶，其在包含於6與10之間的pH下展現與相同pH下的具有親本酯酶中所示之胺基酸序列之酯酶相比增加之活性。在本發明之上下文中，親本酯酶可為SEQ ID NO: 1或SEQ ID NO: 2之酯酶。Therefore, it is an object of the present invention to provide an esterase which exhibits an increased activity compared to an esterase having the amino acid sequence shown in a parent esterase at the same pH at a pH comprised between 6 and 10. In the context of the present invention, the parent esterase may be the esterase of SEQ ID NO: 1 or SEQ ID NO: 2.

特定言之，本案發明人已鑑定出SEQ ID NO: 1及SEQ ID NO: 2中之特定胺基酸取代，其有利地導致在鹼性條件下酯酶對聚合物，尤其對聚酯，更尤其對聚對苯二甲酸伸乙酯(PET)之活性增加。Specifically, the inventors of the present invention have identified specific amino acid substitutions in SEQ ID NO: 1 and SEQ ID NO: 2 that advantageously result in increased activity of the esterase towards polymers, particularly polyesters, and more particularly polyethylene terephthalate (PET), under alkaline conditions.

在本發明之上下文中，術語「增加之活性」或「增加之降解活性」指示酯酶在給定條件(例如溫度、pH、濃度)下使聚酯降解之能力及/或吸附於聚酯上之能力與在相同條件下親本酯酶使同一聚酯降解及/或吸附於同一聚酯上之能力相比增加。特定言之，本發明之酯酶具有增加之PET降解活性。此類增加之活性可比親本酯酶之聚酯降解活性大至少10%，較佳至少15%、20%、30%、40%、50%、60%、70%、80%、90%、100%、110%、120%、130%或更大。特定言之，降解活性為產生聚酯之單體及/或寡聚物的解聚合活性，可進一步重新得到該等單體及/或寡聚物且視情況再使用。在本發明之上下文內，酯酶至少在鹼性條件下，尤其在包含於6與10之間的pH下展現與親本酯酶在相同pH下之降解活性相比增加之降解活性。較佳地，酯酶至少在包含於6.5與9之間的pH下、更佳在包含於7與9之間的pH下、甚至更佳在包含於7.5與9之間的pH下、例如在pH 8下展現增加之活性。In the context of the present invention, the term "increased activity" or "increased degradation activity" indicates that the ability of the esterase to degrade polyester and/or to adsorb on polyester under given conditions (e.g., temperature, pH, concentration) is increased compared to the ability of the parent esterase to degrade and/or adsorb on the same polyester under the same conditions. In particular, the esterase of the present invention has an increased PET degradation activity. Such increased activity may be at least 10% greater than the polyester degradation activity of the parent esterase, preferably at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130% or more. In particular, the degradation activity is a depolymerization activity to produce monomers and/or oligomers of polyester, which can be further recovered and reused as appropriate. In the context of the present invention, the esterase exhibits an increased degradation activity compared to the degradation activity of the parent esterase at the same pH at least under alkaline conditions, in particular at a pH comprised between 6 and 10. Preferably, the esterase exhibits an increased activity at least at a pH comprised between 6.5 and 9, more preferably at a pH comprised between 7 and 9, even more preferably at a pH comprised between 7.5 and 9, for example at pH 8.

酯酶之「降解活性」可藉由熟習此項技術者根據此項技術中本身已知的方法來進行評估。舉例而言，可藉由量測特定聚合物之解聚合活性率、量測使分散於瓊脂培養盤中之固體聚合物化合物降解之速率或量測反應器中之聚合物之解聚合活性率來估計降解活性。特定言之，可藉由量測酯酶之「比降解活性」來評估降解活性。酯酶針對PET之「比降解活性」對應於在反應之初始時段(亦即，前24小時)期間每毫克酯酶每分鐘水解的PET之微莫耳數或每小時產生的當量TA之毫克數，且係根據反應之水解曲線的線性部分確定，該曲線係藉由在前24小時期間之不同時間處進行的若干取樣建立。作為另一實例，可藉由以下方式評估「降解活性」：當使聚合物或含有聚合物之塑膠產品與降解酶接觸時，在規定時段之後(例如在24 h、48 h或72 h之後)，量測在適合之溫度、pH及緩衝液條件下釋放的寡聚物及/或單體之速率及/或產率。The "degradation activity" of the esterase can be evaluated by a person skilled in the art according to methods known per se in the art. For example, the degradation activity can be estimated by measuring the depolymerization activity rate of a specific polymer, measuring the rate of degradation of a solid polymer compound dispersed in an agar culture dish, or measuring the depolymerization activity rate of a polymer in a reactor. Specifically, the degradation activity can be evaluated by measuring the "specific degradation activity" of the esterase. The "specific degradation activity" of the esterase against PET corresponds to the micromoles of PET hydrolyzed per minute per milligram of esterase or the milligrams of equivalent TA produced per hour during the initial period of the reaction (i.e., the first 24 hours), and is determined based on the linear portion of the hydrolysis curve of the reaction, which is established by a number of samples taken at different times during the first 24 hours. As another example, "degradation activity" can be assessed by measuring the rate and/or yield of oligomers and/or monomers released under suitable temperature, pH and buffer conditions after a specified period of time (e.g., after 24 h, 48 h, or 72 h) when a polymer or a plastic product containing a polymer is contacted with a degrading enzyme.

酶吸附在基質上之能力可藉由熟習此項技術者根據此項技術中本身已知的方法來進行評估。舉例而言，酶吸附在基質上之能力可自含有該酶且其中該酶先前已與基質在合適條件下一起培育的溶液量測。The ability of an enzyme to be adsorbed on a substrate can be assessed by a person skilled in the art according to methods known per se in the art. For example, the ability of an enzyme to be adsorbed on a substrate can be measured from a solution containing the enzyme in which the enzyme has previously been incubated with the substrate under suitable conditions.

本案發明人亦鑑定出親本酯酶中之標靶胺基酸殘基，其可有利地經修飾以在高溫下在鹼性條件下改良對應酯酶之穩定性(亦即，改良之熱穩定性)，且有利地在50℃或以上及90℃或以下、較佳在60℃或以上及80℃或以下、更佳在65℃或以上及75℃或以下之溫度下。The inventors of the present invention have also identified target amino acid residues in the parent esterase that can be advantageously modified to improve the stability of the corresponding esterase under alkaline conditions at high temperatures (i.e., improved thermal stability), and advantageously at a temperature of 50°C or above and 90°C or below, preferably at 60°C or above and 80°C or below, and more preferably at 65°C or above and 75°C or below.

因此，本發明之目標為提供新穎之酯酶，其在鹼性條件下所展現的熱穩定性與具有親本酯酶中所示之胺基酸序列的酯酶在相同pH下之熱穩定性相比增加。Therefore, an object of the present invention is to provide novel esterases which exhibit increased thermostability under alkaline conditions compared to the thermostability of an esterase having the amino acid sequence shown in a parent esterase at the same pH.

在本發明之上下文中，術語「增加之熱穩定性」指示與親本酯酶相比，酯酶在高溫下，且尤其在50℃與90℃之間的溫度下抵抗其化學及/或物理結構之變化的能力增加。在特定實施例中，在50℃與90℃之間、50℃與80℃之間、50℃與75℃之間、50℃與70℃之間、50℃與65℃之間、55℃與90℃之間、55℃與80℃之間、55℃與75℃之間、55℃與70℃之間、55℃與65℃之間、60℃與90℃之間、60℃與80℃之間、60℃與75℃之間、60℃與70℃之間、60℃與65℃之間、65℃與90℃之間、65℃與80℃之間、65℃與75℃之間、65℃與70℃之間的溫度下，酯酶之熱穩定性在鹼性條件下與親本酯酶之熱穩定性相比得到改良。特定言之，在40℃與80℃之間、50℃與72℃之間、55℃與60℃之間、50℃與55℃之間、60℃與72℃之間的溫度下，酯酶之熱穩定性在酸性條件下與親本酯酶之熱穩定性相比得到改良。在特定實施例中，酯酶之熱穩定性至少在50℃與65℃之間的溫度下與親本酯酶之熱穩定性相比得到改良。在本發明之上下文中，以+/-1℃給定溫度。In the context of the present invention, the term "increased thermostability" indicates an increased ability of the esterase to resist changes in its chemical and/or physical structure at high temperatures, and in particular at temperatures between 50°C and 90°C, compared to the parent esterase. In particular embodiments, the thermostability of the esterase is improved under alkaline conditions compared to the thermostability of the parent esterase at a temperature of between 50°C and 90°C, between 50°C and 80°C, between 50°C and 75°C, between 50°C and 70°C, between 50°C and 65°C, between 55°C and 90°C, between 55°C and 80°C, between 55°C and 75°C, between 55°C and 70°C, between 55°C and 65°C, between 60°C and 90°C, between 60°C and 80°C, between 60°C and 75°C, between 60°C and 70°C, between 60°C and 65°C, between 65°C and 90°C, between 65°C and 80°C, between 65°C and 75°C, between 65°C and 70°C. In particular, the thermostability of the esterase is improved under acidic conditions compared to the thermostability of the parent esterase at temperatures between 40°C and 80°C, between 50°C and 72°C, between 55°C and 60°C, between 50°C and 55°C, between 60°C and 72°C. In a particular embodiment, the thermostability of the esterase is improved compared to the thermostability of the parent esterase at least at temperatures between 50°C and 65°C. In the context of the present invention, temperatures are given as +/-1°C.

特定言之，可經由估計酯酶之解鏈溫度(Tm)來評估熱穩定性。在本發明之上下文中，「解鏈溫度」係指所考慮的酶群體中之一半展開或錯誤摺疊的溫度。通常，與親本酯酶在酸性條件下，尤其在包含於6與10之間的pH下之Tm相比，本發明之酯酶顯示Tm增加了約0.8℃、1℃、2℃、3℃、4℃、5℃、10℃或更高。特定言之，在包含於6與10之間的pH下，與親本酯酶相比，本發明之酯酶可在50℃與90℃之間的溫度下具有增加之半衰期。特定言之，與SEQ ID NO: 1之酯酶相比，本發明之酯酶可在50℃與90℃之間、50℃與80℃之間、50℃與75℃之間、50℃與70℃之間、50℃與65℃之間、55℃與90℃之間、55℃與80℃之間、55℃與75℃之間、55℃與70℃之間、55℃與65℃之間、60℃與90℃之間、60℃與80℃之間、60℃與75℃之間、60℃與70℃之間、60℃與65℃之間、65℃與90℃之間、65℃與80℃之間、65℃與75℃之間、65℃與70℃之間的溫度下具有增加之半衰期。較佳地，與親本酯酶相比，本發明之酯酶至少在50℃與65℃之間的溫度下具有增加之半衰期。In particular, thermal stability can be assessed by estimating the melting temperature (Tm) of the esterase. In the context of the present invention, "melting temperature" refers to the temperature at which one half of the enzyme population under consideration is unfolded or misfolded. Typically, the esterase of the present invention shows an increase in Tm of about 0.8°C, 1°C, 2°C, 3°C, 4°C, 5°C, 10°C or more compared to the Tm of the parent esterase under acidic conditions, especially at a pH comprised between 6 and 10. In particular, at a pH comprised between 6 and 10, the esterase of the present invention may have an increased half-life at a temperature between 50°C and 90°C compared to the parent esterase. Specifically, the esterases of the present invention may have an increased half-life at temperatures between 50°C and 90°C, between 50°C and 80°C, between 50°C and 75°C, between 50°C and 70°C, between 50°C and 65°C, between 55°C and 90°C, between 55°C and 80°C, between 55°C and 75°C, between 55°C and 70°C, between 55°C and 65°C, between 60°C and 90°C, between 60°C and 80°C, between 60°C and 75°C, between 60°C and 70°C, between 60°C and 65°C, between 65°C and 90°C, between 65°C and 80°C, between 65°C and 75°C, between 65°C and 70°C. Preferably, the esterase of the invention has an increased half-life at least at a temperature between 50°C and 65°C compared to the parent esterase.

在本發明之上下文內，本發明之酯酶至少在包含於6與10之間的pH下與具有SEQ ID NO: 1或SEQ ID NO: 2中所示之胺基酸序列之酯酶(亦即親本酯酶)相比展現增加之熱穩定性。較佳地，酯酶至少在包含於6.5與9之間的pH下、更佳在包含於7與9之間的pH下、甚至更佳在包含於7.5與9之間的pH下、例如在pH 8下展現增加之熱穩定性。In the context of the present invention, the esterase of the present invention exhibits increased thermostability compared to the esterase having the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 (i.e. the parent esterase) at least at a pH comprised between 6 and 10. Preferably, the esterase exhibits increased thermostability at least at a pH comprised between 6.5 and 9, more preferably at a pH comprised between 7 and 9, even more preferably at a pH comprised between 7.5 and 9, for example at pH 8.

酯酶之解鏈溫度(Tm)可藉由熟習此項技術者根據此項技術中本身已知的方法來進行量測。舉例而言，可使用DSF來量化酯酶之熱變性溫度之變化，且由此判定該酯酶之Tm。替代地，可藉由使用圓二色性來分析蛋白質摺疊而估計Tm。較佳地，使用DSF或圓二色性來量測Tm，如實驗部分中所揭露。在本發明之上下文中，將Tm與在相同條件(例如，聚酯之pH、性質及量等)下量測之Tm進行比較。The melting temperature (Tm) of an esterase can be measured by a person skilled in the art according to methods known per se in the art. For example, DSF can be used to quantify the change in the thermal denaturation temperature of an esterase, and the Tm of the esterase can be determined thereby. Alternatively, Tm can be estimated by analyzing protein folding using circular dichroism. Preferably, Tm is measured using DSF or circular dichroism, as disclosed in the experimental section. In the context of the present invention, Tm is compared to Tm measured under the same conditions (e.g., pH, properties and amount of polyester, etc.).

替代地，可藉由量測在不同溫度下培育之後酯酶之酯酶活性及/或聚酯解聚合活性且與親本酯酶之酯酶活性及/或聚酯解聚合活性進行比較來評估熱穩定性。亦可評估在不同溫度下進行多輪聚酯解聚合分析之能力。快速且有價值的測試可由藉由暈圈直徑量測來評估酯酶在不同溫度下培育之後使分散於瓊脂培養盤中之固體聚酯化合物降解的能力組成。Alternatively, thermal stability can be assessed by measuring the esterase activity and/or polyester depolymerization activity of the esterase after incubation at different temperatures and comparing it to the esterase activity and/or polyester depolymerization activity of the parent esterase. The ability to perform multiple rounds of polyester depolymerization analysis at different temperatures can also be assessed. A rapid and valuable test can consist of assessing the ability of the esterase to degrade solid polyester compounds dispersed in agar culture plates after incubation at different temperatures by halo diameter measurement.

本發明之一目標為提供一種酯酶，其：(i)與SEQ ID NO: 1中所示之全長胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%一致性；(ii)與SEQ ID NO: 1相比，具有至少一種選自以下之胺基酸取代：H183Q/L/A/Y/N/D/E/W、R30G、A64V、A68N、T109S、L124G、S145G、L152M、E158A、K159R、S193L/Q、W228R/S、V242E、G7A、N9T、A24Q、Y26H、T27A、G39S、G46V、T50M、A64T、R73H、L74V、Y106F、R108L、V167L、I169M、P179N、I185V、F187L/Q、P192S、P213A、L227N、Q237L、L239M、R251S、S66T、F90C/V、P151V、A246R、A215E/D、D230E、L15K/R、N143D、V200I及N253D，或至少一種選自以下之取代的組合：N204G + M208L + N211E、F90E + N204G + N211E、F90N + N204G + N211E、F90Q + N204G + N211E、F90R + N204G + N211E、F90W + N204G + N211E、F187I + N204G + N211E、S98R + E173Q、M208Q + N211M及M208N + N211M，其中參考SEQ ID NO: 1中所示之胺基酸序列對位置進行編號；(iii)具有聚酯降解活性；及(iv)與SEQ ID NO: 1之酯酶相比，在包含於6與10之間的pH下展現增加之熱穩定性及/或增加之聚酯降解活性。An object of the present invention is to provide an esterase which: (i) has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the full-length amino acid sequence shown in SEQ ID NO: 1; (ii) has at least one amino acid substitution selected from the group consisting of H183Q/L/A/Y/N/D/E/W, R30G, A64V, A68N, T109S, L124G, S145G, L152M, E158A, K159R, S193L/Q, W228R/S, V242E, G7A, N9T, A24Q, Y26H, T27A, G39S, G46V, T50M, A64T, R73H, L74 V, Y106F, R108L, V167L, I169M, P179N, I185V, F187L/Q, P192S, P213A, L227N, Q237L, L239M, R251S, S66T, F90C/V, P151V, A246R, A215E/D, D230E, L15K/R, N143D, V200I, and N253D, or a combination of at least one substitution selected from the following: N204G + M208L + N211E, F90E + N204G + N211E, F90N + N204G + N211E, F90Q + N204G + N211E, F90R + N204G + N211E, F90W + N204G + N211E, F187I + N204G + N211E, S98R + E173Q, M208Q + N211M and M208N + N211M, wherein the positions are numbered with reference to the amino acid sequence shown in SEQ ID NO: 1; (iii) have polyester degradation activity; and (iv) exhibit increased thermostability and/or increased polyester degradation activity at a pH comprised between 6 and 10 compared to the esterase of SEQ ID NO: 1.

在實施例中，酯酶包含選自以下之至少一種胺基酸取代或至少一種取代之組合：H183Q/L/A/Y/N/D/E/W、R30G、A64V、A68N、T109S、L124G、S145G、L152M、E158A、K159R、S193L/Q、W228R/S、V242E、G7A、N9T、A24Q、Y26H、T27A、G39S、G46V、T50M、A64T、R73H、L74V、Y106F、R108L、V167L、I169M、P179N、I185V、F187L/Q、P192S、P213A、L227N、Q237L、L239M、R251S、S66T、F90C/V、P151V、A246R、A215E/D、D230E、L15K/R、N253D、N204G + M208L + N211E、F90E + N204G + N211E、F90N + N204G + N211E、F90Q + N204G + N211E、F90R + N204G + N211E、F90W + N204G + N211E、F187I + N204G + N211E、S98R + E173Q及M208N + N211M。In an embodiment, the esterase comprises at least one amino acid substitution or a combination of at least one substitution selected from the group consisting of H183Q/L/A/Y/N/D/E/W, R30G, A64V, A68N, T109S, L124G, S145G, L152M, E158A, K159R, S193L/Q, W228R/S, V242E, G7A, N9T, A24Q, Y26H, T27A, G39S, G4 6V, T50M, A64T, R73H, L74V, Y106F, R108L, V167L, I169M, P179N, I185V, F187L/Q, P192S, P213A, L227N, Q237L, L239M, R251S, S66T, F90C/V, P151V, A246R, A215E/D, D230E, L15K/R, N253D, N204G + M208L + N211E, F90E + N204G + N211E, F90N + N204G + N211E, F90Q + N204G + N211E, F90R + N204G + N211E, F90W + N204G + N211E, F187I + N204G + N211E, S98R + E173Q and M208N + N211M.

在實施例中，酯酶包含至少一種選自以下之胺基酸取代：H183Q/L/A/Y/N/D/E/W、R30G、A64V、A68N、T109S、L124G、S145G、L152M、E158A、K159R、S193L/Q、W228R/S、V242E、G7A、N9T、A24Q、Y26H、T27A、G39S、G46V、T50M、A64T、R73H、L74V、Y106F、R108L、V167L、I169M、P179N、I185V、F187L/Q、P192S、P213A、L227N、Q237L、L239M、R251S、S66T、F90C/V、P151V、A246R、A215E/D、D230E、L15K/R，或至少一種選自以下之取代的組合：N204G + M208L + N211E、F90E + N204G + N211E、F90N + N204G + N211E、F90Q + N204G + N211E、F90R + N204G + N211E、F90W + N204G + N211E、F187I + N204G + N211E、S98R + E173Q。In an embodiment, the esterase comprises at least one amino acid substitution selected from the group consisting of H183Q/L/A/Y/N/D/E/W, R30G, A64V, A68N, T109S, L124G, S145G, L152M, E158A, K159R, S193L/Q, W228R/S, V242E, G7A, N9T, A24Q, Y26H, T27A, G39S, G46V, T50M, A6 4T, R73H, L74V, Y106F, R108L, V167L, I169M, P179N, I185V, F187L/Q, P192S, P213A, L227N, Q237L, L239M, R251S, S66T, F90C/V, P151V, A246R, A215E/D, D230E, L15K/R, or a combination of at least one substitution selected from the following: N204G + M208L + N211E, F90E + N204G + N211E, F90N + N204G + N211E, F90Q + N204G + N211E, F90R + N204G + N211E, F90W + N204G + N211E, F187I + N204G + N211E, S98R + E173Q.

在實施例中，酯酶包含至少一種選自以下之取代：R30G、A64V、A68N、T109S、L124G、S145G、L152M、E158A、K159R、H183Q/L/A/Y/N/D/E/W、S193L/Q、W228R/S、V242E、G7A、N9T、A24Q、Y26H、T27A、G39S、G46V、T50M、A64T、R73H、L74V、Y106F、R108L、V167L、I169M、P179N、I185V、F187L/Q、P192S、P213A、L227N、Q237L、L239M及R251S，較佳選自R30G、A64V、A68N、T109S、L124G、S145G、L152M、E158A、K159R、H183Q/L/A/Y/N/D/E/W、S193L/Q、W228R/S、V242E，更佳選自R30G、A64V、T109S、S145G、E158A、H183Q/N、W228S及V242E。In embodiments, the esterase comprises at least one substitution selected from the group consisting of R30G, A64V, A68N, T109S, L124G, S145G, L152M, E158A, K159R, H183Q/L/A/Y/N/D/E/W, S193L/Q, W228R/S, V242E, G7A, N9T, A24Q, Y26H, T27A, G39S, G46V, T50M, A64T, R73H, L74V, Y106F, R108L, V167L, I169M, P179N, I185V, F187L/Q, P192S, P213A, L227N, Q237L, L239M and R251S, preferably R30G, A64V, A68N, T109S, L124G, S145G, L152M, E158A, K159R, H183Q/L/A/Y/N/D/E/W, S193L/Q, W228R/S, V242E, more preferably R30G, A64V, T109S, S145G, E158A, H183Q/N, W228S and V242E.

在實施例中，酯酶包含選自以下之至少一種取代，較佳為至少兩種取代：R30G、A64V、A68N、T109S、L124G、S145G、L152M、E158A、K159R、H183Q/L/A/Y/N/D/E/W、S193L/Q、W228R/S、V242E、G7A、N9T、A24Q、Y26H、T27A、G39S、G46V、T50M、A64T、S66N、R73H、L74V、Y106F、R108L、V167L、I169M、P179N、I185V、F187L、P192S、P213A、L227N、Q237L、L239M及R251S。In an embodiment, the esterase comprises at least one substitution selected from the following, preferably at least two substitutions: R30G, A64V, A68N, T109S, L124G, S145G, L152M, E158A, K159R, H183Q/L/A/Y/N/D/E/W, S193L/Q, W228R/S, V242E, G7A, N 9T, A24Q, Y26H, T27A, G39S, G46V, T50M, A64T, S66N, R73H, L74V, Y106F, R108L, V167L, I169M, P179N, I185V, F187L, P192S, P213A, L227N, Q237L, L239M and R251S.

在另一實施例中，酯酶包含至少一種選自以下之取代：R30G、A64V、L124G、S145G、H183Q/L/A/Y/N/D/E/W、S193L/Q、W228S及V242E，較佳選自R30G、A64V、L124G、S145G、H183Q/N、S193L/Q、W228S及V242E。In another embodiment, the esterase comprises at least one substitution selected from the group consisting of R30G, A64V, L124G, S145G, H183Q/L/A/Y/N/D/E/W, S193L/Q, W228S and V242E, preferably selected from the group consisting of R30G, A64V, L124G, S145G, H183Q/N, S193L/Q, W228S and V242E.

根據本發明，酯酶可進一步包含在至少一個選自以下之胺基酸位置處的取代：S1、Y4、Q5、R6、N9、P10、T11、R12、L13、A14、L15、T16、A17、D18、S22、T25、Y26、T27、V28、S29、R30、L31、S32、V33、S34、G35、F36、G37、G38、G39、Y43、S48、T50、G53、I54、M56、P58、G59、Y60、T61、A62、D63、A64、S65、S66、L67、A68、W69、L70、R72、R73、L74、L82、I84、N85、T86、N87、S88、R89、F90、D91、G92、P93、D94、S95、R96、S98、Q99、A103、L104、N105、L107、R108、S113、V115、L119、A121、N122、L124、A125、A127、G128、H129、M131、G132、G133、G134、G135、R138、A140、Q142、N143、S145、K147、A149、V150、L152、T153、P154、W155、H156、T157、E158、K159、T160、N162、S164、V167、L168、I170、A172、E173、A174、T176、V177、A178、P179、S181、Q182、H183、F187、Q189、N190、S193、T194、P196、V198、V200、L202、C203、N204、A205、S206、M208、A209、P210、N211、S212、P213、N214、A215、A216、I217、S218、V219、Y220、T221、S223、W224、M225、N231、T233、R236、Q237、F238、L239、N241、V242、N243、D244、P245、A246、L247、C248、T252、N253、N254、R255、H256、Q258、F161及T163，其中參考SEQ ID NO: 1中所示之胺基酸序列對位置進行編號，較佳在至少一個選自以下之位置處：S1、Y4、Q5、R6、P10、T11、R12、L13、A14、L15、T16、A17、D18、S22、T25、V28、S29、L31、S32、V33、S34、G35、F36、G37、G38、Y43、S48、、G53、I54、M56、P58、G59、Y60、T61、A62、D63、S65、L67、W69、L70、R72、L82、I84、N85、T86、N87、S88、R89、F90、D91、G92、P93、D94、S95、R96、S98、Q99、A103、L104、N105、L107、S113、V115、L119、A121、N122、A125、A127、G128、H129、M131、G132、G133、G134、G135、R138、A140、Q142、N143、K147、A149、V150、T153、P154、W155、H156、T157、T160、N162、S164、L168、I170、A172、E173、A174、T176、V177、A178、S181、Q182、Q189、N190、T194、P196、V198、V200、L202、C203、N204、A205、S206、M208、A209、P210、N211、S212、N214、A215、A216、I217、S218、V219、Y220、T221、S223、W224、M225、N231、T233、R236、Q237、F238、N241、N243、D244、P245、A246、L247、C248、T252、N253、N254、R255、H256、Q258、F161及T163，更佳選自G35、W69、F90、N162、S181、N204、A216、N243、A246、V28、T157、M208、N211、A17、V200、I170、N122、Q142、Q237、Q258、N85、N105、L13、E158，甚至更佳選自G35、W69、F90、V115I、N162、S181、N204、A216、N243、A246、V28及T157。較佳地，酯酶進一步包含至少一種選自以下之取代：G35A、W69L、F90L/Y、N162S、S181K、N204G、A216C、N243Y、A246C、V28I、T157P、T16K/R、Y4K/R、V219E/D、M208N、N211M、A17F、 M208L、M208T、V200I、I170V、N122D、Q142E、Q237E、Q258E、N85D、N105D、L13S、E158D，較佳選自G35A、W69L、F90L/Y、V115I、N162S、S181K、N204G、A216C、N243Y、A246C、V28I、T157P、T16K/R、Y4K/R及V219E/D，更佳選自G35A、W69L、F90L/Y、V115I、N162S、S181K、N204G、A216C、N243Y、A246C、V28I及T157P。舉例而言，酯酶進一步包含至少一種選自S66N + M208T及A62T + M208T之取代的組合。在特定實施例中，酯酶進一步至少包含選自L13S + E158D之取代的組合。According to the present invention, the esterase may further comprise a substitution at at least one amino acid position selected from the group consisting of: S1, Y4, Q5, R6, N9, P10, T11, R12, L13, A14, L15, T16, A17, D18, S22, T25, Y26, T27, V28, S29, R30, L31, S32, V33, S34, G35, F36, G37, G38, G39, Y43, S48, T50, G53, I54, M56, P58, G59, Y60, T61, A62, D63, A64, S65, S66 6. L67, A68, W69, L70, R72, R73, L74, L82, I84, N85, T86, N87, S88, R89, F90, D91, G92, P93, D94, S95, R96, S98, Q99, A103, L104 , N105, L107, R108, S113, V115, L119, A121, N122, L124, A125, A127, G128, H129, M131, G132, G133, G134, G135, R138, A140, Q14 2. N143, S145, K147, A149, V150, L152, T153, P154, W155, H156, T157, E158, K159, T160, N162, S164, V167, L168, I170, A172, E 173, A174, T176, V177, A178, P179, S181, Q182, H183, F187, Q189, N190, S193, T194, P196, V198, V200, L202, C203, N204, A205, , S206, M208, A209, P210, N211, S212, P213, N214, A215, A216, I217, S218, V219, Y220, T221, S223, W224, M225, N231, T233, R236, Q237, F238, L239, N241, V242, N243, D244, P245, A246, L247, C248, T252, N253, N254, R255, H256, Q258, F161, and T163, with reference to SEQ ID NO: The amino acid sequence shown in 1 is numbered for positions, preferably at least one position selected from the following: S1, Y4, Q5, R6, P10, T11, R12, L13, A14, L15, T16, A17, D18, S22, T25, V28, S29, L31, S32, V33, S34, G35, F36, G37, G38, Y43, S48,, G53, I54, M56, P58, G59, Y60, T61, A62, D63, S65, L67, W69, L70, R72, L82, I84, N85, T86, N87, S88, R89 , F90, D91, G92, P93, D94, S95, R96, S98, Q99, A103, L104, N105, L107, S113, V115, L119, A121, N122, A125, A127, G128, H129, M131, G132 , G133, G134, G135, R138, A140, Q142, N143, K147, A149, V150, T153, P154, W155, H156, T157, T160, N162, S164, L168, I170, A172, E173, A174, T176, V177, A178, S181, Q182, Q189, N190, T194, P196, V198, V200, L202, C203, N204, A205, S206, M208, A209, P210, N211, S212, N 214, A215, A216, I217, S218, V219, Y220, T221, S223, W224, M225, N231, T233, R236, Q237, F238, N241, N243, D244, P245, A246, L247, C2 48, T252, N253, N254, R255, H256, Q258, F161 and T163, more preferably selected from G35, W69, F90, N162, S181, N204, A216, N243, A246, V28, T157, M208, N211, A17, V200, I170, N122, Q142, Q237, Q258, N85, N105, L13, E158, even more preferably selected from G35, W69, F90, V115I, N162, S181, N204, A216, N243, A246, V28 and T157. Preferably, the esterase further comprises at least one substitution selected from the group consisting of G35A, W69L, F90L/Y, N162S, S181K, N204G, A216C, N243Y, A246C, V28I, T157P, T16K/R, Y4K/R, V219E/D, M208N, N211M, A17F, M208L, M208T, V200I, I170V, N122D, Q142E, Q237E, Q258E, N85D, N105D, L13S, E158D, preferably G35A, W69L, F90L/Y, V115I, N162S, S181K, N204G, A216C, N243Y, A246C, V28I, T157P, T16K/R, Y4K/R and V219E/D, more preferably G35A, W69L, F90L/Y, V115I, N162S, S181K, N204G, A216C, N243Y, A246C, V28I and T157P. For example, the esterase further comprises at least one combination of substitutions selected from S66N + M208T and A62T + M208T. In a specific embodiment, the esterase further comprises at least a combination of substitutions selected from L13S + E158D.

在實施例中，酯酶包含至少一種選自A215E/D、D230E及L15K/R之取代，且進一步包含至少一種選自T16K/R、Y4K/R及V219E/D之取代。特定言之，酯酶變異體包含選自A215E + T16K/R、A215D + T16R、D230E + Y4K/R及V219E/D + L15K/R之取代的組合。In an embodiment, the esterase comprises at least one substitution selected from A215E/D, D230E and L15K/R, and further comprises at least one substitution selected from T16K/R, Y4K/R and V219E/D. In particular, the esterase variant comprises a combination of substitutions selected from A215E+T16K/R, A215D+T16R, D230E+Y4K/R and V219E/D+L15K/R.

在實施例中，酯酶包含至少一種選自以下之取代：R30G、A64V、A68N、T109S、L124G、S145G、L152M、E158A、K159R、H183Q/L/A/Y/N/D/E/W、S193L/Q、W228R/S、V242E、N143D及N253D，較佳選自R30G、A64V、A68N、T109S、L124G、S145G、L152M、E158A、K159R、H183Q/L/A/Y/N/D/E/W、S193L/Q、W228R/S、V242E及N253D，更佳選自R30G、A64V、A68N、T109S、L124G、S145G、L152M、E158A、K159R、H183Q/L/A/Y/N/D/E/W、S193L/Q、W228R/S、V242E，或至少一種選自以下之取代的組合：H183L + F187I、H183A + F187I、H183Y + F187I、H183D + F187I、H183W + F187I、H183E + F187I、A62T + S66N + M208T、F90A + H183S + F187L、F90L + H183E + F187L、F90S + H183S + F187L、F90Y + H183L + F187L S22R + G39S + P179N + L239M、M208Q + N211M及M208N + N211M，較佳選自H183L + F187I、H183A + F187I、H183Y + F187I、H183D + F187I、H183W + F187I、H183E + F187I、A62T + S66N + M208T、F90A + H183S + F187L、F90L + H183E + F187L、F90S + H183S + F187L、F90Y + H183L + F187L及S22R + G39S + P179N + L239M。In an embodiment, the esterase comprises at least one substitution selected from the group consisting of R30G, A64V, A68N, T109S, L124G, S145G, L152M, E158A, K159R, H183Q/L/A/Y/N/D/E/W, S193L/Q, W228R/S, V242E, N143D, and N253D, preferably selected from the group consisting of R30G, A64V, A68N, T109S, L124G, S145G, L152M, E158A, K159R, H183Q/L/A/Y/N/D/E/W, S193L/Q, W228R/S, V242E, N143D, and N253D. A, K159R, H183Q/L/A/Y/N/D/E/W, S193L/Q, W228R/S, V242E and N253D, more preferably selected from R30G, A64V, A68N, T109S, L124G, S145G, L152M, E158A, K159R, H183Q/L/A/Y/N/D/E/W, S193L/Q, W228R/S, V242E, or at least one substituted combination selected from the following: H183L + F187I, H183A + F187I, H183Y + F187I, H183D + F187I, H183W + F187I, H183E + F187I, A62T + S66N + M208T, F90A + H183S + F187L, F90L + H183E + F187L, F90S + H183S + F187L, F90Y + H183L + F187L S22R + G39S + P179N + L239M, M208Q + N211M and M208N + N211M, preferably H183L + F187I, H183A + F187I, H183Y + F187I, H183D + F187I, H183W + F187I, H183E + F187I, A62T + S66N + M208T, F90A + H183S + F187L, F90L + H183E + F187L, F90S + H183S + F187L, F90Y + H183L + F187L and S22R + G39S + P179N + L239M.

較佳地，酯酶包含至少一種選自以下之取代或至少一種取代的組合：R30G、A64V、A68N、T109S、L124G、S145G、L152M、E158A、K159R、H183Q/N、 S193L/Q、W228R/S、V242E、H183L + F187I、H183A + F187I、H183Y + F187I、H183D + F187I、H183W + F187I、H183E + F187I、A62T + S66N + M208T、F90A + H183S + F187L、F90L + H183E +  F187L、F90S + H183S + F187L、F90Y + H183L + F187L及S22R + G39S + P179N + L239M，更佳選自R30G、A64V、T109S、S145G、E158A、H183Q/N、W228S、V242E、H183A + F187I、H183Y + F187I、H183D + F187I、H183W + F187I、H183E + F187I、、F90A + H183S + F187L、F90L + H183E + F187L、F90S + H183S + F187L及F90Y + H183L + F187L。Preferably, the esterase comprises at least one substitution or a combination of at least one substitution selected from the group consisting of R30G, A64V, A68N, T109S, L124G, S145G, L152M, E158A, K159R, H183Q/N, S193L/Q, W228R/S, V242E, H183L + F187I, H183A + F187I, H183Y + F187I, H183D + F187I, H183W + F187I, H183E + F187I, A62T + S66N + M208T, F90A + H183S + F187L, F90L + H183E +  F187L, F90S + H183S + F187L, F90Y + H183L + F187L and S22R + G39S + P179N + L239M, preferably R30G, A64V, T109S, S145G, E158A, H183Q/N, W228S, V242E, H183A + F187I, H183Y + F187I, H183D + F187I, H183W + F187I, H183E + F187I, F90A + H183S + F187L, F90L + H183E + F187L, F90S + H183S + F187L and F90Y + H183L + F187L.

在實施例中，酯酶包含至少一種選自以下之取代：R30G、A64V、L124G、S145G、H183Q/N、S193L/Q、W228S及V242E，且進一步包含至少一種選自以下之取代：G35A、W69L、F90L/Y、V115I、N162S、S181K、N204G、A216C、N243Y、A246C、V28I、T157P、A17F、F90Q、M208L、N211M、N122D、S193E、Q142E、N143D、Q237E、N253D、Q258E、V200I、I170V、N85D、N105D、N211E、F187I、M208N、M208T、L13S、E158D，較佳選自G35A、W69L、F90L/Y、V115I、N162S、S181K、N204G、A216C、N243Y、A246C、V28I及T157P。In an embodiment, the esterase comprises at least one substitution selected from the group consisting of R30G, A64V, L124G, S145G, H183Q/N, S193L/Q, W228S, and V242E, and further comprises at least one substitution selected from the group consisting of G35A, W69L, F90L/Y, V115I, N162S, S181K, N204G, A216C, N243Y, A246C, V28I, T157P, A17F, F90Q, M208L, N2 11M, N122D, S193E, Q142E, N143D, Q237E, N253D, Q258E, V200I, I170V, N85D, N105D, N211E, F187I, M208N, M208T, L13S, E158D, preferably G35A, W69L, F90L/Y, V115I, N162S, S181K, N204G, A216C, N243Y, A246C, V28I and T157P.

在實施例中，酯酶包含取代R30G，且進一步包含至少一種選自H183Q、N204G、F187I、T27A、S145G、W228S及V242E之取代。特定言之，酯酶包含至少一種選自R30G + H183Q、N204G + H183Q + F187I + R30G及T27A + R30G + S145G + W228S + V242E之取代的組合。In an embodiment, the esterase comprises the substitution R30G and further comprises at least one substitution selected from H183Q, N204G, F187I, T27A, S145G, W228S and V242E. Specifically, the esterase comprises at least one combination of substitutions selected from R30G + H183Q, N204G + H183Q + F187I + R30G and T27A + R30G + S145G + W228S + V242E.

在實施例中，酯酶至少包含選自V200I + I170V之取代的組合或由具有V200I + I170V之取代的組合的SEQ ID NO: 1中所示之胺基酸序列組成。In an embodiment, the esterase comprises at least a combination of substitutions selected from V200I + I170V or consists of the amino acid sequence shown in SEQ ID NO: 1 having a combination of substitutions V200I + I170V.

在實施例中，酯酶包含取代A64V，且進一步包含至少一種選自N243Y、V115I、N204G、H183Q及F187I之取代。特定言之，酯酶包含至少一種選自A64V + N243Y、A64V + V115I + N243Y及N204G + H183Q + F187I + A64V之取代的組合。In an embodiment, the esterase comprises the substitution A64V and further comprises at least one substitution selected from N243Y, V115I, N204G, H183Q and F187I. Specifically, the esterase comprises at least one combination of substitutions selected from A64V + N243Y, A64V + V115I + N243Y and N204G + H183Q + F187I + A64V.

在實施例中，酯酶包含取代S145G，且進一步包含至少一種選自T27A、R30G、W228S及V242E之取代。特定言之，酯酶至少包含取代T27A + R30G + S145G + W228S + V242E之組合。In an embodiment, the esterase comprises the substitution S145G and further comprises at least one substitution selected from T27A, R30G, W228S and V242E. Specifically, the esterase comprises at least the combination of substitutions T27A + R30G + S145G + W228S + V242E.

在實施例中，酯酶包含至少一種選自H183Q/L/A/Y/N/D/E/W之取代，且進一步包含至少一種選自以下之取代：F187I、F187L、A24Q、R30G、L74V、F187Q、F187I、L227N、R251S、Y26H、A64T、G35A、A62T、G46V、R73H、T50M、I169M、Y106F、I185V、R108L、V167L、N204G、G39S、A64V、L124G、N162S、S193Q、Q237L、M208T、S181K、W69L、A216C、A246C、F90I、F90Y、A17F、F90Q、M208L、N211M、N122D、S193E、Q142E、N143D、Q237E、N253D、Q258E、V200I、I170V、N85D、N105D、N211E、M208N、M208T、L13S及E158D，較佳選自F187I、F187L、A24Q、R30G、L74V、F187Q、F187I、L227N、R251S、Y26H、A64T、G35A、A62T、G46V、R73H、T50M、I169M、Y106F、I185V、R108L、V167L、N204G、G39S、A64V、L124G、N162S、S193Q、Q237L、M208T、S181K、W69L、A216C、A246C、F90I及F90Y。特定言之，酯酶包含至少一種選自以下之取代的組合或由具有選自以下之取代的組合的SEQ ID NO: 1中所示之胺基酸序列組成：H183L + F187I、H183A + F187I、H183Y + F187I、H183D + F187I、H183W + F187I、H183E + F187I、F90L + H183E + F187L、F90Y + H183L + F187L、A24Q + H183Q、R30G + H183Q、L74V + H183Q、H183Q + F187Q、H183Q + F187I、L227N + H183Q、R251S + H183Q、Y26H + A64T + H183Q、G35A + A62T + H183Q、G46V + R73H + H183Q、T50M + I169M + H183Q、Y106F + I185V + H183Q、R108L + V167L + H183Q、H183Q + F187I + N204G、N204G + H183Q + F187I + R30G、N204G + H183Q + F187I + G39S、N204G + H183Q + F187I + A62T、N204G + H183Q + F187I + A64V、N204G + H183Q + F187I + L124G、N204G + H183Q + F187I + N162S、N204G + H183Q + F187I + S193Q、N204G + H183Q + F187I + Q237L、M208T + N204G + H183Q + F187I、N204G + H183Q + F187I + N162S + S181K、N204G + H183Q + F187I + N162S + S181K + S193Q及N204G + H183Q + F187I + W69L + A216C + A246C、A17F + F90Q + H183N + N204G + M208L + N211M、H183Q + F187I + N204G + M208N + N211M + S193E、M208T + N204G + H183Q + F187I + N211M + S193E、H183Q + F187I + N204G + N211M + S193E + V200I + I170V、A17F + F90Q + N122D + H183N + S193E + N204G + M208L + N211M、A17F + F90Q + Q142E + H183N + S193E + N204G + M208L + N211M、A17F + F90Q + N143D + H183N + S193E + N204G + M208L + N211M、A17F + F90Q + H183N + S193E + N204G + M208L + N211M + Q237E、A17F + F90Q + H183N + S193E + N204G + M208L + N211M + N253D、A17F + F90Q + H183N + S193E + N204G + M208L + N211M + Q258E、H183Q + F187I + N204G + M208N + N211M + S193E + V200I + I170V、M208T + N204G + H183Q + F187I + N211M + S193E + V200I + I170V、A17F + N85D + F90Q + H183N + S193E + N204G + M208L + N211M、A17F + F90Q + N105D + H183N + S193E + N204G + M208L + N211E、H183Q + F187I + N204G + L13S + E158D、M208T + N204G + H183Q + F187I + L13S + E158D、A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I、A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + H183Q + F187I + N204G、A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I + L13S + E158D，較佳選自H183L + F187I、H183A + F187I、H183Y + F187I、H183D + F187I、H183W + F187I、H183E + F187I、F90L + H183E + F187L、F90Y + H183L +  F187L、A24Q + H183Q、R30G + H183Q、L74V + H183Q、H183Q + F187Q、H183Q + F187I、L227N + H183Q、R251S + H183Q、Y26H + A64T + H183Q、G35A + A62T + H183Q、G46V + R73H + H183Q、T50M + I169M + H183Q、Y106F + I185V + H183Q、R108L + V167L + H183Q、H183Q + F187I + N204G、N204G + H183Q + F187I + R30G、N204G + H183Q + F187I + G39S、N204G + H183Q + F187I + A62T、N204G + H183Q + F187I + A64V、N204G + H183Q + F187I + L124G、N204G + H183Q + F187I + N162S、N204G + H183Q + F187I + S193Q、N204G + H183Q + F187I + Q237L、M208T + N204G + H183Q + F187I、N204G + H183Q + F187I + N162S + S181K、N204G + H183Q + F187I + N162S + S181K + S193Q及N204G + H183Q + F187I + W69L + A216C + A246C。In an embodiment, the esterase comprises at least one substitution selected from H183Q/L/A/Y/N/D/E/W, and further comprises at least one substitution selected from F187I, F187L, A24Q, R30G, L74V, F187Q, F187I, L227N, R251S, Y26H, A64T, G35A, A62T, G46V, R73H, T50 M, I169M, Y106F, I185V, R108L, V167L, N204G, G39S, A64V, L124G, N162S, S193Q, Q237L , M208T, S181K, W69L, A216C, A246C, F90I, F90Y, A17F, F90Q, M208L, N211M, N122D, S19 3E, Q142E, N143D, Q237E, N253D, Q258E, V200I, I170V, N85D, N105D, N211E, M208N, M208T, L13S and E158D, preferably F187I, F187L, A24Q, R30G, L74V, F187Q, F187I, L227N, R251S, Y2 6H, A64T, G35A, A62T, G46V, R73H, T50M, I169M, Y106F, I185V, R108L, V167L, N204G, G3 9S, A64V, L124G, N162S, S193Q, Q237L, M208T, S181K, W69L, A216C, A246C, F90I and F90Y. In particular, the esterase comprises at least one combination of substitutions selected from the group consisting of or consists of the amino acid sequence shown in SEQ ID NO: 1 having a combination of substitutions selected from the group consisting of H183L + F187I, H183A + F187I, H183Y + F187I, H183D + F187I, H183W + F187I, H183E + F187I, F90L + H183E + F187L, F90Y + H183L + F187L, A24Q + H183Q, R30G + H183Q, L74V + H183Q, H183Q + F187Q, H183Q + F187I, L227N + H183Q, R251S + H183Q, Y26H + A64T + H183Q, G35A + A62T + H183Q, G46V + R73H + H183Q, T50M + I169M + H183Q, Y106F + I185V + H183Q, R108L + V167L + H183Q, H183Q + F187I + N204G, N204G + H183Q + F187I + R30G, N204G + H183Q + F187I + G39S, N204G + H183Q + F187I + A62T, N204G + H183Q + F187I + A64V, N204G + H183Q + F187I + L124G, N204G + H183Q + F187I + N162S, N204G + H183Q + F187I + S193Q, N204G + H183Q + F187I + Q237L, M208T + N204G + H183Q + F187I, N204G + H183Q + F187I + N162S + S181K, N204G + H183Q + F187I + N162S + S181K + S193Q and N204G + H183Q + F187I + W69L + A216C + A246C, A17F + F90Q + H183N + N204G + M208L + N211M, H183Q + F187I + N204G + M208N + N211M + S193E, M208T + N204G + H183Q + F187I + N211M + S193E, H183Q + F187I + N204G + N211M + S193E + V200I + I170V, A17F + F90Q + N122D + H183N + S193E + N204G + M208L + N211M, A17F + F90Q + Q142E + H183N + S193E + N204G + M208L + N211M, A17F + F90Q + N143D + H183N + S193E + N204G + M208L + N211M, A17F + F90Q + H183N + S193E + N204G + M208L + N211M + Q237E, A17F + F90Q + H183N + S193E + N204G + M208L + N211M + N253D, A17F + F90Q + H183N + S193E + N204G + M208L + N211M + Q258E, H183Q + F187I + N204G + M208N + N211M + S193E + V200I + I170V, M208T + N204G + H183Q + F187I + N211M + S193E + V200I + I170V, A17F + N85D + F90Q + H183N + S193E + N204G + M208L + N211M, A17F + F90Q + N105D + H183N + S193E + N204G + M208L + N211E, H183Q + F187I + N204G + L13S + E158D, M208T + N204G + H183Q + F187I + L13S + E158D, A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I, A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + H183Q + F187I + N204G, A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I + L13S + E158D, preferably H183L + F187I, H183A + F187I, H183Y + F187I, H183D + F187I, H183W + F187I, H183E + F187I, F90L + H183E + F187L, F90Y + H183L + F187L, A24Q + H183Q, R30G + H183Q, L74V + H183Q, H183Q + F187Q, H183Q + F187I, L227N + H183Q, R251S + H183Q, Y26H + A64T + H183Q, G35A + A62T + H183Q, G46V + R73H + H183Q, T50M + I169M + H183Q, Y106F + I185V + H183Q, R108L + V167L + H183Q, H183Q + F187I + N204G, N204G + H183Q + F187I + R30G, N204G + H183Q + F187I + G39S, N204G + H183Q + F187I + A62T, N204G + H183Q + F187I + A64V, N204G + H183Q + F187I + L124G, N204G + H183Q + F187I + N162S, N204G + H183Q + F187I + S193Q, N204G + H183Q + F187I + Q237L, M208T+ N204G + H183Q + F187I, N204G + H183Q + F187I + N162S + S181K, N204G + H183Q + F187I + N162S + S181K + S193Q and N204G + H183Q + F187I + W69L + A216C + A246C.

在特定實施例中，酯酶包含取代H183Q，且進一步包含選自以下之至少一種取代、至少兩種取代、至少三種取代：A24Q、R30G、L74V、F187Q、F187I、L227N、R251S、Y26H、A64T、G35A、A62T、G46V、R73H、T50M、I169M、Y106F、I185V、R108L、V167L、N204G、G39S、A64V、L124G、N162S、S193Q、Q237L、M208T、S181K、W69L、A216C、A246C、N204G、M208N、N211M、S193E、V200I、I170V、L13S、E158D，較佳選自A24Q、R30G、L74V、F187Q、F187I、L227N、R251S、Y26H、A64T、G35A、A62T、G46V、R73H、T50M、I169M、Y106F、I185V、R108L、V167L、N204G、G39S、A64V、L124G、N162S、S193Q、Q237L、M208T、S181K、W69L、A216C及A246C。特定言之，酯酶包含至少一種選自以下之取代的組合或由具有選自以下之取代的組合的SEQ ID NO: 1中所示之胺基酸序列組成：A24Q + H183Q、R30G + H183Q、L74V + H183Q、H183Q + F187Q、H183Q + F187I、L227N + H183Q、R251S + H183Q、Y26H + A64T + H183Q、G35A + A62T + H183Q、G46V + R73H + H183Q、T50M + I169M + H183Q、Y106F + I185V + H183Q、R108L + V167L + H183Q、H183Q + F187I + N204G、N204G + H183Q + F187I + R30G、N204G + H183Q + F187I + G39S、N204G + H183Q + F187I + A62T、N204G + H183Q + F187I + A64V、N204G + H183Q + F187I + L124G、N204G + H183Q + F187I + N162S、N204G + H183Q + F187I + S193Q、N204G + H183Q + F187I + Q237L、M208T + N204G + H183Q + F187I、N204G + H183Q + F187I + N162S + S181K、N204G + H183Q + F187I + N162S + S181K + S193Q、N204G + H183Q + F187I + W69L + A216C + A246C、H183Q + F187I + N204G + M208N + N211M + S193E、M208T + N204G + H183Q + F187I + N211M + S193E、H183Q + F187I + N204G + N211M + S193E + V200I + I170V、H183Q + F187I + N204G + M208N + N211M + S193E + V200I + I170V、M208T + N204G + H183Q + F187I + N211M + S193E + V200I + I170V、H183Q + F187I + N204G + N211M + S193E、N204G + H183Q + F187I + L13S + E158D、N204G + H183Q + F187I、M208T + N204G + H183Q + F187I + L13S + E158D、H183Q + F187I + N204G + L13S + E158D、M208T + N204G + H183Q + F187I + L13S + E158D、A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I、 A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + H183Q + F187I + N204G、A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I + L13S + E158D，較佳選自A24Q + H183Q、R30G + H183Q、L74V + H183Q、H183Q + F187Q、H183Q + F187I、L227N + H183Q、R251S + H183Q、Y26H + A64T + H183Q、G35A + A62T + H183Q、G46V + R73H + H183Q、T50M + I169M + H183Q、Y106F + I185V + H183Q、R108L + V167L + H183Q、H183Q + F187I + N204G、N204G + H183Q + F187I + R30G、N204G + H183Q + F187I + G39S、N204G + H183Q + F187I + A62T、N204G + H183Q + F187I + A64V、N204G + H183Q + F187I + L124G、N204G + H183Q + F187I + N162S、N204G + H183Q + F187I + S193Q、N204G + H183Q + F187I + Q237L、M208T + N204G + H183Q + F187I、N204G + H183Q + F187I + N162S + S181K、N204G + H183Q + F187I + N162S + S181K + S193Q及N204G + H183Q + F187I + W69L + A216C + A246C。In certain embodiments, the esterase comprises the substitution H183Q and further comprises at least one substitution, at least two substitutions, at least three substitutions selected from the group consisting of A24Q, R30G, L74V, F187Q, F187I, L227N, R251S, Y26H, A64T, G35A, A62T, G46V, R73H, T50M, I169M, Y106F, I185V, R108L, V167L, N204G, G39S, A64V, L124G, N162S, S193Q, Q237L, M208T, S181K, W69L, A216C, A246C , N204G, M208N, N211M, S193E, V200I, I170V, L13S, E158D, preferably A24Q, R30G, L74V, F187Q, F187I, L227N, R251S, Y26H, A64T, G35A, A62T, G46V, R73H, T50M, I169M, Y106F, I185V, R108L, V167L, N204G, G39S, A64V, L124G, N162S, S193Q, Q237L, M208T, S181K, W69L, A216C and A246C. In particular, the esterase comprises at least one combination of substitutions selected from the group consisting of or consists of the amino acid sequence shown in SEQ ID NO: 1 having a combination of substitutions selected from the group consisting of A24Q + H183Q, R30G + H183Q, L74V + H183Q, H183Q + F187Q, H183Q + F187I, L227N + H183Q, R251S + H183Q, Y26H + A64T + H183Q, G35A + A62T + H183Q, G46V + R73H + H183Q, T50M + I169M + H183Q, Y106F + I185V + H183Q, R108L + V167L + H183Q, H183Q + F187I + N204G, N204G + H183Q + F187I + R30G, N204G + H183Q + F187I + G39S, N204G + H183Q + F187I + A62T, N204G + H183Q + F187I + A64V, N204G + H183Q + F187I + L124G, N204G + H183Q + F187I + N162S, N204G + H183Q + F187I + S193Q, N204G + H183Q + F187I + Q237L, M208T + N204G + H183Q + F187I, N204G + H183Q + F187I+N162S+ S181K, N204G + H183Q + F187I + N162S + S181K + S193Q, N204G + H183Q + F187I + W69L + A216C + A246C, H183Q + F187I + N204G + M208N + N211M + S193E, M208T + N204G + H183Q + F187I + N211M + S193E, H183Q + F187I + N204G + N211M + S193E + V200I + I170V, H183Q + F187I + N204G + M208N + N211M + S193E + V200I + I170V, M208T + N204G + H183Q + F187I + N211M + S193E + V200I + I170V, H183Q + F187I + N204G + N211M + S193E, N204G + H183Q + F187I + L13S + E158D, N204G + H183Q + F187I, M208T + N204G + H183Q + F187I + L13S + E158D, H183Q + F187I + N204G + L13S + E158D, M208T + N204G + H183Q + F187I + L13S + E158D, A17T + T27S + S48T+L82I+F90L+ G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I, A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + H183Q + F187I + N204G, A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I + L13S + E158D, preferably A24Q + H183Q, R30G + H183Q, L74V + H183Q, H183Q + F187Q, H183Q + F187I, L227N + H183Q, R251S + H183Q, Y26H + A64T + H183Q, G35A + A62T + H183Q, G46V + R73H + H183Q, T50M + I169M + H183Q, Y106F + I185V + H183Q, R108L + V167L + H183Q, H183Q + F187I + N204G, N204G + H183Q + F187I + R30G, N204G + H183Q + F187I + G39S, N204G + H183Q + F187I + A62T, N204G + H183Q + F187I + A64V, N204G + H183Q + F187I + L124G, N204G + H183Q + F187I + N162S, N204G + H183Q + F187I + S193Q, N204G + H183Q + F187I + Q237L, M208T + N204G + H183Q + F187I, N204G + H183Q + F187I + N162S + S181K, N204G + H183Q + F187I + N162S + S181K + S193Q and N204G + H183Q + F187I + W69L + A216C + A246C.

在另一特定實施例中，酯酶包含取代H183N，且進一步包含至少一種選自以下之取代：F187I、F90L、F187L、F90Y、A17F、F90Q、N204G、M208L、N211M、N122D、S193E、Q142E、N143D、Q237E、N253D、Q258E、V200I、I170V、N85D、N105D、N211E、L13S、E158D，較佳選自F187I、F90L、F187L及F90Y。特定言之，酯酶包含至少一種選自以下之取代的組合或由具有選自以下之取代的組合的SEQ ID NO: 1中所示之胺基酸序列組成：H183N + F187I、F90L + H183N +  F187L、F90Y + H183N、A17F + F90Q + H183N + N204G + M208L + N211M、A17F + F90Q + N122D + H183N + S193E + N204G + M208L + N211M、A17F + F90Q + Q142E + H183N + S193E + N204G + M208L + N211M、A17F + F90Q + N143D + H183N + S193E + N204G + M208L + N211M、A17F + F90Q + H183N + S193E + N204G + M208L + N211M + Q237E、A17F + F90Q + H183N + S193E + N204G + M208L + N211M + N253D、A17F + F90Q + H183N + S193E + N204G + M208L + N211M + Q258E、A17F + F90Q + H183N + S193E + N204G + M208L + N211M + V200I + I170V、A17F + N85D + F90Q + H183N + S193E + N204G + M208L + N211M及A17F + F90Q + N105D + H183N + S193E + N204G + M208L + N211E，較佳選自H183N + F187I、F90L + H183N + F187L及F90Y + H183N。In another specific embodiment, the esterase comprises the substitution H183N and further comprises at least one substitution selected from the group consisting of F187I, F90L, F187L, F90Y, A17F, F90Q, N204G, M208L, N211M, N122D, S193E, Q142E, N143D, Q237E, N253D, Q258E, V200I, I170V, N85D, N105D, N211E, L13S, E158D, preferably selected from F187I, F90L, F187L and F90Y. In particular, the esterase comprises at least one combination of substitutions selected from the following or consists of the amino acid sequence shown in SEQ ID NO: 1 having a combination of substitutions selected from the following. + M208L + N211M, A17F + F90Q + H183N + S193E + N204G + M208L + N211M + Q237E, A17F + F90Q + H183N + S193E + N204G + M208L + N211M + N253D, A17F + F90Q + H183N + S193E + N204G + M208L + N211M + Q258E, A17F + F90Q + H183N + S193E + N204G + M208L + N211M + V200I + I170V, A17F + N85D + F90Q + H183N + S193E + N204G+M208L+ N211M and A17F + F90Q + N105D + H183N + S193E + N204G + M208L + N211E. H183N + F187I, F90L + H183N + F187L and F90Y + H183N are better choices.

在實施例中，酯酶包含取代S193L，且進一步至少包含取代S181K。特定言之，酯酶至少包含取代S181K + S193L之組合。In an embodiment, the esterase comprises the substitution S193L and further comprises at least the substitution S181K. In particular, the esterase comprises at least the combination of substitutions S181K + S193L.

在另一實施例中，酯酶包含取代S193Q，且進一步包含至少一種選自N162S、S181K、N204G、H183Q及F187I之取代。特定言之，酯酶包含至少一種選自以下之取代的組合：N162S + S193Q、N162S + S181K + S193Q、N204G + H183Q + F187I + S193Q及N204G + H183Q + F187I + N162S + S181K + S193Q。In another embodiment, the esterase comprises the substitution S193Q and further comprises at least one substitution selected from N162S, S181K, N204G, H183Q and F187I. Specifically, the esterase comprises at least one combination of substitutions selected from N162S + S193Q, N162S + S181K + S193Q, N204G + H183Q + F187I + S193Q and N204G + H183Q + F187I + N162S + S181K + S193Q.

在實施例中，酯酶包含取代W228S，且進一步包含至少一種選自T27A、R30G、S145G及V242E之取代。特定言之，酯酶至少包含選自T27A + R30G + S145G + W228S + V242E之取代的組合。In an embodiment, the esterase comprises the substitution W228S and further comprises at least one substitution selected from T27A, R30G, S145G and V242E. In particular, the esterase comprises at least a combination of substitutions selected from T27A + R30G + S145G + W228S + V242E.

在實施例中，酯酶包含取代V242E，且進一步包含至少一種選自T27A、R30G、S145G及W228S之取代。特定言之，酯酶至少包含選自T27A + R30G + S145G + W228S + V242E之取代的組合。In an embodiment, the esterase comprises the substitution V242E and further comprises at least one substitution selected from T27A, R30G, S145G and W228S. In particular, the esterase comprises at least a combination of substitutions selected from T27A + R30G + S145G + W228S + V242E.

在實施例中，酯酶包含至少一種選自以下之取代的組合或由具有選自以下之取代的組合的SEQ ID NO: 1中所示之胺基酸序列組成：H183L + F187I、H183A + F187I、H183Y + F187I、H183D + F187I、H183W + F187I、H183E + F187I、F90A + H183S + F187L、F90L + H183E + F187L、F90S + H183S + F187L、F90Y + H183L + F187L、S22R + G39S + P179N + L239M、A24Q + H183Q、R30G + H183Q、A64V + N243Y、L74V + H183Q、N162S + S193Q、S181K + S193L、H183Q + F187Q、H183Q + F187I、H183N + F187I、L227N + H183Q、R251S + H183Q、Y26H + A64T + H183Q、G35A + A62T + H183Q、G46V + R73H + H183Q、T50M + I169M + H183Q、A64V + V115I + N243Y、F90L + H183N + F187L、F90Y + H183N、Y106F + I185V + H183Q、R108L + V167L + H183Q、N162S + S181K + S193Q、H183Q + F187I + N204G、N204G + H183Q + F187I + R30G、N204G + H183Q + F187I + G39S、N204G + H183Q + F187I + A62T、N204G + H183Q + F187I + A64V、N204G + H183Q + F187I + L124G、N204G + H183Q + F187I + N162S、N204G + H183Q + F187I + S193Q、N204G + H183Q + F187I + Q237L、M208T + N204G + H183Q + F187I、T27A + R30G + S145G + W228S + V242E、N204G + H183Q + F187I + N162S + S181K、G7A + N9T + P192S + P213A + W228R + Q237L、N204G + H183Q + F187I + N162S + S181K + S193Q及N204G + H183Q + F187I + W69L + A216C + A246C。In an embodiment, the esterase comprises at least one combination of substitutions selected from the following or consists of the amino acid sequence shown in SEQ ID NO: 1 having a combination of substitutions selected from the following. + H183Q, N162S + S193Q, S181K + S193L, H183Q + F187Q, H183Q + F187I, H183N + F187I, L227N + H183Q, R251S + H183Q, Y26H + A64T + H183Q, G35A + A62T + H183Q, G46V + R73H + H183Q, T50M + I169M + H183Q, A64V + V115I + N243Y, F90L + H183N + F187L, F90Y + H183N, Y106F + I185V + H183Q, R108L + V167L + H183Q, N162S+ S181K + S193Q, H183Q + F187I + N204G, N204G + H183Q + F187I + R30G, N204G + H183Q + F187I + G39S, N204G + H183Q + F187I + A62T, N204G + H183Q + F187I + A64V, N204G + H183Q + F187I + L124G, N204G + H183Q + F187I + N162S, N204G + H183Q + F187I + S193Q, N204G + H183Q + F187I + Q237L, M208T + N204G + H183Q + F187I, T27A + R30G + S145G + W228S + V242E, N204G + H183Q + F187I + N162S + S181K, G7A + N9T + P192S + P213A + W228R + Q237L, N204G + H183Q + F187I + N162S + S181K + S193Q and N204G + H183Q + F187I + W69L + A216C + A246C.

在特定實施例中，酯酶至少包含N204G + H183Q + F187I之取代的組合，且視情況進一步包含至少一種選自以下之取代：R30G、G39S、A62T、A64V、W69L、L124G、N162S、S181K、S193Q、M208T、A216C、Q237L、A246C、M208N、N211M、S193E、V200I、I170V、L13S、E158D，較佳選自R30G、G39S、A62T、A64V、W69L、L124G、N162S、S181K、S193Q、M208T、A216C、Q237L及A246C。In a specific embodiment, the esterase comprises at least a combination of substitutions N204G + H183Q + F187I, and optionally further comprises at least one substitution selected from the following: R30G, G39S, A62T, A64V, W69L, L124G, N162S, S181K, S193Q, M208T, A216C, Q237L, A246C, M208N, N211M, S193E, V200I, I170V, L13S, E158D, preferably selected from R30G, G39S, A62T, A64V, W69L, L124G, N162S, S181K, S193Q, M208T, A216C, Q237L and A246C.

特定言之，酯酶包含至少一種選自以下之取代的組合或由具有選自以下之取代的組合的SEQ ID NO: 1中所示之胺基酸序列組成：N204G + H183Q + F187I + R30G、N204G + H183Q + F187I + G39S、N204G + H183Q + F187I + A62T、N204G + H183Q + F187I + A64V、N204G + H183Q + F187I + L124G、N204G + H183Q + F187I + N162S、N204G + H183Q + F187I + S193Q、N204G + H183Q + F187I + Q237L、M208T + N204G + H183Q + F187I、N204G + H183Q + F187I + N162S + S181K、N204G + H183Q + F187I + N162S + S181K + S193Q、N204G + H183Q + F187I + W69L + A216C + A246C、H183Q + F187I + N204G + M208N + N211M + S193E、H183Q + F187I + N204G + M208N + N211M + S193E + V200I + I170V、M208T + N204G + H183Q + F187I + N211M + S193E、M208T + N204G + H183Q + F187I + N211M + S193E + V200I + I170V、H183Q + F187I + N204G + N211M + S193E、H183Q + F187I + N204G + N211M + S193E + V200I + I170V、H183Q + F187I + N204G + L13S + E158D、M208T + N204G + H183Q + F187I + L13S + E158D、A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I、A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + H183Q + F187I + N204G、A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I + L13S + E158D，較佳選自N204G + H183Q + F187I + R30G、N204G + H183Q + F187I + G39S、N204G + H183Q + F187I + A62T、N204G + H183Q + F187I + A64V、N204G + H183Q + F187I + L124G、N204G + H183Q + F187I + N162S、N204G + H183Q + F187I + S193Q、N204G + H183Q + F187I + Q237L、M208T + N204G + H183Q + F187I、N204G + H183Q + F187I + N162S + S181K、N204G + H183Q + F187I + N162S + S181K + S193Q、N204G + H183Q + F187I + W69L + A216C + A246C、H183Q + F187I + N204G + M208N + N211M + S193E、H183Q + F187I + N204G + M208T + N211M + S193E、H183Q + F187I + N204G + N211M + S193E + V200I + I170V、H183Q + F187I + N204G + M208N + N211M + S193E + V200I + I170V、H183Q + F187I + N204G + M208T + N211M + S193E + V200I + I170V，更佳選自N204G + H183Q + F187I + A62T、N204G + H183Q + F187I + A64V、N204G + H183Q + F187I + L124G、N204G + H183Q + F187I + N162S、N204G + H183Q + F187I + S193Q、N204G + H183Q + F187I + Q237L、M208T + N204G + H183Q + F187I、N204G + H183Q + F187I + N162S + S181K + S193Q、H183Q + F187I + N204G + M208N + N211M + S193E、H183Q + F187I + N204G + M208T + N211M + S193E及H183Q + F187I + N204G + M208N + N211M + S193E + V200I + I170V。In particular, the esterase comprises at least one combination of substitutions selected from the group consisting of or consists of the amino acid sequence shown in SEQ ID NO: 1 having a combination of substitutions selected from the group consisting of N204G + H183Q + F187I + R30G, N204G + H183Q + F187I + G39S, N204G + H183Q + F187I + A62T, N204G + H183Q + F187I + A64V, N204G + H183Q + F187I + L124G, N204G + H183Q + F187I + N162S, N204G + H183Q + F187I + S193Q, N204G + H183Q + F187I + Q237L, M208T + N204G + H183Q + F187I, N204G + H183Q + F187I + N162S + S181K, N204G + H183Q + F187I + N162S + S181K + S193Q, N204G + H183Q + F187I + W69L + A216C + A246C, H183Q + F187I + N204G + M208N + N211M + S193E, H183Q + F187I + N204G + M208N + N211M + S193E + V200I + I170V, M208T + N204G + H183Q + F187I + N211M + S193E, M208T + N204G + H183Q + F187I + N211M + S193E + V200I + I170V, H183Q + F187I + N204G + N211M + S193E, H183Q + F187I + N204G + N211M + S193E + V200I + I170V, H183Q + F187I + N204G + L13S + E158D, M208T + N204G + H183Q + F187I + L13S + E158D, A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I, A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + H183Q + F187I + N204G, A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I + L13S + E158D, preferably N204G + H183Q + F187I + R30G, N204G + H183Q + F187I + G39S, N204G + H183Q + F187I + A62T, N204G + H183Q + F187I + A64V, N204G + H183Q + F187I + L124G, N204G + H183Q + F187I + N162S, N204G + H183Q + F187I + S193Q, N204G + H183Q + F187I + Q237L, M208T + N204G + H183Q + F187I, N204G + H183Q + F187I + N162S + S181K, N204G + H183Q + F187I + N162S + S181K + S193Q, N204G + H183Q + F187I + W69L + A216C + A246C, H183Q + F187I + N204G + M208N + N211M + S193E, H183Q + F187I + N204G + M208T + N211M + S193E, H183Q + F187I + N204G + N211M + S193E + V200I + I170V, H183Q + F187I+N204G+M208N+ N211M + S193E + V200I + I170V, H183Q + F187I + N204G + M208T + N211M + S193E + V200I + I170V, preferably N204G + H183Q + F187I + A62T, N204G + H183Q + F187I + A64V, N204G + H183Q + F187I + L124G, N204G + H183Q + F187I + N162S, N204G + H183Q + F187I + S193Q, N204G + H183Q + F187I + Q237L, M208T + N204G + H183Q + F187I, N204G + H183Q + F187I + N162S + S181K + S193Q, H183Q + F187I + N204G + M208N + N211M + S193E, H183Q + F187I + N204G + M208T + N211M + S193E and H183Q + F187I + N204G + M208N + N211M + S193E + V200I + I170V.

特定言之，酯酶至少包含N204G + H183Q + F187I之取代的組合，且視情況進一步包含至少一種選自M208T、L13S及E158D之取代。特定言之，酯酶包含至少一種選自以下之取代的組合或由具有選自以下之取代的組合的SEQ ID NO: 1中所示之胺基酸序列組成：N204G + H183Q + F187I + M208T、N204G + H183Q + F187I + M208T + L13S + E158D、N204G + H183Q + F187I + L13S + E158D。Specifically, the esterase comprises at least a combination of substitutions of N204G + H183Q + F187I, and optionally further comprises at least one substitution selected from M208T, L13S and E158D. Specifically, the esterase comprises at least one combination of substitutions selected from the following or consists of the amino acid sequence shown in SEQ ID NO: 1 having a combination of substitutions selected from the following: N204G + H183Q + F187I + M208T, N204G + H183Q + F187I + M208T + L13S + E158D, N204G + H183Q + F187I + L13S + E158D.

在特定實施例中，酯酶至少包含M208N + N211M之取代的組合。In certain embodiments, the esterase comprises at least the substitution combination of M208N + N211M.

在實施例中，酯酶至少包含選自H183Q + F187I + N204G + N211M + S193E之取代的組合，且進一步包含至少一種選自M208N/T、V200I及I170V之取代。在較佳實施例中，酯酶包含至少一種選自N204G + H183Q + F187I + M208N/T + N211M + S193E之取代的組合，較佳為組合H183Q + F187I + N204G + M208N + N211M + S193E。In an embodiment, the esterase comprises at least a combination of substitutions selected from H183Q + F187I + N204G + N211M + S193E, and further comprises at least one substitution selected from M208N/T, V200I and I170V. In a preferred embodiment, the esterase comprises at least one combination of substitutions selected from N204G + H183Q + F187I + M208N/T + N211M + S193E, preferably the combination H183Q + F187I + N204G + M208N + N211M + S193E.

在實施例中，酯酶包含至少一種選自以下之取代或取代之組合：R30G、A64V、A68N、T109S、L124G、S145G、L152M、E158A、K159R、H183Q/L/A/Y/N/D/E/W、S193L/Q、W228R/S、V242E、H183L + F187I、H183A + F187I、H183Y + F187I、H183D + F187I、H183W + F187I、H183E + F187I、F90A + H183S + F187L、F90L + H183E + F187L、F90S + H183S + F187L、F90Y + H183L + F187L、S22R + G39S + P179N + L239M、A24Q + H183Q、R30G + H183Q、A64V + N243Y、L74V + H183Q、N162S + S193Q、S181K + S193L、H183Q + F187Q、H183Q + F187I、H183N + F187I、L227N + H183Q、R251S + H183Q、Y26H + A64T + H183Q、G35A + A62T + H183Q、G46V + R73H + H183Q、T50M + I169M + H183Q、A64V + V115I + N243Y、F90L + H183N + F187L、F90Y + H183N、Y106F + I185V + H183Q、R108L + V167L + H183Q、N162S + S181K + S193Q、H183Q + F187I + N204G、N204G + H183Q + F187I + R30G、N204G + H183Q + F187I + G39S、N204G + H183Q + F187I + A62T、N204G + H183Q + F187I + A64V、N204G + H183Q + F187I + L124G、N204G + H183Q + F187I + N162S、N204G + H183Q + F187I + S193Q、N204G + H183Q + F187I + Q237L、M208T + N204G + H183Q + F187I、T27A + R30G + S145G + W228S + V242E、N204G + H183Q + F187I + N162S + S181K、G7A + N9T + P192S + P213A + W228R + Q237L、N204G + H183Q + F187I + N162S + S181K + S193Q、N204G + H183Q + F187I + W69L + A216C + A246C。In an embodiment, the esterase comprises at least one substitution or combination of substitutions selected from the group consisting of R30G, A64V, A68N, T109S, L124G, S145G, L152M, E158A, K159R, H183Q/L/A/Y/N/D/E/W, S193L/Q, W228R/S, V242E, H183L + F187I, H183A + F187I, H183Y + F187I, H183D + F187I, H183W + F187I, H183E + F187I, F90A + H183S + F187L, F90L + H183E + F187L, F90S + H183S + F187L, F90Y + H183L + F187L, S22R + G39S + P179N + L239M, A24Q + H183Q, R30G + H183Q, A64V + N243Y, L74V + H183Q, N162S + S193Q, S181K + S193L, H183Q + F187Q, H183Q + F187I, H183N + F187I, L227N + H183Q, R251S + H183Q, Y26H + A64T + H183Q, G35A + A62T + H183Q, G46V + R73H + H183Q, T50M + I169M + H183Q, A64V + V115I + N243Y, F90L + H183N + F187L, F90Y + H183N, Y106F + I185V + H183Q, R108L + V167L + H183Q, N162S + S181K + S193Q, H183Q + F187I + N204G, N204G + H183Q + F187I + R30G, N204G + H183Q + F187I + G39S, N204G + H183Q + F187I + A62T, N204G + H183Q + F187I + A64V, N204G + H183Q + F187I + L124G, N204G + H183Q + F187I+ N162S, N204G + H183Q + F187I + S193Q, N204G + H183Q + F187I + Q237L, M208T + N204G + H183Q + F187I, T27A + R30G + S145G + W228S + V242E, N204G + H183Q + F187I + N162S + S181K, G7A + N9T + P192S + P213A + W228R + Q237L, N204G + H183Q + F187I + N162S + S181K + S193Q, N204G + H183Q + F187I + W69L + A216C + A246C.

在另一實施例中，酯酶係由具有選自以下之取代或取代之組合的SEQ ID NO: 1中所示之胺基酸序列組成：R30G、A64V、A68N、T109S、L124G、S145G、L152M、E158A、K159R、H183Q/L/A/Y/N/D/E/W、S193L/Q、W228R/S、V242E、H183L + F187I、H183A + F187I、H183Y + F187I、H183D + F187I、H183W + F187I、H183E + F187I、H183S + F187I、A62T + S66N + M208T、F90A + H183S + F187L、F90L + H183E + F187L、F90S + H183S + F187L、F90Y + H183L + F187L、S22R + G39S + P179N + L239M、A24Q + H183Q、R30G + H183Q、A64V + N243Y、L74V + H183Q、N162S + S193Q、S181K + S193L、H183Q + F187Q、H183Q + F187I、H183N + F187I、L227N + H183Q、R251S + H183Q、Y26H + A64T + H183Q、G35A + A62T + H183Q、G46V + R73H + H183Q、T50M + I169M + H183Q、A64V + V115I + N243Y、F90L + H183N + F187L、F90Y + H183N、Y106F + I185V + H183Q、R108L + V167L + H183Q、N162S + S181K + S193Q、H183Q + F187I + N204G、N204G + H183Q + F187I + R30G、N204G + H183Q + F187I + G39S、N204G + H183Q + F187I + A62T、N204G + H183Q + F187I + A64V、N204G + H183Q + F187I + L124G、N204G + H183Q + F187I + N162S、N204G + H183Q + F187I + S193Q、N204G + H183Q + F187I + Q237L、M208T + N204G + H183Q + F187I、T27A + R30G + S145G + W228S + V242E、N204G + H183Q + F187I + N162S + S181K、G7A + N9T + P192S + P213A + W228R + Q237L、N204G + H183Q + F187I + N162S + S181K + S193Q、N204G + H183Q + F187I + W69L + A216C + A246C，其中參考SEQ ID NO: 1中所示之胺基酸序列對位置進行編號。In another embodiment, the esterase consists of the amino acid sequence shown in SEQ ID NO: 1 having a substitution or combination of substitutions selected from the group consisting of R30G, A64V, A68N, T109S, L124G, S145G, L152M, E158A, K159R, H183Q/L/A/Y/N/D/E/W, S193L/Q, W228R/S, V242E, H183L + F187I, H183A + F187I, H183Y + F187I, H183D + F187I, H183W + F187I, H183E + F187I, H183S + F187I, A62T + S66N + M208T, F90A + H183S + F187L, F90L + H183E + F187L, F90S + H183S + F187L, F90Y + H183L + F187L, S22R + G39S + P179N + L239M, A24Q + H183Q, R30G + H183Q, A64V + N243Y, L74V + H183Q, N162S + S193Q, S181K + S193L, H183Q + F187Q, H183Q + F187I, H183N + F187I, L227N + H183Q, R251S + H183Q, Y26H + A64T + H183Q, G35A + A62T + H183Q, G46V+ R73H + H183Q, T50M + I169M + H183Q, A64V + V115I + N243Y, F90L + H183N + F187L, F90Y + H183N, Y106F + I185V + H183Q, R108L + V167L + H183Q, N162S + S181K + S193Q, H183Q + F187I + N204G, N204G + H183Q + F187I + R30G, N204G + H183Q + F187I + G39S, N204G + H183Q + F187I + A62T, N204G + H183Q + F187I + A64V, N204G + H183Q + F187I + L124G, N204G + H183Q + F187I + N162S, N204G + H183Q + F187I + S193Q, N204G + H183Q + F187I + Q237L, M208T + N204G + H183Q + F187I, T27A + R30G + S145G + W228S + V242E, N204G + H183Q + F187I + N162S + S181K, G7A + N9T + P192S + P213A + W228R + Q237L, N204G + H183Q + F187I + N162S + S181K + S193Q, N204G + H183Q + F187I + W69L + A216C + A246C, wherein the positions are numbered with reference to the amino acid sequence shown in SEQ ID NO: 1.

在實施例中，酯酶包含至少一種選自以下之取代的組合或由具有一種選自以下之取代的組合的SEQ ID NO: 1中所示之胺基酸序列組成：M208N + N211M、H183Q + F187I + N204G + M208N + N211M + S193E、H183Q + F187I + N204G、H183Q + F187I + N204G + L13S + E158D、M208T + N204G + H183Q + F187I、M208T + N204G + H183Q + F187I + L13S + E158D、A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I、 A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + H183Q + F187I + N204G、A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I + L13S + E158D。In an embodiment, the esterase comprises at least one combination of substitutions selected from the following or consists of the amino acid sequence shown in SEQ ID NO: 1 having a combination of substitutions selected from the following: M208N + N211M, H183Q + F187I + N204G + M208N + N211M + S193E, H183Q + F187I + N204G, H183Q + F187I + N204G + L13S + E158D, M208T + N204G + H183Q + F187I, M208T + N204G + H183Q + F187I + L13S + E158D, A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I, A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + H183Q + F187I + N204G, A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q+S206T+T252S+I170V+M208T+N204G+H183Q+F187I+L13S+E158D.

有利的是，與SEQ ID NO: 1之酯酶相比，本發明之酯酶在6與10之間的pH範圍內展現增加之聚酯降解活性及/或增加之熱穩定性。特定言之，與SEQ ID NO: 1之酯酶相比，本發明之酯酶在pH 6至10、6至9、6.5至9、7至9、7.5至8.5、7至8.5、7.5至9範圍內展現增加之聚酯降解活性及/或增加之熱穩定性。根據本發明，pH範圍之名稱包括該範圍之下限及上限。Advantageously, the esterase of the present invention exhibits increased polyester degradation activity and/or increased thermostability in a pH range between 6 and 10 compared to the esterase of SEQ ID NO: 1. Specifically, the esterase of the present invention exhibits increased polyester degradation activity and/or increased thermostability in a pH range of 6 to 10, 6 to 9, 6.5 to 9, 7 to 9, 7.5 to 8.5, 7 to 8.5, 7.5 to 9 compared to the esterase of SEQ ID NO: 1. According to the present invention, the designation of a pH range includes the lower and upper limits of the range.

在實施例中，與SEQ ID NO: 1之酯酶相比，酯酶至少在鹼性條件下展現增加之聚酯降解活性。較佳地，酯酶至少在7.5與9之間的pH下，諸如在pH 8下展現增加之聚酯降解活性。In embodiments, the esterase exhibits increased polyester degradation activity at least under alkaline conditions compared to the esterase of SEQ ID NO: 1. Preferably, the esterase exhibits increased polyester degradation activity at least at a pH between 7.5 and 9, such as at pH 8.

特定言之，與SEQ ID NO: 1之酯酶相比，酯酶可在規定時段之後，例如在24 h、48 h或72 h之後展現增加之比降解活性及/或增加之PET解聚合產率。In particular, the esterase may exhibit increased specific degradation activity and/or increased PET depolymerization yield after a specified period of time, for example after 24 h, 48 h or 72 h, compared to the esterase of SEQ ID NO: 1.

在實施例中，酯酶包含至少一種上文所描述之取代或取代之組合，且與SEQ ID NO: 1之酯酶相比，在包含於6與10之間、較佳7與9之間、更佳7.5與9之間的pH下，甚至更佳在pH 8下展現增加之聚酯降解活性。In an embodiment, the esterase comprises at least one substitution or combination of substitutions described above and exhibits increased polyester degradation activity at a pH comprised between 6 and 10, preferably between 7 and 9, more preferably between 7.5 and 9, even more preferably at pH 8 compared to the esterase of SEQ ID NO: 1.

根據實施例，酯酶包含至少一種上文所描述之取代或取代之組合，且與SEQ ID NO: 1相比，在24 h之後PET解聚合產率增加。According to an embodiment, the esterase comprises at least one substitution or combination of substitutions described above and has an increased PET depolymerization yield after 24 h compared to SEQ ID NO: 1.

特定言之，酯酶包含至少一種上文所描述之取代或取代之組合，且與SEQ ID NO: 1之酯酶相比，在包含於6與10之間、較佳7與9之間、更佳7.5與9之間的pH下，甚至更佳在pH 8下展現增加之比降解活性。In particular, the esterase comprises at least one substitution or combination of substitutions described above and exhibits an increased specific degradation activity compared to the esterase of SEQ ID NO: 1 at a pH comprised between 6 and 10, preferably between 7 and 9, more preferably between 7.5 and 9, even more preferably at pH 8.

在另一特定實施例中，酯酶至少包含上文所描述之取代或取代之組合，且與SEQ ID NO: 1之酯酶相比，在包含於6與10之間、較佳7與9之間、更佳7.5與9之間的pH下，甚至更佳在pH 8下，在48 h之後展現增加之PET解聚合產率。In another particular embodiment, the esterase comprises at least a substitution or a combination of substitutions described above and exhibits an increased PET depolymerization yield after 48 h at a pH comprised between 6 and 10, preferably between 7 and 9, more preferably between 7.5 and 9, even more preferably at pH 8, compared to the esterase of SEQ ID NO: 1.

替代地或另外，與SEQ ID NO: 1之酯酶相比，酯酶可展現增加之熱穩定性。較佳地，酯酶至少在7與9之間的pH下，諸如在pH 8下展現增加之聚酯降解活性。Alternatively or additionally, the esterase may exhibit increased thermostability compared to the esterase of SEQ ID NO: 1. Preferably, the esterase exhibits increased polyester degradation activity at least at a pH between 7 and 9, such as at pH 8.

在特定實施例中，酯酶包含至少一種選自F187I及S193Q之取代，或至少包含取代S181K + S193L之組合，且與SEQ ID NO: 1之酯酶相比，在包含於6與10之間的pH下、更佳在包含於7與9之間的pH下、更佳在pH 8下展現增加之熱穩定性。In a particular embodiment, the esterase comprises at least one substitution selected from F187I and S193Q, or at least the combination of substitutions S181K + S193L, and exhibits increased thermostability at a pH comprised between 6 and 10, more preferably at a pH comprised between 7 and 9, more preferably at pH 8, compared to the esterase of SEQ ID NO: 1.

在實施例中，與SEQ ID NO: 1之酯酶相比，本發明之酯酶在包含於3與6之間的pH下、較佳在包含於5與6之間的pH下、更佳在包含於5與5.5之間的pH下、甚至更佳在pH 5.2下進一步展現增加之熱穩定性及/或增加之聚酯降解活性。In embodiments, the esterase of the present invention further exhibits increased thermostability and/or increased polyester degradation activity at a pH comprised between 3 and 6, preferably at a pH comprised between 5 and 6, more preferably at a pH comprised between 5 and 5.5, even more preferably at pH 5.2, compared to the esterase of SEQ ID NO: 1.

特定言之，酯酶包含至少一種上文所描述之取代或至少一種取代之組合，且與SEQ ID NO: 1之酯酶相比，在包含於6與10之間的pH下、尤其在包含於7.5與9之間的pH下展現增加之熱穩定性及/或增加之聚酯降解活性，且與SEQ ID NO: 1之酯酶相比，在3與6之間的pH下、較佳在包含於5與6之間的pH下、更佳在包含於5與5.5之間的pH下、甚至更佳在pH 5.2下進一步展現增加之熱穩定性及/或增加之聚酯降解活性。In particular, the esterase comprises at least one substitution or a combination of at least one substitution described above and exhibits increased thermostability and/or increased polyester degradation activity compared to the esterase of SEQ ID NO: 1 at a pH comprised between 6 and 10, in particular at a pH comprised between 7.5 and 9, and further exhibits increased thermostability and/or increased polyester degradation activity compared to the esterase of SEQ ID NO: 1 at a pH comprised between 3 and 6, preferably at a pH comprised between 5 and 6, more preferably at a pH comprised between 5 and 5.5, even more preferably at pH 5.2.

根據本發明，酯酶與SEQ ID NO: 1之親本酯酶一樣可包含至少一種選自S130、D175、H207、C240或C275之胺基酸殘基，亦即，本發明之酯酶在此等位置中之一者、兩者、三者等或全部處未經修飾。According to the present invention, the esterase may comprise at least one amino acid residue selected from S130, D175, H207, C240 or C275 like the parent esterase of SEQ ID NO: 1, i.e., the esterase of the present invention is not modified at one, two, three, etc. or all of these positions.

特定言之，酯酶與親本酯酶一樣至少可展現形成酯酶催化位點之胺基酸S130、D175及H207，及/或形成二硫鍵之胺基酸C240及C275。較佳地，酯酶與親本酯酶一樣至少包含選自S130 + D175 + H207、C240 + C275及S130 + D175 + H207 + C240 + C275之胺基酸殘基的組合，更佳與親本酯酶一樣包含組合S130 + D175 + H207 + C240 + C275。Specifically, the esterase can at least exhibit the amino acids S130, D175 and H207 that form the esterase catalytic site, and/or the amino acids C240 and C275 that form disulfide bonds, like the parent esterase. Preferably, the esterase at least comprises a combination of amino acid residues selected from S130 + D175 + H207, C240 + C275, and S130 + D175 + H207 + C240 + C275, like the parent esterase, and more preferably comprises the combination S130 + D175 + H207 + C240 + C275, like the parent esterase.

另外，酯酶與SEQ ID NO: 1之親本酯酶一樣可進一步包含至少一種選自C203及C248之胺基酸殘基。舉例而言，酯酶與親本酯酶一樣可包含胺基酸殘基C203 + C248之組合。替代地，酯酶與親本酯酶一樣至少包含取代C203K/R，較佳為C203K及胺基酸殘基C248。In addition, the esterase may further comprise at least one amino acid residue selected from C203 and C248, like the parent esterase of SEQ ID NO: 1. For example, the esterase may comprise the combination of amino acid residues C203 + C248, like the parent esterase. Alternatively, the esterase comprises at least the substitution C203K/R, preferably C203K and amino acid residue C248, like the parent esterase.

本發明之另一目標為提供一種酯酶變異體，其：(i)與SEQ ID NO: 1中所示之全長胺基酸序列具有至少97%、98%或99%一致性；(ii)與SEQ ID NO: 1之胺基酸序列相比，具有至少一種選自以下之胺基酸取代：H183Q/L/A/Y/N/D/E/W、R30G、A64V、A68N、T109S、L124G、S145G、L152M、K159R、S193L/Q、W228R/S、V242E、G7A、N9T、A24Q、Y26H、T27A、G39S、G46V、T50M、A64T、R73H、L74V、Y106F、R108L、V167L、I169M、P179N、I185V、F187L/Q、P192S、L227N、Q237L、L239M、R251S、N9E、V28I、A62T、F90Y/R/W/L、V115I、S181K、H183S、F187I、M208T、A246K、S66N、N243Y、S66T、F90C/V、P151V、A246R、A215E/D、D230E、L15K/R、T16K/R、Y4K/R、V219E/D、N143D、N253D、E182D、M208G、T16E、Q142E、Q237E、M208K、N105D、N85D、M208A、M208D，或至少一種選自以下之取代的組合：N204G + M208L + N211E、F90E + N204G + N211E、F90N + N204G + N211E、F90Q + N204G + N211E、F90R + N204G + N211E、F90W + N204G + N211E、F187I + N204G + N211E、S98R + E173Q、M208Q + N211M及M208N + N211M，其中參考SEQ ID NO: 1中所示之胺基酸序列對位置進行編號；(iii)與SEQ ID NO: 1之親本酯酶一樣至少具有胺基酸C240、C275、I170、G92、P213、E182、L13及E158；(iv)具有聚酯降解活性；及(v)與SEQ ID NO: 1之酯酶相比，在包含於6與10之間的pH下展現增加之熱穩定性及/或增加之聚酯降解活性。Another object of the present invention is to provide an esterase variant which: (i) has at least 97%, 98% or 99% identity to the full-length amino acid sequence shown in SEQ ID NO: 1; (ii) has at least 97%, 98% or 99% identity to the full-length amino acid sequence shown in SEQ ID NO: 1, has at least one amino acid substitution selected from the following: H183Q/L/A/Y/N/D/E/W, R30G, A64V, A68N, T109S, L124G, S145G, L152M, K159R, S193L/Q, W228R/S, V242E, G7A, N9T, A24Q, Y26H, T27A, G39S, G46V, T50M, A64T, R73H, L74V, Y106F, R108L, V167L, I169M, P179N, I185V, F187L/Q, P192S, L227N, Q237L, L239M, R251S, N9E, V28I, A62T, F90Y/R/W/L, V115I, S181K, H183S, F187I, M208T, A246K, S66N, N243Y, S66T, F90C/V, P151V, A246R, A215E/D, D230E, L15K/R, T16K/R, Y4K/R, V219E/D, N143D, N253D, E182D, M208G, T16E, Q142E, Q237E, M208K, N105D, N85D, M208A, M208D, or a combination of at least one substitution selected from the following: N204G + M208L + N211E, F90E + N204G + N211E, F90N + N204G + N211E, F90Q + N204G + N211E, F90R + N204G + N211E, F90W + N204G + N211E, F187I + N204G + N211E, S98R + E173Q, M208Q + N211M and M208N + N211M, wherein the positions are numbered with reference to the amino acid sequence shown in SEQ ID NO: 1; (iii) with SEQ ID NO: (iv) having polyester degradation activity; and (v) exhibiting increased thermostability and/or increased polyester degradation activity at a pH comprised between 6 and 10 compared to the esterase of SEQ ID NO: 1.

在實施例中，酯酶包含至少一種選自以下之胺基酸取代：H183Q/L/A/Y/N/D/E/W、R30G、A64V、A68N、T109S、L124G、S145G、L152M、K159R、S193L/Q、W228R/S、V242E、G7A、N9T、A24Q、Y26H、T27A、G39S、G46V、T50M、A64T、R73H、L74V、Y106F、R108L、V167L、I169M、P179N、I185V、F187L/Q、P192S、L227N、Q237L、L239M、R251S、N9E、V28I、A62T、F90Y/R/W/L、V115I、S181K、H183S、F187I、M208T、A246K、S66N、N243Y、S66T、F90C/V、P151V、A246R、A215E/D、D230E、L15K/R、T16K/R、Y4K/R、V219E/D，或至少一種選自以下之取代的組合：N204G + M208L + N211E、F90E + N204G + N211E、F90N + N204G + N211E、F90Q + N204G + N211E、F90R + N204G + N211E、F90W + N204G + N211E、F187I + N204G + N211E及S98R + E173Q。In an embodiment, the esterase comprises at least one amino acid substitution selected from the group consisting of H183Q/L/A/Y/N/D/E/W, R30G, A64V, A68N, T109S, L124G, S145G, L152M, K159R, S193L/Q, W228R/S, V242E, G7A, N9T, A24Q, Y26H, T27A, G39S, G46V, T50M, A64T, R73H, L74V, Y106F, R108L, V167L, I169M, P179N, I185 V, F187L/Q, P192S, L227N, Q237L, L239M, R251S, N9E, V28I, A62T, F90Y/R/W/L, V115I, S181K, H183S, F187I, M208T, A246K, S66N, N243Y, S66T, F90C/V, P151V, A246R, A215E/D, D230E, L15K/R, T16K/R, Y4K/R, V219E/D, or a combination of at least one substitution selected from the following: N204G + M208L + N211E, F90E + N204G + N211E, F90N + N204G + N211E, F90Q + N204G + N211E, F90R + N204G + N211E, F90W + N204G + N211E, F187I + N204G + N211E and S98R + E173Q.

在實施例中，酯酶包含至少一種選自以下之取代：N9E、V28I、A62T、F90Y、V115I、S181K、H183S、F187I、M208T、S66N、N243Y、E182D、M208G、T16E、Q142E、Q237E、M208K、N105D、N85D、M208A、M208D，較佳選自N9E、V28I、A62T、F90Y、V115I、S181K、H183S、F187I、M208T、S66N、N243Y、E182D、M208G、T16E、Q142E、N105D、N85D及M208A，更佳選自N9E、H183S、M208T、N243Y、M208G、T16E、Q142E、M208A及F187I。In an embodiment, the esterase comprises at least one substitution selected from the group consisting of N9E, V28I, A62T, F90Y, V115I, S181K, H183S, F187I, M208T, S66N, N243Y, E182D, M208G, T16E, Q142E, Q237E, M208K, N105D, N85D, M208A, M208D, preferably selected from the group consisting of N9E, V28I, A62T, F90Y, V115I, S181K, H183S, F187I, M208T, S66N, N243Y, E182D, M208G, T16E, Q142E, Q237E, M208K, N105D, N85D, M208A, M208D. 28I, A62T, F90Y, V115I, S181K, H183S, F187I, M208T, S66N, N243Y, E182D, M208G, T16E, Q142E, N105D, N85D and M208A, more preferably selected from N9E, H183S, M208T, N243Y, M208G, T16E, Q142E, M208A and F187I.

在較佳實施例中，與SEQ ID NO: 1之酯酶相比，酯酶包含至少一種選自以下之取代：R30G、A64V、A68N、T109S、L124G、S145G、L152M、K159R、H183Q/L/A/Y/N/D/E/W、S193L/Q、W228R/S、V242E、 G7A、N9T、A24Q、Y26H、T27A、G39S、G46V、T50M、A64T、R73H、L74V、Y106F、R108L、V167L、I169M、P179N、I185V、F187L/Q、P192S、L227N、Q237L、L239M、R251S、N9E、V28I、A62T、F90Y、V115I、S181K、H183S、F187I、M208T、S66N及N243Y，較佳選自R30G、A64V、A68N、T109S、L124G、S145G、L152M、K159R、H183Q/L/A/Y/N/D/E/W、S193L/Q、W228R/S、V242E、N9E、V28I、A62T、F90Y、V115I、S181K、F187I、M208T及N243Y，更佳選自R30G、A64V、T109S、S145G、H183Q/N、M208T、W228R/S、V242E、N9E及N243Y。在較佳實施例中，酯酶包含至少一種選自H183Q/L/A/Y/N/D/E/W，更佳選自H183Q/N之取代。In a preferred embodiment, the esterase comprises at least one substitution selected from the group consisting of R30G, A64V, A68N, T109S, L124G, S145G, L152M, K159R, H183Q/L/A/Y/N/D/E/W, S193L/Q, W228R/S, V242E, G7A, N9T, A24Q, Y26H, T27A, G39S, G46V, T50M, A64T, R73H, L74V, Y106F, R108L, V167L, I169M, P179N, I185V, F187L/Q, P192S, L227N, Q237L, L239M, R251S, N9E, V28I, A62T, F90Y, V115I, S181K, H183S, F187I, M208T, S66N and N243Y, preferably R30G , A64V, A68N, T109S, L124G, S145G, L152M, K159R, H183Q/L/A/Y/N/D/E/W, S193L/Q, W228R/S, V242E, N9E, V28I, A62T, F90Y, V115I, S181K, F187I, M208T and N243Y, preferably R30G, A64V, T109S, S145G, H183Q/N, M208T, W228R/S, V242E, N9E and N243Y. In a preferred embodiment, the esterase comprises at least one substitution selected from H183Q/L/A/Y/N/D/E/W, more preferably selected from H183Q/N.

在實施例中，酯酶包含一或兩種選自A215E/D、D230E、L15K/R、T16K/R、Y4K/R及V219E/D之取代。特定言之，酯酶包含選自A215E + T16K/R、A215D + T16R、D230E + Y4K/R及V219E/D + L15K/R之取代的組合。In an embodiment, the esterase comprises one or two substitutions selected from A215E/D, D230E, L15K/R, T16K/R, Y4K/R and V219E/D. In particular, the esterase comprises a combination of substitutions selected from A215E+T16K/R, A215D+T16R, D230E+Y4K/R and V219E/D+L15K/R.

在另一實施例中，酯酶包含選自以下之至少一種、較佳至少兩種取代：R30G、A64V、A68N、T109S、L124G、S145G、L152M、K159R、H183Q/L/A/Y/N/D/E/W、S193L/Q、W228R/S、V242E、G7A、N9T、A24Q、Y26H、T27A、G39S、G46V、T50M、A64T、R73H、L74V、Y106F、R108L、V167L、I169M、P179N、I185V、F187L/Q、P192S、L227N、Q237L、L239M、R251S、N9E、V28I、A62T、F90Y、V115I、S181K、H183S、F187I、M208T、S66N及N243Y。特定言之，酯酶包含選自S66N + M208T、A62T + M208T之取代的組合。In another embodiment, the esterase comprises at least one, preferably at least two substitutions selected from the group consisting of R30G, A64V, A68N, T109S, L124G, S145G, L152M, K159R, H183Q/L/A/Y/N/D/E/W, S193L/Q, W228R/S, V242E, G7A, N9T, A24Q, Y26H, T27A, G39S, G46V, T 50M, A64T, R73H, L74V, Y106F, R108L, V167L, 1169M, P179N, 1185V, F187L/Q, P192S, L227N, Q237L, L239M, R251S, N9E, V28I, A62T, F90Y, V115I, S181K, H183S, F187I, M208T, S66N and N243Y. Specifically, the esterase comprises a combination of substitutions selected from S66N + M208T, A62T + M208T.

在實施例中，酯酶包含至少一種選自以下之取代：R30G、A62T、A64V、F90Y、V115I、L124G、S145G、L152M、K159R、H183Q/N/D/E、F187I、S193L/Q、M208T、W228R/S、V242E及N243Y，較佳選自R30G、A62T、A64V、F90Y、V115I、L124G、S145G、L152M、K159R、H183Q/N、F187I、S193L/Q、M208T、W228R/S、V242E及N243Y。In an embodiment, the esterase comprises at least one substitution selected from the group consisting of R30G, A62T, A64V, F90Y, V115I, L124G, S145G, L152M, K159R, H183Q/N/D/E, F187I, S193L/Q, M208T, W228R/S, V242E and N243Y, preferably selected from the group consisting of R30G, A62T, A64V, F90Y, V115I, L124G, S145G, L152M, K159R, H183Q/N, F187I, S193L/Q, M208T, W228R/S, V242E and N243Y.

根據本發明，酯酶可進一步包含在選自以下胺基酸位置處之一種至七種取代：S1、Y4、Q5、R6、N9、P10、T11、R12、L13、A14、L15、T16、A17、D18、S22、T25、Y26、T27、V28、S29、R30、L31、S32、V33、S34、G35、F36、G37、G38、G39、Y43、S48、T50、G53、I54、M56、P58、G59、Y60、T61、A62、D63、A64、S65、S66、L67、A68、W69、L70、R72、R73、L74、L82、I84、N85、T86、N87、S88、R89、F90、D91、G92、P93、D94、S95、R96、S98、Q99、A103、L104、N105、L107、R108、S113、V115、L119、A121、N122、L124、A125、A127、G128、H129、M131、G132、G133、G134、G135、R138、A140、N143、S145、K147、A149、V150、L152、T153、P154、W155、H156、T157、E158、K159、T160、N162、S164、V167、L168、I170、A172、E173、A174、T176、V177、A178、P179、S181、Q182、H183、F187、Q189、N190、S193、T194、P196、V198、V200、L202、C203、N204、A205、S206、M208、A209、P210、N211、S212、P213、N214、A215、A216、I217、S218、V219、Y220、T221、S223、W224、M225、N231、T233、R236、Q237、F238、L239、N241、V242、N243、D244、P245、A246、L247、C248、T252、N253、N254、R255、H256、Q258、F161及T163，較佳選自S1、Y4、Q5、R6、P10、T11、R12、A14、L15、T16、A17、D18、S22、T25、S29、L31、S32、V33、S34、G35、F36、G37、G38、Y43、S48、G53、I54、M56、P58、G59、Y60、T61、D63、S65、L67、W69、L70、R72、L82、I84、N85、T86、N87、S88、R89、D91、P93、D94、S95、R96、S98、Q99、A103、L104、N105、L107、S113、L119、A121、N122、A125、A127、G128、H129、M131、G132、G133、G134、G135、R138、A140、N143、K147、A149、V150、T153、P154、W155、H156、T157、T160、N162、S164、L168、A172、E173、A174、T176、V177、A178、Q189、N190、T194、P196、V198、V200、L202、C203、N204、A205、S206、A209、P210、N211、S212、N214、A215、A216、I217、S218、V219、Y220、T221、S223、W224、M225、N231、T233、R236、F238、N241、D244、P245、A246、L247、C248、T252、N253、N254、R255、H256、Q258、F161及T163，更佳選自N162、G35、N204、W69、A216、A246、T157、F90、N243、V28、N211、A17、N122及Q258，甚至更佳選自N162、G35、N204、W69、A216、A246及T157。較佳地，酯酶進一步包含選自以下之一種至七種取代：N162S、G35A、N204G、W69L、A216C、A246C、T157P、M208N、N211M、A17F、M208L、M208T、N122D、Q142E、Q237E及Q258E，較佳選自N162S、G35A、N204G、W69L、A216C、A246C及T157P。According to the present invention, the esterase may further comprise one to seven substitutions at the amino acid positions selected from the group consisting of S1, Y4, Q5, R6, N9, P10, T11, R12, L13, A14, L15, T16, A17, D18, S22, T25, Y26, T27, V28, S29, R30, L31, S32, V33, S34, G35, F36, G37, G38, G39, Y43, S48, T50, G53, I54, M56, P58, G59, Y60, T61, A62 , D63, A64, S65, S66, L67, A68, W69, L70, R72, R73, L74, L82, I84, N85, T86, N87, S88, R89, F90, D91, G92, P93, D94, S95 , R96, S98, Q99, A103, L104, N105, L107, R108, S113, V115, L119, A121, N122, L124, A125, A127, G128, H129, M131, G132 , G133, G134, G135, R138, A140, N143, S145, K147, A149, V150, L152, T153, P154, W155, H156, T157, E158, K159, T160, N162, S164, V167, L168, I170, A172, E173, A174, T176, V177, A178, P179, S181, Q182, H183, F187, Q189, N190, S193, T1 94. P196, V198, V200, L202, C203, N204, A205, S206, M208, A209, P210, N211, S212, P213, N214, A215, A216, I217, S21 8. V219, Y220, T221, S223, W224, M225, N231, T233, R236, Q237, F238, L239, N241, V242, N243, D244, P245, A246, L247, C248, T252, N253, N254, R255, H256, Q258, F161 and T163, preferably S1, Y4, Q5, R6, P10, T11, R12, A14, L15, T16, A17, D18, S22, T25, S29, L31, S32, V33, S34, G35, F36, G37, G38, Y43, S48, G53, I54, M56, P58, G59, Y60, T61, D63, S65, L67, W69, L70 , R72, L82, I84, N85, T86, N87, S88, R89, D91, P93, D94, S95, R96, S98, Q99, A103, L104, N105, L107, S113, L119, A121, N122, A125, A127, G128, H129, M131, G132, G133, G134, G135, R138, A140, N143, K147, A149, V150, T153, P154, W155, H1 56. T157, T160, N162, S164, L168, A172, E173, A174, T176, V177, A178, Q189, N190, T194, P196, V198, V200, L202, C20 3. N204, A205, S206, A209, P210, N211, S212, N214, A215, A216, I217, S218, V219, Y220, T221, S223, W224, M225, N231, T233, R236, F238, N241, D244, P245, A246, L247, C248, T252, N253, N254, R255, H256, Q258, F161 and T163, preferably selected from N162, G35, N204, W69, A216, A246, T157, F90, N243, V28, N211, A17, N122 and Q258, even more preferably selected from N162, G35, N204, W69, A216, A246 and T157. Preferably, the esterase further comprises one to seven substitutions selected from the group consisting of N162S, G35A, N204G, W69L, A216C, A246C, T157P, M208N, N211M, A17F, M208L, M208T, N122D, Q142E, Q237E and Q258E, preferably selected from N162S, G35A, N204G, W69L, A216C, A246C and T157P.

根據實施例，酯酶包含取代F90Y，且進一步包含至少一種選自H183L/N之取代。特定言之，酯酶包含選自F90Y + H183L + F187L及F90Y + H183N之取代的組合。According to an embodiment, the esterase comprises the substitution F90Y and further comprises at least one substitution selected from H183L/N. In particular, the esterase comprises a combination of substitutions selected from F90Y + H183L + F187L and F90Y + H183N.

根據另一實施例，酯酶包含取代V115I，且進一步包含至少一種選自A64V及N243Y之取代。特定言之，酯酶包含取代A64V + V115I + N243Y之組合。According to another embodiment, the esterase comprises the substitution V115I and further comprises at least one substitution selected from A64V and N243Y. In particular, the esterase comprises the combination of substitutions A64V + V115I + N243Y.

根據另一實施例，酯酶包含取代F187I，且進一步包含選自以下之一種、兩種或更多種取代：R30G、G39S、A62T、A64V、W69L、L124G、N162S、S181K、H183L/A/Y/Q/N/D/W/E/S、S193Q、N204G、M208T、A216C、Q237L、A246C、M208N、N211M、S193E、V200I及I170V，較佳選自R30G、G39S、A62T、A64V、W69L、L124G、N162S、S181K、H183L/A/Y/Q/N/D/W/E/S、S193Q、N204G、M208T、A216C、Q237L、A246C。特定言之，酯酶包含至少一種選自以下之取代的組合或由具有選自以下之取代的組合的SEQ ID NO: 1中所示之胺基酸序列組成：H183L + F187I、H183A + F187I、H183Y + F187I、H183Q + F187I、H183N + F187I、H183D + F187I、H183W + F187I、H183E + F187I、H183S + F187I、H183Q + F187I + N204G、N204G + H183Q + F187I + R30G、N204G + H183Q + F187I + G39S、N204G + H183Q + F187I + A62T、N204G + H183Q + F187I + A64V、N204G + H183Q + F187I + L124G、N204G + H183Q + F187I + N162S、N204G + H183Q + F187I + S193Q、N204G + H183Q + F187I + Q237L、M208T + N204G + H183Q + F187I、N204G + H183Q + F187I + N162S + S181K、N204G + H183Q + F187I + N162S + S181K + S193Q、N204G + H183Q + F187I + W69L + A216C + A246C、H183Q + F187I + N204G + M208N + N211M + S193E、M208T + N204G + H183Q + F187I + N211M + S193E、H183Q + F187I + N204G + N211M + S193E 、H183Q + F187I + N204G + N211M + S193E + V200I + I170V、M208T + N204G + H183Q + F187I，較佳選自H183L + F187I、H183A + F187I、H183Y + F187I、H183Q + F187I、H183N + F187I、H183D + F187I、H183W + F187I、H183E + F187I、H183S + F187I、H183Q + F187I + N204G、N204G + H183Q + F187I + R30G、N204G + H183Q + F187I + G39S、N204G + H183Q + F187I + A62T、N204G + H183Q + F187I + A64V、N204G + H183Q + F187I + L124G、N204G + H183Q + F187I + N162S、N204G + H183Q + F187I + S193Q、N204G + H183Q + F187I + Q237L、M208T + N204G + H183Q + F187I、N204G + H183Q + F187I + N162S + S181K、N204G + H183Q + F187I + N162S + S181K + S193Q及N204G + H183Q + F187I + W69L + A216C + A246C。According to another embodiment, the esterase comprises substitution F187I and further comprises one, two or more substitutions selected from the group consisting of R30G, G39S, A62T, A64V, W69L, L124G, N162S, S181K, H183L/A/Y/Q/N/D/W/E/S, S193Q, N204G, M208T, A216C, Q237L, A 246C, M208N, N211M, S193E, V200I and I170V, preferably R30G, G39S, A62T, A64V, W69L, L124G, N162S, S181K, H183L/A/Y/Q/N/D/W/E/S, S193Q, N204G, M208T, A216C, Q237L, A246C. In particular, the esterase comprises at least one combination of substitutions selected from the group consisting of or consists of the amino acid sequence shown in SEQ ID NO: 1 having a combination of substitutions selected from the group consisting of H183L + F187I, H183A + F187I, H183Y + F187I, H183Q + F187I, H183N + F187I, H183D + F187I, H183W + F187I, H183E + F187I, H183S + F187I, H183Q + F187I + N204G, N204G + H183Q + F187I + R30G, N204G + H183Q + F187I + G39S, N204G + H183Q + F187I + A62T, N204G + H183Q + F187I + A64V, N204G + H183Q + F187I + L124G, N204G + H183Q + F187I + N162S, N204G + H183Q + F187I + S193Q, N204G + H183Q + F187I + Q237L, M208T + N204G + H183Q + F187I, N204G + H183Q + F187I + N162S + S181K, N204G + H183Q + F187I + N162S + S181K + S193Q, N204G + H183Q + F187I + W69L + A216C + A246C, H183Q + F187I + N204G + M208N + N211M + S193E, M208T + N204G + H183Q + F187I + N211M + S193E, H183Q + F187I + N204G + N211M + S193E, H183Q + F187I + N204G + N211M + S193E + V200I + I170V, M208T + N204G + H183Q + F187I, preferably H183L + F187I, H183A + F187I, H183Y + F187I, H183Q + F187I, H183N + F187I, H183D + F187I, H183W + F187I, H183E + F187I, H183S + F187I, H183Q + F187I + N204G, N204G + H183Q + F187I + R30G, N204G + H183Q + F187I + G39S, N204G + H183Q + F187I + A62T, N204G + H183Q + F187I + A64V, N204G + H183Q + F187I + L124G, N204G + H183Q + F187I + N162S, N204G + H183Q + F187I + S193Q, N204G + H183Q + F187I + Q237L, M208T + N204G + H183Q + F187I, N204G + H183Q + F187I + N162S + S181K, N204G + H183Q + F187I + N162S + S181K + S193Q and N204G + H183Q + F187I + W69L + A216C + A246C.

根據另一實施例，酯酶包含取代M208T，且進一步包含至少一種選自A62T、S66N、H183Q、F187I、N204G、N211M、S193E、V200I、I170V，較佳選自A62T、S66N、H183Q、F187I及N204G之取代。特定言之，酯酶至少包含選自以下之取代的組合或由具有選自以下之取代的組合的SEQ ID NO: 1中所示之胺基酸序列組成：A62T + S66N + M208T、M208T + N204G + H183Q + F187I、M208T + N204G + H183Q + F187I + N211M + S193E及M208T + N204G + H183Q + F187I，較佳選自A62T + S66N + M208T及M208T + N204G + H183Q + F187I。According to another embodiment, the esterase comprises the substitution M208T and further comprises at least one substitution selected from A62T, S66N, H183Q, F187I, N204G, N211M, S193E, V200I, I170V, preferably selected from A62T, S66N, H183Q, F187I and N204G. Specifically, the esterase comprises at least a combination of substitutions selected from the following or consists of the amino acid sequence shown in SEQ ID NO: 1 having a combination of substitutions selected from the following: A62T + S66N + M208T, M208T + N204G + H183Q + F187I, M208T + N204G + H183Q + F187I + N211M + S193E and M208T + N204G + H183Q + F187I, preferably selected from A62T + S66N + M208T and M208T + N204G + H183Q + F187I.

根據另一實施例，酯酶包含取代N243Y，且進一步包含至少一種選自A64V及V115I之取代。特定言之，酯酶至少包含選自A64V + N243Y及A64V + V115I + N243Y之取代的組合。According to another embodiment, the esterase comprises the substitution N243Y and further comprises at least one substitution selected from A64V and V115I. In particular, the esterase comprises at least a combination of substitutions selected from A64V + N243Y and A64V + V115I + N243Y.

在實施例中，酯酶包含至少一種選自以下之取代的組合或由具有選自以下之取代的組合的SEQ ID NO: 1中所示之胺基酸序列組成：S66N + M208T、A62T + M208T、F90A + H183S + F187L、F90L + H183E + F187L、F90S + H183S + F187L、S22R + G39S + P179N + L239M、A24Q + H183Q、R30G + H183Q、A64V + N243Y、L74V + H183Q、N162S + S193Q、S181K + S193L、H183Q + F187Q、H183L + F187I、H183A + F187I、H183Y + F187I、H183Q + F187I、H183N + F187I、H183D + F187I、H183W + F187I、H183E + F187I、H183S + F187I、L227N + H183Q、R251S + H183Q、Y26H + A64T + H183Q、G35A + A62T + H183Q、G46V + R73H + H183Q、T50M + I169M + H183Q、A62T + S66N + M208T、A64V + V115I + N243Y、F90L + H183N +  F187L、F90Y + H183L + F187L、F90Y + H183N、Y106F + I185V + H183Q、R108L + V167L + H183Q、N162S + S181K + S193Q、H183Q + F187I + N204G、N204G + H183Q + F187I + R30G、N204G + H183Q + F187I + G39S、N204G + H183Q + F187I + A62T、N204G + H183Q + F187I + A64V、N204G + H183Q + F187I + L124G、N204G + H183Q + F187I + N162S、N204G + H183Q + F187I + S193Q、N204G + H183Q + F187I + Q237L、M208T + N204G + H183Q + F187I、T27A + R30G + S145G + W228S + V242E、N204G + H183Q + F187I + N162S + S181K、G7A + N9T + P192S + P213A + W228R + Q237L、N204G + H183Q + F187I + N162S + S181K + S193Q及N204G + H183Q + F187I + W69L + A216C + A246C。In an embodiment, the esterase comprises at least one combination of substitutions selected from the following or consists of the amino acid sequence shown in SEQ ID NO: 1 having a combination of substitutions selected from the following: S66N + M208T, A62T + M208T, F90A + H183S + F187L, F90L + H183E + F187L, F90S + H183S + F187L, S22R + G39S + P179N + L239M, A24Q + H183Q, R30G + H183Q, A64V + N243Y, L74V + H183Q, N162S + S193Q, S181K + S193L, H183Q + F187Q, H183L + F187I, H183A + F187I, H183Y + F187I, H183Q + F187I, H183N + F187I, H183D + F187I, H183W + F187I, H183E + F187I, H183S + F187I, L227N + H183Q, R251S + H183Q, Y26H + A64T + H183Q, G35A + A62T + H183Q, G46V + R73H + H183Q, T50M + I169M + H183Q, A62T + S66N + M208T, A64V + V115I + N243Y, F90L + H183N + F187L, F90Y + H183L + F187L, F90Y + H183N, Y106F + I185V + H183Q, R108L + V167L + H183Q, N162S + S181K + S193Q, H183Q + F187I + N204G, N204G + H183Q + F187I + R30G, N204G + H183Q + F187I + G39S, N204G + H183Q + F187I + A62T, N204G + H183Q + F187I + A64V, N204G + H183Q + F187I + L124G, N204G + H183Q + F187I + N162S, N204G + H183Q + F187I + S193Q, N204G + H183Q + F187I + Q237L, M208T + N204G + H183Q + F187I, T27A + R30G + S145G + W228S + V242E, N204G + H183Q + F187I + N162S + S181K, G7A + N9T + P192S + P213A + W228R + Q237L, N204G + H183Q + F187I + N162S + S181K + S193Q and N204G + H183Q + F187I + W69L + A216C + A246C.

在特定實施例中，酯酶至少包含N204G + H183Q + F187I之取代的組合，且視情況進一步包含至少一種選自以下之取代：R30G、G39S、A62T、A64V、W69L、L124G、N162S、S181K、S193Q、M208T、A216C、Q237L、A246C、M208N、N211M、S193E，較佳選自R30G、G39S、A62T、A64V、W69L、L124G、N162S、S181K、S193Q、M208T、A216C、Q237L及A246C。特定言之，酯酶包含至少一種選自以下之取代的組合或由具有選自以下之取代的組合的SEQ ID NO: 1中所示之胺基酸序列組成：N204G + H183Q + F187I + R30G、N204G + H183Q + F187I + G39S、N204G + H183Q + F187I + A62T、N204G + H183Q + F187I + A64V、N204G + H183Q + F187I + L124G、N204G + H183Q + F187I + N162S、N204G + H183Q + F187I + S193Q、N204G + H183Q + F187I + Q237L、M208T + N204G + H183Q + F187I、N204G + H183Q + F187I + N162S + S181K、N204G + H183Q + F187I + N162S + S181K + S193Q、N204G + H183Q + F187I + W69L + A216C + A246C、H183Q + F187I + N204G + M208N + N211M + S193E、M208T + N204G + H183Q + F187I + N211M + S193E、H183Q + F187I + N204G + N211M + S193E，較佳選自N204G + H183Q + F187I + R30G、N204G + H183Q + F187I + G39S、N204G + H183Q + F187I + A62T、N204G + H183Q + F187I + A64V、N204G + H183Q + F187I + L124G、N204G + H183Q + F187I + N162S、N204G + H183Q + F187I + S193Q、N204G + H183Q + F187I + Q237L、M208T + N204G + H183Q + F187I、N204G + H183Q + F187I + N162S + S181K、N204G + H183Q + F187I + N162S + S181K + S193Q及N204G + H183Q + F187I + W69L + A216C + A246C、H183Q + F187I + N204G + M208N + N211M + S193E、H183Q + F187I + N204G + M208T + N211M + S193E、H183Q + F187I + N204G + N211M + S193E + V200I + I170V，更佳選自N204G + H183Q + F187I + A62T、N204G + H183Q + F187I + A64V、N204G + H183Q + F187I + L124G、N204G + H183Q + F187I + N162S、N204G + H183Q + F187I + S193Q、N204G + H183Q + F187I + Q237L、M208T + N204G + H183Q + F187I、N204G + H183Q + F187I + N162S + S181K + S193Q、H183Q + F187I + N204G + M208N + N211M + S193E及H183Q + F187I + N204G + M208T + N211M + S193E。In a specific embodiment, the esterase comprises at least a combination of substitutions N204G + H183Q + F187I, and optionally further comprises at least one substitution selected from the following: R30G, G39S, A62T, A64V, W69L, L124G, N162S, S181K, S193Q, M208T, A216C, Q237L, A246C, M208N, N211M, S193E, preferably selected from R30G, G39S, A62T, A64V, W69L, L124G, N162S, S181K, S193Q, M208T, A216C, Q237L, A246C and R30G. In particular, the esterase comprises at least one combination of substitutions selected from the group consisting of or consists of the amino acid sequence shown in SEQ ID NO: 1 having a combination of substitutions selected from the group consisting of N204G + H183Q + F187I + R30G, N204G + H183Q + F187I + G39S, N204G + H183Q + F187I + A62T, N204G + H183Q + F187I + A64V, N204G + H183Q + F187I + L124G, N204G + H183Q + F187I + N162S, N204G + H183Q + F187I + S193Q, N204G + H183Q + F187I + Q237L, M208T + N204G + H183Q + F187I, N204G + H183Q + F187I + N162S + S181K, N204G + H183Q + F187I + N162S + S181K + S193Q, N204G + H183Q + F187I + W69L + A216C + A246C, H183Q + F187I + N204G + M208N + N211M + S193E, M208T + N204G + H183Q + F187I + N211M + S193E, H183Q + F187I + N204G + N211M + S193E, the best choice is N204G + H183Q + F187I + R30G, N204G + H183Q + F187I + G39S, N204G + H183Q + F187I + A62T, N204G + H183Q + F187I + A64V, N204G + H183Q + F187I + L124G, N204G + H183Q + F187I + N162S, N204G + H183Q + F187I + S193Q, N204G + H183Q + F187I + Q237L, M208T + N204G + H183Q + F187I, N204G + H183Q + F187I + N162S + S181K, N204G + H183Q + F187I + N162S + S181K + S193Q and N204G + H183Q + F187I + W69L + A216C + A246C, H183Q + F187I + N204G + M208N + N211M + S193E, H183Q + F187I + N204G + M208T + N211M + S193E, H183Q + F187I + N204G + N211M + S193E + V200I + I170V, preferably N204G + H183Q + F187I + A62T, N204G + H183Q + F187I + A64V, N204G + H183Q + F187I + L124G, N204G + H183Q + F187I + N162S, N204G + H183Q + F187I + S193Q, N204G + H183Q + F187I + Q237L, M208T + N204G + H183Q + F187I, N204G + H183Q + F187I + N162S + S181K + S193Q, H183Q + F187I + N204G + M208N + N211M + S193E and H183Q + F187I + N204G + M208T + N211M + S193E.

在特定實施例中，酯酶至少包含M208N + N211M之取代的組合。In certain embodiments, the esterase comprises at least the substitution combination of M208N + N211M.

在實施例中，酯酶包含至少一種選自以下之取代或取代之組合或由具有選自以下之取代的組合的SEQ ID NO: 1中所示之胺基酸序列組成：R30G、A64V、A68N、T109S、L124G、S145G、L152M、K159R、H183Q/L/A/Y/N/D/E/W、S193L、S193Q、W228R/S、V242E、N9E、V28I、A62T、F90Y、V115I、S181K、F187I、M208T、N243Y、S66N + M208T、A62T + M208T、F90A + H183S + F187L、F90L + H183E + F187L、F90S + H183S + F187L、S22R + G39S + P179N + L239M、A24Q + H183Q、R30G + H183Q、A64V + N243Y、L74V + H183Q、N162S + S193Q、S181K + S193L、H183Q + F187Q、H183L + F187I、H183A + F187I、H183Y + F187I、H183Q + F187I、H183N + F187I、H183D + F187I、H183W + F187I、H183E + F187I、H183S + F187I、L227N + H183Q、R251S + H183Q、Y26H + A64T + H183Q、G35A + A62T + H183Q、G46V + R73H + H183Q、T50M + I169M + H183Q、A62T + S66N + M208T、A64V + V115I + N243Y、F90L + H183N + F187L、F90Y + H183L + F187L、F90Y + H183N、Y106F + I185V + H183Q、R108L + V167L + H183Q、N162S + S181K + S193Q、H183Q + F187I + N204G、N204G + H183Q + F187I + R30G、N204G + H183Q + F187I + G39S、N204G + H183Q + F187I + A62T、N204G + H183Q + F187I + A64V、N204G + H183Q + F187I + L124G、N204G + H183Q + F187I + N162S、N204G + H183Q + F187I + S193Q、N204G + H183Q + F187I + Q237L、M208T + N204G + H183Q + F187I、T27A + R30G + S145G + W228S + V242E、N204G + H183Q + F187I + N162S + S181K、G7A + N9T + P192S + P213A + W228R + Q237L、N204G + H183Q + F187I + N162S + S181K + S193Q及N204G + H183Q + F187I + W69L + A216C + A246C。In embodiments, the esterase comprises at least one substitution or combination of substitutions selected from or consists of the amino acid sequence shown in SEQ ID NO: 1 having a combination of substitutions selected from the group consisting of R30G, A64V, A68N, T109S, L124G, S145G, L152M, K159R, H183Q/L/A/Y/N/D/E/W, S193L, S193Q, W228R/S, V242E, N9E, V28I, A62T, F90Y, V115I, S181K, F187I, M208T, N243Y, S66N+M208T, A62T+M208T, F90A+H183S+F187L, F90L+H183E+ F187L, F90S + H183S + F187L, S22R + G39S + P179N + L239M, A24Q + H183Q, R30G + H183Q, A64V + N243Y, L74V + H183Q, N162S + S193Q, S181K + S193L, H183Q + F187Q, H183L + F187I, H183A + F187I, H183Y + F187I, H183Q + F187I, H183N + F187I, H183D + F187I, H183W + F187I, H183E + F187I, H183S + F187I, L227N + H183Q, R251S + H183Q, Y26H + A64T + H183Q, G35A + A62T + H183Q, G46V + R73H + H183Q, T50M + I169M + H183Q, A62T + S66N + M208T, A64V + V115I + N243Y, F90L + H183N + F187L, F90Y + H183L + F187L, F90Y + H183N, Y106F + I185V + H183Q, R108L + V167L + H183Q, N162S + S181K + S193Q, H183Q + F187I + N204G, N204G + H183Q + F187I + R30G, N204G + H183Q + F187I + G39S, N204G + H183Q + F187I + A62T, N204G + H183Q + F187I + A64V, N204G + H183Q + F187I + L124G, N204G + H183Q + F187I + N162S, N204G + H183Q + F187I + S193Q, N204G + H183Q + F187I + Q237L, M208T + N204G + H183Q + F187I, T27A + R30G + S145G + W228S + V242E, N204G + H183Q + F187I + N162S + S181K, G7A + N9T + P192S + P213A + W228R + Q237L, N204G + H183Q + F187I + N162S + S181K + S193Q and N204G + H183Q + F187I + W69L + A216C + A246C.

在實施例中，酯酶包含至少一種選自F187I及S193Q之取代，或至少一種選自S181K + S193L之取代的組合，且與SEQ ID NO: 1之酯酶相比，在包含於6與10之間的pH下、更佳在包含於7與9之間的pH下、更佳在pH 8下展現增加之熱穩定性。In an embodiment, the esterase comprises at least one substitution selected from F187I and S193Q, or a combination of at least one substitution selected from S181K + S193L, and exhibits increased thermostability at a pH comprised between 6 and 10, more preferably at a pH comprised between 7 and 9, more preferably at pH 8, compared to the esterase of SEQ ID NO: 1.

根據本發明，酯酶與SEQ ID NO: 1之親本酯酶一樣可包含至少一種選自S130、D175、H207、C240或C275之胺基酸殘基，亦即，本發明之酯酶在此等位置中之一者、兩者、三者等或全部處未經修飾。According to the present invention, the esterase may comprise at least one amino acid residue selected from S130, D175, H207, C240 or C275 like the parent esterase of SEQ ID NO: 1, i.e., the esterase of the present invention is not modified at one, two, three, etc. or all of these positions.

特定言之，酯酶與親本酯酶一樣至少可展現形成酯酶催化位點之胺基酸S130、D175及H207，及/或形成二硫鍵之胺基酸C240及C275。較佳地，酯酶與親本酯酶一樣至少包含選自S130 + D175 + H207、C240 + C275及S130 + D175 + H207 + C240 + C275之胺基酸殘基的組合，更佳與親本酯酶一樣包含組合S130 + D175 + H207 + C240 + C275。Specifically, the esterase can at least exhibit the amino acids S130, D175 and H207 that form the esterase catalytic site, and/or the amino acids C240 and C275 that form disulfide bonds, like the parent esterase. Preferably, the esterase at least comprises a combination of amino acid residues selected from S130 + D175 + H207, C240 + C275, and S130 + D175 + H207 + C240 + C275, like the parent esterase, and more preferably comprises the combination S130 + D175 + H207 + C240 + C275, like the parent esterase.

另外，酯酶與SEQ ID NO: 1之親本酯酶一樣可進一步包含至少一種選自C203及C248之胺基酸殘基。舉例而言，酯酶與親本酯酶一樣可包含胺基酸殘基C203 + C248之組合。替代地，酯酶與親本酯酶一樣至少包含取代C203K/R，較佳為C203K及胺基酸殘基C248。In addition, the esterase may further comprise at least one amino acid residue selected from C203 and C248, like the parent esterase of SEQ ID NO: 1. For example, the esterase may comprise the combination of amino acid residues C203 + C248, like the parent esterase. Alternatively, the esterase comprises at least the substitution C203K/R, preferably C203K and amino acid residue C248, like the parent esterase.

在特定實施例中，酯酶係由具有選自以下之取代的組合的SEQ ID NO: 1中所示之胺基酸序列組成：A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + H183Q + F187I + C203K + N204G + S206T + M208T + C248S + T252S、A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + H183Q + F187I + C203K + N204G + S206T + C248S + T252S、A17T + T27S + S48T + L82I + F90L + G92Y + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + H183Q + F187I + C203K + N204G + M208T + S206T + C248S + T252S、A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + H183Q + F187I + N204G + S206T + M208T + T252S或A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + H183Q + F187I + N204G + S206T + T252S。In a specific embodiment, the esterase consists of the amino acid sequence shown in SEQ ID NO: 1 having a combination of substitutions selected from the group consisting of A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + H183Q + F187I + C203K + N204G + S206T + M208T + C248S + T252S, A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + H183Q + F187I + C203K + N204G + S206T + C248S + T252S, A17T + T27S + S48T + L82I + F90L + G92Y + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + H183Q + F187I + C203K + N204G + M208T + S206T + C248S + T252S, A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + H183Q + F187I + N204G + S206T + M208T + T252S or A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + H183Q + F187I + N204G + S206T + T252S.

在特定實施例中，本發明之酯酶係衍生自SEQ ID NO: 2，其對應於SEQ ID NO: 1之胺基酸序列，與SEQ ID NO: 1相比，該胺基酸序列具有A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + I170V + S206T + T252S之取代的組合。換言之，衍生自SEQ ID NO: 2之變異體包含與SEQ ID NO: 1相比此等取代中之至少一者及本申請案中所描述之一或多種其他取代。In a specific embodiment, the esterase of the present invention is derived from SEQ ID NO: 2, which corresponds to the amino acid sequence of SEQ ID NO: 1, and the amino acid sequence has a combination of substitutions of A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + I170V + S206T + T252S compared to SEQ ID NO: 1. In other words, the variant derived from SEQ ID NO: 2 comprises at least one of these substitutions compared to SEQ ID NO: 1 and one or more other substitutions described in the present application.

本發明之另一目標為提供一種酯酶變異體，其：(i)與SEQ ID NO: 2中所示之全長胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%一致性；(ii)與SEQ ID NO: 2之胺基酸序列相比，包含至少一種選自以下之胺基酸取代：H183Q/L/A/Y/N/D/E/W、R30G、A64V、A68N、T109S、L124G、T145G、L152M、E158A、K159R、S193L/Q、W228R/S、V242E、G7A、N9T、A24Q、Y26H、S27A、G39S、G46V、T50M、A64T、R73H、L74V、Y106F、R108L、Q167L、I169M、P179N、I185V、F187L/Q、P192S、P213A、L227N、Q237L、L239M、R251S、S66T、L90C/V、F92K、P151V、A246R、A215E/D、D230E、L15K/R、I143D、N253D及V200I，或至少一種選自以下之取代的組合：N204G + M208L + N211E、L90E + N204G + N211E、L90N + N204G + N211E、L90Q + N204G + N211E、L90R + N204G + N211E、L90W + N204G + N211E、F187I + N204G + N211E、S98R + E173Q、M208A + N211M、M208G + N211M、M208K + N211M、M208N + N211M、M208P + N211M、M208Q + N211M、M208H + N211M及M208T + N211M，其中參考SEQ ID NO: 2中所示之胺基酸序列對位置進行編號；(iii)具有聚酯降解活性；及(iv)與SEQ ID NO: 2之酯酶相比，在包含於6與10之間的pH下展現增加之熱穩定性及/或增加之聚酯降解活性。Another object of the present invention is to provide an esterase variant which: (i) has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the full-length amino acid sequence shown in SEQ ID NO: 2; (ii) has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the full-length amino acid sequence shown in SEQ ID NO: 2, comprising at least one amino acid substitution selected from the following: H183Q/L/A/Y/N/D/E/W, R30G, A64V, A68N, T109S, L124G, T145G, L152M, E158A, K159R, S193L/Q, W228R/S, V242E, G7A, N9T, A24Q, Y26H, S27A, G39S, G46V, T50M, A64T, R73H, L74 V, Y106F, R108L, Q167L, I169M, P179N, I185V, F187L/Q, P192S, P213A, L227N, Q237L, L239M, R251S, S66T, L90C/V, F92K, P151V, A246R, A215E/D, D230E, L15K/R, I143D, N253D, and V200I, or a substituted combination of at least one selected from the following: N204G + M208A + N211M, M208G + N211M, M208K + N211M, M208N + N211M, M208P + N211M, M208Q + N211M, M208H + N211M and M208T + N211M, with reference to SEQ ID NO: (iii) having polyester degradation activity; and (iv) exhibiting increased thermostability and/or increased polyester degradation activity at a pH comprised between 6 and 10 compared to the esterase of SEQ ID NO: 2.

在實施例中，酯酶包含至少一種選自以下之取代：R30G、A64V、A68N、T109S、L124G、T145G、L152M、E158A、K159R、H183Q/L/A/Y/N/D/E/W、S193L/Q、W228R/S、V242E、G7A、N9T、A24Q、Y26H、S27A、G39S、G46V、T50M、A64T、S66N、R73H、L74V、Y106F、R108L、Q167L、I169M、P179N、I185V、F187L/Q、P192S、P213A、L227N、Q237L、L239M及R251S，較佳選自R30G、A64V、A68N、T109S、L124G、T145G、L152M、E158A、K159R、H183Q/L/A/Y/N/D/E/W、S193L/D、W228R/S及V242E，較佳選自R30G、A64V、T109S、T145G、E158A、H183Q/N、W228S及V242E。In embodiments, the esterase comprises at least one substitution selected from the group consisting of R30G, A64V, A68N, T109S, L124G, T145G, L152M, E158A, K159R, H183Q/L/A/Y/N/D/E/W, S193L/Q, W228R/S, V242E, G7A, N9T, A24Q, Y26H, S27A, G39S, G46V, T50M, A64T, S66N, R73H, L74V, Y106F, R108L, Q167L, I169M, P179 N, I185V, F187L/Q, P192S, P213A, L227N, Q237L, L239M and R251S, preferably R30G, A64V, A68N, T109S, L124G, T145G, L152M, E158A, K159R, H183Q/L/A/Y/N/D/E/W, S193L/D, W228R/S and V242E, preferably R30G, A64V, T109S, T145G, E158A, H183Q/N, W228S and V242E.

在較佳實施例中，酯酶包含至少一種選自A64V、S66N、H183N、I143D、N253D，較佳選自H183N及S66N之取代。In a preferred embodiment, the esterase comprises at least one substitution selected from A64V, S66N, H183N, I143D, N253D, preferably selected from H183N and S66N.

在實施例中，酯酶包含至少一種選自R30G、A64V、L124G、T145G、H183Q/L/A/Y/N/D/E/W、S193L/Q、W228S及V242E，較佳選自R30G、A64V、L124G、T145G、H183Q/N、S193L/Q、W228S及V242E，更佳選自H183Q/N之取代。In an embodiment, the esterase comprises at least one substitution selected from R30G, A64V, L124G, T145G, H183Q/L/A/Y/N/D/E/W, S193L/Q, W228S and V242E, preferably selected from R30G, A64V, L124G, T145G, H183Q/N, S193L/Q, W228S and V242E, more preferably selected from H183Q/N.

根據本發明，與SEQ ID NO: 2之胺基酸序列相比，酯酶可進一步包含在至少一個選自以下之胺基酸位置處之取代：S1、Y4、Q5、R6、N9、P10、T11、R12、L13、A14、L15、T16、T17、D18、S22、T25、Y26、S27、V28、S29、R30、L31、S32、V33、S34、G35、F36、G37、G38、G39、Y43、T48、T50、G53、I54、M56、P58、G59、Y60、T61、A62、D63、A64、S65、S66、L67、A68、W69、L70、R72、R73、L74、I82、I84、N85、T86、N87、S88、R89、L90、D91、F92、P93、D94、S95、R96、S98、Q99、A103、L104、N105、L107、R108、S113、V115、L119、A121、N122、L124、A125、A127、G128、H129、M131、G132、G133、G134、A135、R138、S140、I143、T145、K147、G149、V150、L152、T153、P154、W155、H156、T157、E158、K159、T160、N162、P164、Q167、L168、V170、A172、E173、A174、T176、V177、A178、P179、S181、Q182、H183、F187、Q189、N190、S193、T194、P196、V198、V200、L202、C203、N204、A205、T206、M208、A209、P210、N211、S212、P213、N214、A215、A216、I217、S218、V219、Y220、T221、S223、W224、M225、N231、T233、R236、Q237、F238、L239、N241、V242、N243、D244、P245、A246、L247、C248、S252、N253、N254、R255、H256、Q258、F161及T163，較佳在至少一個選自以下之位置：S1、Y4、Q5、R6、P10、T11、R12、L13、A14、L15、T16、T17、D18、S22、T25、V28、S29、L31、S32、V33、S34、G35、F36、G37、G38、Y43、T48、G53、I54、M56、P58、G59、Y60、T61、A62、D63、S65、L67、W69、L70、R72、I82、I84、N85、T86、N87、S88、R89、L90、D91、F92、P93、D94、S95、R96、S98、Q99、A103、L104、N105、L107、S113、V115、L119、A121、N122、A125、A127、G128、H129、M131、G132、G133、G134、A135、R138、S140、I143、K147、G149、V150、T153、P154、W155、H156、T157、T160、N162、P164、L168、V170、A172、E173、A174、T176、V177、A178、S181、Q182、Q189、N190、T194、P196、V198、V200、L202、C203、N204、A205、T206、M208、A209、P210、N211、S212、N214、A215、A216、I217、S218、V219、Y220、T221、S223、W224、M225、N231、T233、R236、F238、N241、N243、D244、P245、A246、L247、C248、S252、N253、N254、R255、H256、Q258、F161及T163，更佳選自G35、W69、L90、V115I、N162、S181、N204、A216、N243、A246、V28、T157、H183、F187、M208、N211、S193、V200、T17、N85、N122、Q142、I145、Q237、N253、Q258、S98、N105、L13、E158，較佳選自G35、W69、L90、V115I、N162、S181、N204、A216、N243、A246、V28及T157。較佳地，酯酶進一步包含至少一種選自以下之取代：G35A、W69L、L90L/Y、V115I、N162S、S181K、N204G、A216C、N243Y、A246C、V28I、T157P、T16K/R、Y4K/R、V219E/D、F187I、L90Q、H183D、H183E、H183N、H183Q、I145D、L90Q、M208A、M208L、M208N、M208T、N105D、N122D、N211M、N253D、N85D、Q142E、Q237E、Q258E、S193E、S98R、T17F、V200I、L13S、E158D，較佳選自G35A、W69L、L90L/Y、V115I、N162S、S181K、N204G、A216C、N243Y、A246C、V28I、T157P、T16K/R、Y4K/R及V219E/D，更佳選自G35A、W69L、L90Y、V115I、N162S、S181K、N204G、A216C、N243Y、A246C、V28I及T157P。舉例而言，酯酶進一步包含至少一種選自S66N + M208T及A62T + M208T之取代的組合。According to the present invention, the esterase may further comprise a substitution at at least one amino acid position selected from the group consisting of S1, Y4, Q5, R6, N9, P10, T11, R12, L13, A14, L15, T16, T17, D18, S22, T25, Y26, S27, V28, S29, R30, L31, S32, V33, S34, G35, F36, G37, G38, G39, Y43, T48, T50, G53, I54, M56, P58, G59, Y60, T61, A62, D63, A64, S65, S66, L67, A68, W69, L70, R72, R73, L74, I82, I84, N85, T86, N87, S88, R89, L90, D91, F92, P93, D94, S95, R96, S98, Q99, A103, L104, N105 , L107, R108, S113, V115, L119, A121, N122, L124, A125, A127, G128, H129, M131, G132, G133, G134, A135, R138, S140, I143, T145, K 147, G149, V150, L152, T153, P154, W155, H156, T157, E158, K159, T160, N162, P164, Q167, L168, V170, A172, E173, A174, T176, V1 77. A178, P179, S181, Q182, H183, F187, Q189, N190, S193, T194, P196, V198, V200, L202, C203, N204, A205, T206, M208, A209, P210 , N211, S212, P213, N214, A215, A216, I217, S218, V219, Y220, T221, S223, W224, M225, N231, T233, R236, Q237, F238, L239, N241, V242, N243, D244, P245, A246, L247, C248, S252, N253, N254, R255, H256, Q258, F161 and T163, preferably in at least one position selected from the following: S1, Y4, Q5, R6, P10, T1 1. R12, L13, A14, L15, T16, T17, D18, S22, T25, V28, S29, L31, S32, V33, S34, G35, F36, G37, G38, Y43, T48, G53, I54, M56, P58, G59, Y60, T61, A62, D63, S65, L67, W69, L70, R72, I82, I84, N85, T86, N87, S88, R89, L90, D91, F92, P93, D94, S95, R96, S98, Q99, A103, L1 04, N105, L107, S113, V115, L119, A121, N122, A125, A127, G128, H129, M131, G132, G133, G134, A135, R138, S140, I143, K147, G149 , V150, T153, P154, W155, H156, T157, T160, N162, P164, L168, V170, A172, E173, A174, T176, V177, A178, S181, Q182, Q189, N190, T 194, P196, V198, V200, L202, C203, N204, A205, T206, M208, A209, P210, N211, S212, N214, A215, A216, I217, S218, V219, Y220, T2 21. S223, W224, M225, N231, T233, R236, F238, N241, N243, D244, P245, A246, L247, C248, S252, N253, N254, R255, H256, Q258, F161 and T163, more preferably selected from G35, W69, L90, V115I, N162, S181, N204, A216, N243, A246, V28, T157, H183, F187, M208, N211, S193, V200, T17, N85, N122, Q142, I145, Q237, N253, Q258, S98, N105, L13, E158, preferably selected from G35, W69, L90, V115I, N162, S181, N204, A216, N243, A246, V28 and T157. Preferably, the esterase further comprises at least one substitution selected from the group consisting of G35A, W69L, L90L/Y, V115I, N162S, S181K, N204G, A216C, N243Y, A246C, V28I, T157P, T16K/R, Y4K/R, V219E/D, F187I, L90Q, H183D, H183E, H183N, H183Q, I145D, L90Q, M208A, M208L, M208N, M208T, N105D, N122D, N211M, N253D, N85D, Q1 42E, Q237E, Q258E, S193E, S98R, T17F, V200I, L13S, E158D, preferably G35A, W69L, L90L/Y, V115I, N162S, S181K, N204G, A216C, N243Y, A246C, V28I, T157P, T16K/R, Y4K/R and V219E/D, more preferably G35A, W69L, L90Y, V115I, N162S, S181K, N204G, A216C, N243Y, A246C, V28I and T157P. For example, the esterase further comprises at least one combination of substitutions selected from S66N + M208T and A62T + M208T.

在實施例中，與SEQ ID NO: 2之胺基酸序列相比，酯酶包含至少一種選自A215E/D、D230E及L15K/R之取代，且進一步包含至少一種選自T16K/R、Y4K/R及V219E/D之取代。特定言之，酯酶變異體包含選自A215E + T16K/R、A215D + T16R、D230E + Y4K/R及V219E/D + L15K/R之取代的組合。In an embodiment, the esterase comprises at least one substitution selected from A215E/D, D230E and L15K/R, and further comprises at least one substitution selected from T16K/R, Y4K/R and V219E/D compared to the amino acid sequence of SEQ ID NO: 2. Specifically, the esterase variant comprises a combination of substitutions selected from A215E+T16K/R, A215D+T16R, D230E+Y4K/R and V219E/D+L15K/R.

在實施例中，與SEQ ID NO: 2之胺基酸序列相比，酯酶包含至少一種選自R30G、A64V、L124G、T145G、H183Q/L/A/Y/N/D/E/W、S193L/Q、W228S及V242E，較佳選自R30G、A64V、L124G、T145G、H183Q/N、S193L/Q、W228S及V242E之取代，且進一步包含至少一種選自以下之取代：G35A、W69L、L90L/Y、V115I、N162S、S181K、N204G、A216C、N243Y、A246C、V28I、T157P、L90Q、I145D、L90Q、M208A、M208L、M208M、M208N、N105D、N122D、N211M、N253D、N85D、Q142E、Q237E、Q258E、S193E、S98R、T17F及V200I，較佳選自G35A、W69L、L90L/Y、V115I、N162S、S181K、N204G、A216C、N243Y、A246C、V28I及T157P。In an embodiment, the esterase comprises at least one substitution selected from R30G, A64V, L124G, T145G, H183Q/L/A/Y/N/D/E/W, S193L/Q, W228S and V242E, preferably selected from R30G, A64V, L124G, T145G, H183Q/N, S193L/Q, W228S and V242E, and further comprises at least one substitution selected from the following: G35A, W69L, L90L/Y, V115I, N162S, S181K, N204G, A216C, N 243Y, A246C, V28I, T157P, L90Q, I145D, L90Q, M208A, M208L, M208M, M208N, N105D, N122D, N211M, N253D, N85D, Q142E, Q237E, Q258E, S193E, S98R, T17F and V200I, preferably G35A, W69L, L90L/Y, V115I, N162S, S181K, N204G, A216C, N243Y, A246C, V28I and T157P.

根據實施例，與SEQ ID NO: 2之胺基酸序列相比，酯酶包含至少一種選自H183Q/L/A/Y/N/D/E/W之取代，且進一步包含至少一種選自以下之取代：F187I/L/Q/I、A24Q、R30G、L74V、L227N、R251S、Y26H、A64T、G35A、A62T、G46V、R73H、T50M、I169M、Y106F、I185V、R108L、Q167L、N204G、G39S、A64V、L124G、N162S、S193Q、Q237L、M208T、S181K、W69L、A216C/C、L90I/Y。特定言之，酯酶包含至少一種選自以下之取代的組合：H183L + F187I、H183A + F187I、H183Y + F187I、H183D + F187I、H183W + F187I、H183E + F187I、H183E + F187L、L90Y + H183L + F187L、A24Q + H183Q、R30G + H183Q、L74V + H183Q、H183Q + F187Q、H183Q + F187I、L227N + H183Q、R251S + H183Q、Y26H + A64T + H183Q、G35A + A62T + H183Q、G46V + R73H + H183Q、T50M + I169M + H183Q、Y106F + I185V + H183Q、R108L + Q167L + H183Q、H183Q + F187I + N204G、N204G + H183Q + F187I + R30G、N204G + H183Q + F187I + G39S、N204G + H183Q + F187I + A62T、N204G + H183Q + F187I + A64V、N204G + H183Q + F187I + L124G、N204G + H183Q + F187I + N162S、N204G + H183Q + F187I + S193Q、N204G + H183Q + F187I + Q237L、M208T + N204G + H183Q + F187I、N204G + H183Q + F187I + N162S + S181K、N204G + H183Q + F187I + N162S + S181K + S193Q及N204G + H183Q + F187I + W69L + A216C + A246C。According to an embodiment, the esterase comprises at least one substitution selected from H183Q/L/A/Y/N/D/E/W compared to the amino acid sequence of SEQ ID NO: 2, and further comprises at least one substitution selected from the following: F187I/L/Q/I, A24Q, R30G, L74V, L227N, R251S, Y26H, A64T, G35A, A62T, G46V, R73H, T50M, I169M, Y106F, I185V, R108L, Q167L, N204G, G39S, A64V, L124G, N162S, S193Q, Q237L, M208T, S181K, W69L, A216C/C, L90I/Y. In particular, the esterase comprises at least one combination of substitutions selected from the group consisting of H183L + F187I, H183A + F187I, H183Y + F187I, H183D + F187I, H183W + F187I, H183E + F187I, H183E + F187L, L90Y + H183L + F187L, A24Q + H183Q, R30G + H183Q, L74V + H183Q, H183Q + F187Q, H183Q + F187I, L227N + H183Q, R251S + H183Q, Y26H + A64T + H183Q, G35A + A62T + H183Q, G46V + R73H + H183Q, T50M + I169M + H183Q, Y106F + I185V + H183Q, R108L + Q167L + H183Q, H183Q + F187I + N204G, N204G + H183Q + F187I + R30G, N204G + H183Q + F187I + G39S, N204G + H183Q + F187I + A62T, N204G + H183Q + F187I + A64V, N204G + H183Q + F187I + L124G, N204G + H183Q + F187I + N162S, N204G + H183Q + F187I + S193Q, N204G + H183Q + F187I + Q237L, M208T + N204G + H183Q + F187I, N204G + H183Q + F187I + N162S + S181K, N204G + H183Q + F187I + N162S + S181K + S193Q and N204G + H183Q + F187I + W69L + A216C + A246C.

在較佳實施例中，酯酶包含至少一種選自H183Q/L/A/Y/N/D/E/W之取代，且進一步包含至少一種選自以下之取代：F187I、L90Q、I145D、L90Q、M208A、M208L、M208M、M208N、M208T、N105D、N122D、N204G、N211M、N253D、N85D、Q142E、Q237E、Q258E、S193E、S98R、T17F及V200I。特定言之，酯酶包含至少一種選自以下之取代的組合或由具有選自以下之取代的組合的SEQ ID NO: 2中所示之胺基酸序列組成：H183D + F187I、H183E + F187I、H183N + F187I、M208T + N204G + H183Q + F187I + N211M + S193E、H183Q + F187I + N204G + N211M + S193E + V200I、T17F + L90Q + H183N + N204G + M208L + N211M、T17F + L90Q + H183N + S193E + N204G + M208L + N211M、H183Q + F187I + N204G + M208N + N211M  + S193E + V200I、M208T + N204G + H183Q + F187I + N211M + S193E + V200I、T17F + N85D + L90Q + H183N + S193E + N204G + M208L + N211M、T17F + L90Q + N122D + H183N + S193E + N204G + M208L + N211M、T17F + L90Q + Q142E + H183N + S193E + N204G + M208L + N211M、T17F + L90Q + I145D + H183N + S193E + N204G + M208L + N211M、T17F + L90Q + H183N + S193E + N204G + M208L + N211M + Q237E、T17F + L90Q + H183N + S193E + N204G + M208L + N211M + N253D、T17F + L90Q + H183N + S193E + N204G + M208L + N211M + Q258E、T17F + L90Q + H183N + S193E + N204G + M208L + N211M + V200I、T17F + L90Q + S98R + N105D + H183N + S193E + N204G + N211M、T17F + L90Q + H183N + S193E + N204G + M208N + N211M + V200I、T17F + L90Q + H183N + S193E + N204G + M208A + N211M + V200I、T17F + L90Q + H183N + S193E + N204G + M208M + N211M + V200I、H183Q + F187I + N204G + M208N + N211M + S193E、H183Q + F187I + N204G、H183Q + F187I + N204G + M208T、H183Q + F187I + N204G + M208T + L13S + E158D、T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + M208T + L13S + E158D、T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + M208T、T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G、T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + L13S + E158D，較佳選自H183D + F187I、H183E + F187I、H183N + F187I、M208T + N204G + H183Q + F187I + N211M + S193E、H183Q + F187I + N204G + N211M + S193E + V200I、T17F + L90Q + H183N + N204G + M208L + N211M、T17F + L90Q + H183N + S193E + N204G + M208L + N211M、H183Q + F187I + N204G + M208N + N211M + S193E + V200I、M208T + N204G + H183Q + F187I + N211M  + S193E + V200I、T17F + N85D + L90Q + H183N + S193E + N204G + M208L + N211M、T17F + L90Q + N122D + H183N + S193E + N204G + M208L + N211M、T17F + L90Q + Q142E + H183N + S193E + N204G + M208L + N211M、T17F + L90Q + I145D + H183N + S193E + N204G + M208L + N211M、T17F + L90Q + H183N + S193E + N204G + M208L + N211M + Q237E、T17F + L90Q + H183N + S193E + N204G + M208L + N211M + N253D、T17F + L90Q + H183N + S193E + N204G + M208L + N211M + Q258E、T17F + L90Q + H183N + S193E + N204G + M208L + N211M + V200I、T17F + L90Q + S98R + N105D + H183N + S193E + N204G + N211M、T17F + L90Q + H183N + S193E + N204G + M208N + N211M + V200I、T17F + L90Q + H183N + S193E + N204G + M208A + N211M + V200I、T17F + L90Q + H183N + S193E + N204G + M208M + N211M + V200I。In a preferred embodiment, the esterase comprises at least one substitution selected from H183Q/L/A/Y/N/D/E/W, and further comprises at least one substitution selected from the following: F187I, L90Q, I145D, L90Q, M208A, M208L, M208M, M208N, M208T, N105D, N122D, N204G, N211M, N253D, N85D, Q142E, Q237E, Q258E, S193E, S98R, T17F and V200I. In particular, the esterase comprises at least one combination of substitutions selected from the group consisting of or consists of the amino acid sequence shown in SEQ ID NO: 2 having a combination of substitutions selected from the group consisting of H183D + F187I, H183E + F187I, H183N + F187I, M208T + N204G + H183Q + F187I + N211M + S193E, H183Q + F187I + N204G + N211M + S193E + V200I, T17F + L90Q + H183N + N204G + M208L + N211M, T17F + L90Q + H183N + S193E + N204G + M208L + N211M, H183Q + F187I + N204G + M208N + N211M + S193E + V200I, M208T + N204G + H183Q + F187I + N211M + S193E + V200I, T17F + N85D + L90Q + H183N + S193E + N204G + M208L + N211M, T17F + L90Q + N122D + H183N + S193E + N204G + M208L + N211M, T17F + L90Q + Q142E + H183N + S193E + N204G + M208L + N211M, T17F + L90Q + I145D + H183N + S193E + N204G+M208L+ N211M, T17F + L90Q + H183N + S193E + N204G + M208L + N211M + Q237E, T17F + L90Q + H183N + S193E + N204G + M208L + N211M + N253D, T17F + L90Q + H183N + S193E + N204G + M208L + N211M + Q258E, T17F + L90Q + H183N + S193E + N204G + M208L + N211M + V200I, T17F + L90Q + S98R + N105D + H183N + S193E + N204G + N211M, T17F + L90Q + H183N + S193E + N204G + M208N + N211M + V200I, T17F + L90Q + H183N + S193E + N204G + M208A + N211M + V200I, T17F + L90Q + H183N + S193E + N204G + M208M + N211M + V200I, H183Q + F187I + N204G + M208N + N211M + S193E, H183Q + F187I + N204G, H183Q + F187I + N204G + M208T, H183Q + F187I + N204G + M208T + L13S + E158D, T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + M208T + L13S + E158D, T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + M208T, T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G, T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + L13S + E158D, preferably H183D + F187I, H183E + F187I, H183N + F187I, M208T + N204G + H183Q + F187I + N211M + S193E, H183Q + F187I + N204G + N211M + S193E + V200I, T17F + L90Q + H183N + N204G + M208L + N211M, T17F + L90Q + H183N + S193E + N204G + M208L + N211M, H183Q + F187I + N204G + M208N + N211M + S193E + V200I, M208T + N204G + H183Q + F187I + N211M + S193E + V200I, T17F + N85D + L90Q + H183N + S193E + N204G + M208L + N211M, T17F + L90Q + N122D + H183N + S193E + N204G + M208L + N211M, T17F + L90Q + Q142E + H183N + S193E + N204G + M208L + N211M, T17F + L90Q + I145D + H183N + S193E + N204G + M208L + N211M, T17F + L90Q + H183N + S193E + N204G + M208L + N211M + Q237E, T17F + L90Q + H183N + S193E + N204G + M208L + N211M + N253D, T17F + L90Q + H183N + S193E + N204G + M208L + N211M + Q258E, T17F + L90Q + H183N + S193E + N204G + M208L + N211M + V200I, T17F + L90Q + S98R + N105D + H183N + S193E + N204G + N211M, T17F + L90Q + H183N + S193E + N204G + M208N + N211M + V200I, T17F + L90Q + H183N + S193E + N204G + M208A + N211M + V200I, T17F + L90Q + H183N + S193E + N204G + M208M + N211M + V200I.

在特定實施例中，與SEQ ID NO: 2之胺基酸序列相比，酯酶包含取代H183Q，且進一步包含選自以下之至少一種、兩種、三種或更多種取代：A24Q、R30G、L74V、F187Q/I、L227N、R251S、Y26H、A64T、G35A、A62T、G46V、R73H、T50M、I169M、Y106F、I185V、R108L、Q167L、N204G、G39S、A64V、L124G、N162S、S193Q、Q237L、M208T、S181K、W69L、A216C及A246C。特定言之，酯酶包含至少一種選自以下之取代的組合：A24Q + H183Q、R30G + H183Q、L74V + H183Q、H183Q + F187Q、H183Q + F187I、L227N + H183Q、R251S + H183Q、Y26H + A64T + H183Q、G35A + A62T + H183Q、G46V + R73H + H183Q、T50M + I169M + H183Q、Y106F + I185V + H183Q、R108L + Q167L + H183Q、H183Q + F187I + N204G、N204G + H183Q + F187I + R30G、N204G + H183Q + F187I + G39S、N204G + H183Q + F187I + A62T、N204G + H183Q + F187I + A64V、N204G + H183Q + F187I + L124G、N204G + H183Q + F187I + N162S、N204G + H183Q + F187I + S193Q、N204G + H183Q + F187I + Q237L、M208T + N204G + H183Q + F187I、N204G + H183Q + F187I + N162S + S181K、N204G + H183Q + F187I + N162S + S181K + S193Q及N204G + H183Q + F187I + W69L + A216C + A246C。In a specific embodiment, the esterase comprises the substitution H183Q compared to the amino acid sequence of SEQ ID NO: 2, and further comprises at least one, two, three or more substitutions selected from the group consisting of A24Q, R30G, L74V, F187Q/I, L227N, R251S, Y26H, A64T, G35A, A62T, G46V, R73H, T50M, I169M, Y106F, I185V, R108L, Q167L, N204G, G39S, A64V, L124G, N162S, S193Q, Q237L, M208T, S181K, W69L, A216C, and A246C. In particular, the esterase comprises at least one combination of substitutions selected from the group consisting of A24Q + H183Q, R30G + H183Q, L74V + H183Q, H183Q + F187Q, H183Q + F187I, L227N + H183Q, R251S + H183Q, Y26H + A64T + H183Q, G35A + A62T + H183Q, G46V + R73H + H183Q, T50M + I169M + H183Q, Y106F + I185V + H183Q, R108L + Q167L + H183Q, H183Q + F187I + N204G, N204G + H183Q + F187I + R30G, N204G + H183Q + F187I + G39S, N204G + H183Q + F187I + A62T, N204G + H183Q + F187I + A64V, N204G + H183Q + F187I + L124G, N204G + H183Q + F187I + N162S, N204G + H183Q + F187I + S193Q, N204G + H183Q + F187I + Q237L, M208T + N204G + H183Q + F187I, N204G + H183Q + F187I + N162S + S181K, N204G + H183Q + F187I + N162S + S181K + S193Q and N204G + H183Q + F187I + W69L + A216C + A246C.

在特定實施例中，酯酶包含取代H183Q，且進一步包含選自以下之至少一種、兩種、三種、四種、五種、六種或更多種取代：F187I、M208N、M208T、N204G、N211M、S193E、V200I。特定言之，酯酶包含至少一種選自以下之取代的組合或由具有選自以下之取代的組合的SEQ ID NO: 2中所示之胺基酸序列組成：H183Q + F187I + N204G + M208T + N211M + S193E、H183Q + F187I + N204G + M208N + N211M + S193E + V200I、H183Q + F187I + N204G + M208T + N211M + S193E + V200I、H183Q + F187I + N204G + M208N + N211M + S193E、H183Q + F187I + N204G、H183Q + F187I + N204G + M208T、H183Q + F187I + N204G + M208T + L13S + E158D、T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + M208T + L13S + E158D、T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + M208T、T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G、T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + L13S + E158D，較佳選自H183Q + F187I + N204G + M208T + N211M + S193E、H183Q + F187I + N204G + M208N + N211M + S193E + V200I、H183Q + F187I + N204G + M208T + N211M + S193E + V200I。In particular embodiments, the esterase comprises the substitution H183Q and further comprises at least one, two, three, four, five, six or more substitutions selected from the group consisting of F187I, M208N, M208T, N204G, N211M, S193E, V200I. In particular, the esterase comprises at least one combination of substitutions selected from the group consisting of or consists of the amino acid sequence shown in SEQ ID NO: 2 having a combination of substitutions selected from the group consisting of H183Q + F187I + N204G + M208T + N211M + S193E, H183Q + F187I + N204G + M208N + N211M + S193E + V200I, H183Q + F187I + N204G + M208T + N211M + S193E + V200I, H183Q + F187I + N204G + M208N + N211M + S193E, H183Q + F187I + N204G + M208T, H183Q + F187I + N204G + M208T + L13S + E158D, T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + M208T + L13S + E158D, T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + M208T, T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G, T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I+H183Q+F187I+ N204G + L13S + E158D, better choices are H183Q + F187I + N204G + M208T + N211M + S193E, H183Q + F187I + N204G + M208N + N211M + S193E + V200I, H183Q + F187I + N204G + M208T + N211M + S193E + V200I.

在特定實施例中，酯酶包含取代H183N，且進一步包含選自以下之至少一種、兩種、三種、四種、五種、六種、七種或更多種取代：F187I、L90Q、I145D、L90Q、M208A、M208L、M208N、N105D、N122D、N204G、N211M、N253D、N85D、Q142E、Q237E、Q258E、S193E、S98R、T17F、V200I。特定言之，酯酶包含至少一種選自以下之取代的組合或由具有選自以下之取代的組合的SEQ ID NO: 2中所示之胺基酸序列組成：H183N + F187I、T17F + L90Q + H183N + N204G + M208L + N211M、T17F + L90Q + H183N + S193E + N204G + M208L + N211M、T17F + N85D + L90Q + H183N + S193E + N204G + M208L + N211M、T17F + L90Q + N122D + H183N + S193E + N204G + M208L + N211M、T17F + L90Q + Q142E + H183N + S193E + N204G + M208L + N211M、T17F + L90Q + I145D + H183N + S193E + N204G + M208L + N211M、T17F + L90Q + H183N + S193E + N204G + M208L + N211M + Q237E、T17F + L90Q + H183N + S193E + N204G + M208L + N211M + N253D、T17F + L90Q + H183N + S193E + N204G + M208L + N211M + Q258E、T17F + L90Q + H183N + S193E + N204G + M208L + N211M + V200I、T17F + L90Q + S98R + N105D + H183N + S193E + N204G + N211M、T17F + L90Q + H183N + S193E + N204G + M208N + N211M + V200I、T17F + L90Q + H183N + S193E + N204G + M208A + N211M + V200I、T17F + L90Q + H183N + S193E + N204G + M208M + N211M + V200I。In particular embodiments, the esterase comprises the substitution H183N and further comprises at least one, two, three, four, five, six, seven or more substitutions selected from the group consisting of F187I, L90Q, I145D, L90Q, M208A, M208L, M208N, N105D, N122D, N204G, N211M, N253D, N85D, Q142E, Q237E, Q258E, S193E, S98R, T17F, V200I. In particular, the esterase comprises at least one combination of substitutions selected from the group consisting of or consists of the amino acid sequence shown in SEQ ID NO: 2 having a combination of substitutions selected from the group consisting of H183N + F187I, T17F + L90Q + H183N + N204G + M208L + N211M, T17F + L90Q + H183N + S193E + N204G + M208L + N211M, T17F + N85D + L90Q + H183N + S193E + N204G + M208L + N211M, T17F + L90Q + N122D + H183N + S193E + N204G + M208L + N211M, S193E + N204G + M208L + N211M, T17F + L90Q + I145D + H183N + S193E + N204G + M208L + N211M, T17F + L90Q + H183N + S193E + N204G + M208L + N211M + Q237E, T17F + L90Q + H183N + S193E + N204G + M208L + N211M + N253D, T17F + L90Q + H183N + S193E + N204G + M208L + N211M + Q258E, T17F + L90Q + H183N + S193E + N204G + M208L + N211M + V200I, T17F + L90Q + S98R + N105D + H183N + S193E + N204G + N211M, T17F + L90Q + H183N + S193E + N204G + M208N + N211M + V200I, T17F + L90Q + H183N + S193E + N204G + M208A + N211M + V200I, T17F + L90Q + H183N + S193E + N204G + M208M + N211M + V200I.

在特定實施例中，與SEQ ID NO: 2之胺基酸序列相比，酯酶至少包含N204G + H183Q + F187I之取代的組合，且視情況進一步包含至少一種選自以下之取代：R30G、G39S、A62T、A64V、W69L、L124G、N162S、S181K、S193Q、M208T、A216C、Q237L及A246C。特定言之，酯酶包含至少一種選自以下之取代的組合：N204G + H183Q + F187I + R30G、N204G + H183Q + F187I + G39S、N204G + H183Q + F187I + A62T、N204G + H183Q + F187I + A64V、N204G + H183Q + F187I + L124G、N204G + H183Q + F187I + N162S、N204G + H183Q + F187I + S193Q、N204G + H183Q + F187I + Q237L、M208T + N204G + H183Q + F187I、N204G + H183Q + F187I + N162S + S181K、N204G + H183Q + F187I + N162S + S181K + S193Q及N204G + H183Q + F187I + W69L + A216C + A246C。另外，酯酶可進一步至少包含取代C203K。舉例而言，酯酶可包含取代M208T + C203K + N204G + H183Q + F187I之組合。In a specific embodiment, the esterase comprises at least a combination of substitutions N204G + H183Q + F187I compared to the amino acid sequence of SEQ ID NO: 2, and optionally further comprises at least one substitution selected from the following: R30G, G39S, A62T, A64V, W69L, L124G, N162S, S181K, S193Q, M208T, A216C, Q237L and A246C. In particular, the esterase comprises at least one combination of substitutions selected from the group consisting of N204G + H183Q + F187I + R30G, N204G + H183Q + F187I + G39S, N204G + H183Q + F187I + A62T, N204G + H183Q + F187I + A64V, N204G + H183Q + F187I + L124G, N204G + H183Q + F187I + N162S, N204G + H183Q + F187I + S193Q, N204G + H183Q + F187I + Q237L, M208T + N204G + H183Q + F187I, N204G + H183Q + F187I + N162S + S181K, N204G + H183Q + F187I + N162S + S181K + S193Q and N204G + H183Q + F187I + W69L + A216C + A246C. In addition, the esterase may further comprise at least the substitution C203K. For example, the esterase may comprise the combination of the substitutions M208T + C203K + N204G + H183Q + F187I.

在較佳實施例中，酯酶至少包含N204G + H183Q + F187I之取代的組合，且視情況進一步包含至少一種選自M208T、N211M、S193E、V200I、M208N、L13S、E158D之取代。特定言之，酯酶包含至少一種選自以下之取代的組合或由具有選自以下之取代的組合的SEQ ID NO: 2中所示之胺基酸序列組成：M208T + N204G + H183Q + F187I + N211M + S193E、H183Q + F187I + N204G + M208N + N211M + S193E + V200I、M208T + N204G + H183Q + F187I + N211M + S193E + V200I、H183Q + F187I + N204G + M208N + N211M + S193E、H183Q + F187I + N204G、H183Q + F187I + N204G + M208T、H183Q + F187I + N204G + M208T + L13S + E158D、T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + M208T + L13S + E158D、T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + M208T、T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G、T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + L13S + E158D，較佳選自M208T + N204G + H183Q + F187I + N211M + S193E、H183Q + F187I + N204G + M208N + N211M  + S193E + V200I、M208T + N204G + H183Q + F187I + N211M + S193E + V200I。In a preferred embodiment, the esterase comprises at least a combination of substitutions N204G + H183Q + F187I, and optionally further comprises at least one substitution selected from M208T, N211M, S193E, V200I, M208N, L13S, E158D. In particular, the esterase comprises at least one combination of substitutions selected from the group consisting of or consists of the amino acid sequence shown in SEQ ID NO: 2 having a combination of substitutions selected from the group consisting of M208T + N204G + H183Q + F187I + N211M + S193E, H183Q + F187I + N204G + M208N + N211M + S193E + V200I, M208T + N204G + H183Q + F187I + N211M + S193E + V200I, H183Q + F187I + N204G + M208N + N211M + S193E, H183Q + F187I + N204G, H183Q + F187I + N204G + M208T, H183Q + F187I + N204G + M208T + L13S + E158D, T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + M208T + L13S + E158D, T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + M208T, T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G, T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I+H183Q+F187I+ N204G + L13S + E158D, better choices are M208T + N204G + H183Q + F187I + N211M + S193E, H183Q + F187I + N204G + M208N + N211M + S193E + V200I, M208T + N204G + H183Q + F187I + N211M + S193E + V200I.

在特定實施例中，酯酶至少包含M208N + N211M之取代的組合。In certain embodiments, the esterase comprises at least the substitution combination of M208N + N211M.

在實施例中，與SEQ ID NO: 2之胺基酸序列相比，酯酶包含至少一種選自以下之取代或取代之組合：R30G、A64V、A68N、T109S、L124G、T145G、L152M、E158A、K159R、H183Q/L/A/Y/N/D/E/W、S193L/3Q、W228R/S、V242E、H183L + F187I、H183A + F187I、H183Y + F187I、H183D + F187I、H183W + F187I、H183E + F187I、L90A + H183S + F187L、H183E + F187L、L90S + H183S + F187L、L90Y + H183L + F187L、S22R + G39S + P179N + L239M、A24Q + H183Q、R30G + H183Q、A64V + N243Y、L74V + H183Q、N162S + S193Q、S181K + S193L、H183Q + F187Q、H183Q + F187I、H183N + F187I、L227N + H183Q、R251S + H183Q、Y26H + A64T + H183Q、G35A + A62T + H183Q、G46V + R73H + H183Q、T50M + I169M + H183Q、A64V + V115I + N243Y、H183N +  F187L、L90Y + H183N、Y106F + I185V + H183Q、R108L + Q167L + H183Q、N162S + S181K + S193Q、H183Q + F187I + N204G、N204G + H183Q + F187I + R30G、N204G + H183Q + F187I + G39S、N204G + H183Q + F187I + A62T、N204G + H183Q + F187I + A64V、N204G + H183Q + F187I + L124G、N204G + H183Q + F187I + N162S、N204G + H183Q + F187I + S193Q、N204G + H183Q + F187I + Q237L、M208T + N204G + H183Q + F187I、S27A + R30G + T145G + W228S + V242E、N204G + H183Q + F187I + N162S + S181K、G7A + N9T + P192S + P213A + W228R + Q237L、N204G + H183Q + F187I + N162S + S181K + S193Q、N204G + H183Q + F187I + W69L + A216C + A246C。In an embodiment, the esterase comprises at least one substitution or combination of substitutions selected from the group consisting of R30G, A64V, A68N, T109S, L124G, T145G, L152M, E158A, K159R, H183Q/L/A/Y/N/D/E/W, S193L/3Q, W228R/S, V242E, H183L+F187I, H183A+F187I, H183Y+F187I, H183D+F187I, H183W+F187I, H183E+F187I, L90A+H183S+F187L, H183E+F187L, L90S+H183S+ F187L, L90Y + H183L + F187L, S22R + G39S + P179N + L239M, A24Q + H183Q, R30G + H183Q, A64V + N243Y, L74V + H183Q, N162S + S193Q, S181K + S193L, H183Q + F187Q, H183Q + F187I, H183N + F187I, L227N + H183Q, R251S + H183Q, Y26H + A64T + H183Q, G35A + A62T + H183Q, G46V + R73H + H183Q, T50M + I169M + H183Q, A64V + V115I + N243Y, H183N + F187L, L90Y + H183N, Y106F + I185V + H183Q, R108L + Q167L + H183Q, N162S + S181K + S193Q, H183Q + F187I + N204G, N204G + H183Q + F187I + R30G, N204G + H183Q + F187I + G39S, N204G + H183Q + F187I + A62T, N204G + H183Q + F187I + A64V, N204G + H183Q + F187I + L124G, N204G + H183Q + F187I + N162S, N204G + H183Q + F187I + S193Q, N204G + H183Q + F187I + Q237L, M208T + N204G + H183Q + F187I, S27A + R30G + T145G + W228S + V242E, N204G + H183Q + F187I + N162S + S181K, G7A + N9T + P192S + P213A + W228R + Q237L, N204G + H183Q + F187I + N162S + S181K + S193Q, N204G + H183Q + F187I + W69L + A216C + A246C.

在另一實施例中，酯酶包含至少一種選自以下之取代或取代之組合或由具有選自以下之取代的組合的SEQ ID NO: 2中所示之胺基酸序列組成：M208N + N211M、H183Q + F187I + N204G + M208N + N211M + S193E、H183Q + F187I + N204G、H183Q + F187I + N204G + M208T、H183Q + F187I + N204G + M208T + L13S + E158D、T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + M208T + L13S + E158D、T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + M208T、T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G、T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + L13S + E158D。In another embodiment, the esterase comprises at least one substitution or a combination of substitutions selected from the following or consists of the amino acid sequence shown in SEQ ID NO: 2 having a combination of substitutions selected from the following: M208N + N211M, H183Q + F187I + N204G + M208N + N211M + S193E, H183Q + F187I + N204G, H183Q + F187I + N204G + M208T, H183Q + F187I + N204G + M208T + L13S + E158D, T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + M208T + L13S + E158D, T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + M208T, T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G, T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I+H183Q+F187I+N204G+L13S+E158D.

在實施例中，與SEQ ID NO: 2之胺基酸序列相比，酯酶包含至少一種選自F187I及S193Q之取代，或至少包含取代S181K + S193L之組合，且與SEQ ID NO: 2之酯酶相比，在包含於6與10之間的pH下、更佳在包含於7與9之間的pH下、更佳在pH 8下展現增加之熱穩定性。In an embodiment, the esterase comprises at least one substitution selected from F187I and S193Q, or at least a combination of substitutions S181K + S193L, compared to the amino acid sequence of SEQ ID NO: 2, and exhibits increased thermal stability at a pH comprised between 6 and 10, more preferably at a pH comprised between 7 and 9, and more preferably at pH 8, compared to the esterase of SEQ ID NO: 2.

較佳地，酯酶與SEQ ID NO: 2之親本酯酶一樣展現至少一種選自S130、D175、H207、C240或C275之胺基酸殘基，亦即，本發明之酯酶在此等位置中之一者、兩者、三者等或全部處未經修飾。Preferably, the esterase exhibits at least one amino acid residue selected from S130, D175, H207, C240 or C275 like the parent esterase of SEQ ID NO: 2, i.e., the esterase of the present invention is not modified at one, two, three, etc. or all of these positions.

特定言之，酯酶與親本酯酶一樣至少可展現形成酯酶催化位點之胺基酸S130、D175及H207，及/或形成二硫鍵之胺基酸C240及C275。較佳地，酯酶與親本酯酶一樣至少包含選自S130 + D175 + H207、C240 + C275及S130 + D175 + H207 + C240 + C275之胺基酸殘基的組合，更佳與親本酯酶一樣包含組合S130 + D175 + H207 + C240 + C275。Specifically, the esterase can at least exhibit the amino acids S130, D175 and H207 that form the esterase catalytic site, and/or the amino acids C240 and C275 that form disulfide bonds, like the parent esterase. Preferably, the esterase at least comprises a combination of amino acid residues selected from S130 + D175 + H207, C240 + C275, and S130 + D175 + H207 + C240 + C275, like the parent esterase, and more preferably comprises the combination S130 + D175 + H207 + C240 + C275, like the parent esterase.

根據本發明，酯酶與SEQ ID NO: 2之親本酯酶一樣可進一步包含至少一種選自C203及C248之胺基酸殘基。舉例而言，酯酶與親本酯酶一樣可包含胺基酸殘基C203 + C248之組合。替代地，酯酶與親本酯酶一樣至少包含取代C203K/R，較佳為C203K及胺基酸殘基C248。According to the present invention, the esterase may further comprise at least one amino acid residue selected from C203 and C248, like the parent esterase of SEQ ID NO: 2. For example, the esterase may comprise the combination of amino acid residues C203 + C248, like the parent esterase. Alternatively, the esterase may comprise at least the substitution C203K/R, preferably C203K and amino acid residue C248, like the parent esterase.

根據本發明，與SEQ ID NO: 1之酯酶相比，酯酶進一步展現增加之熱穩定性及/或增加之聚酯降解活性。According to the present invention, the esterase further exhibits increased thermostability and/or increased polyester degradation activity compared to the esterase of SEQ ID NO: 1.

本發明之另一目標為提供一種酯酶變異體，其：(i)與SEQ ID NO: 2中所示之全長胺基酸序列具有至少97%、98%或99%一致性；(ii)與SEQ ID NO: 2之胺基酸序列相比，具有至少一種選自L90W、M208A、M208N、M208S、N122D、Q237E、Q258E及S212T之胺基酸取代，或至少M208I + N211M之取代的組合，其中參考SEQ ID NO: 2中所示之胺基酸序列對位置進行編號；(iii)與SEQ ID NO: 2之親本酯酶一樣至少具有胺基酸C240、C275、V170、F92、P213、E182、L13及E158；(iv)具有聚酯降解活性；及(v)與SEQ ID NO: 2之酯酶相比，在包含於6與10之間的pH下展現增加之熱穩定性及/或增加之聚酯降解活性。Another object of the present invention is to provide an esterase variant which: (i) has at least 97%, 98% or 99% identity to the full-length amino acid sequence shown in SEQ ID NO: 2; (ii) has at least one amino acid substitution selected from L90W, M208A, M208N, M208S, N122D, Q237E, Q258E and S212T, or a combination of at least M208I + N211M substitutions compared to the amino acid sequence of SEQ ID NO: 2, wherein the positions are numbered with reference to the amino acid sequence shown in SEQ ID NO: 2; (iii) has at least amino acids C240, C275, V170, F92, P213, E182, L13 and E158 as the parent esterase of SEQ ID NO: 2; (iv) has polyester degradation activity; and (v) has at least one amino acid substitution selected from L90W, M208A, M208N, M208S, N122D, Q237E, Q258E and S212T, or a combination of at least M208I + N211M substitutions compared to the amino acid sequence of SEQ ID NO: 2, wherein the positions are numbered with reference to the amino acid sequence shown in SEQ ID NO: 2; 2, exhibiting increased thermostability and/or increased polyester degradation activity at a pH comprised between 6 and 10.

在實施例中，酯酶包含至少一種選自M208N、M208S、N122D、Q237E、Q258E及S212T之取代。In embodiments, the esterase comprises at least one substitution selected from M208N, M208S, N122D, Q237E, Q258E, and S212T.

在實施例中，酯酶包含至少一種選自M208A、M208N、N122D、Q237E及Q258E之取代。In embodiments, the esterase comprises at least one substitution selected from M208A, M208N, N122D, Q237E, and Q258E.

根據本發明，與SEQ ID NO: 2之胺基酸序列相比，酯酶可進一步包含在至少一個選自以下之胺基酸位置處之至少一種取代：S1、Y4、Q5、R6、N9、P10、T11、R12、L13、A14、L15、T16、T17、D18、S22、T25、Y26、S27、V28、S29、R30、L31、S32、V33、S34、G35、F36、G37、G38、G39、Y43、T48、T50、G53、I54、M56、P58、G59、Y60、T61、A62、D63、A64、S65、S66、L67、A68、W69、L70、R72、R73、L74、I82、I84、N85、T86、N87、S88、R89、L90、D91、F92、P93、D94、S95、R96、S98、Q99、A103、L104、N105、L107、R108、S113、V115、L119、A121、N122、L124、A125、A127、G128、H129、M131、G132、G133、G134、A135、R138、S140、I143、T145、K147、G149、V150、L152、T153、P154、W155、H156、T157、E158、K159、T160、N162、P164、Q167、L168、V170、T172、E173、T174、T176、V177、T178、P179、S181、Q182、H183、F187、Q189、N190、S193、T194、P196、V198、V200、L202、C203、N204、A205、T206、M208、A209、P210、N211、S212、P213、N214、A215、A216、I217、S218、V219、Y220、T221、S223、W224、M225、N231、T233、R236、Q237、F238、L239、N241、V242、N243、D244、P245、A246、L247、C248、S252、N253、N254、R255、H256、Q258、F161及T163。According to the present invention, the esterase may further comprise at least one substitution at at least one amino acid position selected from the group consisting of S1, Y4, Q5, R6, N9, P10, T11, R12, L13, A14, L15, T16, T17, D18, S22, T25, Y26, S27, V28, S29, R30, L31, S32, V33, S34, G35, F36, G37, G38, G39, Y43, T48, T50, G53, I54, M56, P58, G59, Y60, T61, A62, D63, A64, 64, S65, S66, L67, A68, W69, L70, R72, R73, L74, I82, I84, N85, T86, N87, S88, R89, L90, D91, F92, P93, D94, S95, R96, S98, Q99, A103, L104, N105, L107, R108, S113, V115, L119, A121, N122, L124, A125, A127, G128, H129, M131, G132, G133, G134, A135, R13 8. S140, I143, T145, K147, G149, V150, L152, T153, P154, W155, H156, T157, E158, K159, T160, N162, P164, Q167, L168, V170, T 172, E173, T174, T176, V177, T178, P179, S181, Q182, H183, F187, Q189, N190, S193, T194, P196, V198, V200, L202, C203, N204 , A205, T206, M208, A209, P210, N211, S212, P213, N214, A215, A216, I217, S218, V219, Y220, T221, S223, W224, M225, N231, T233, R236, Q237, F238, L239, N241, V242, N243, D244, P245, A246, L247, C248, S252, N253, N254, R255, H256, Q258, F161 and T163.

在實施例中，酯酶包含至少一種選自M208A、M208N、N122D、Q237E及Q258E之取代，且進一步包含至少一種選自N211M、H183Q、F187I、N204G、S193E、V200I、T17F、L90Q、H183N及M208L之取代。In embodiments, the esterase comprises at least one substitution selected from M208A, M208N, N122D, Q237E, and Q258E, and further comprises at least one substitution selected from N211M, H183Q, F187I, N204G, S193E, V200I, T17F, L90Q, H183N, and M208L.

在實施例中，酯酶至少包含選自M208A + N211M或M208N + N211M之取代的組合或由具有選自M208A + N211M或M208N + N211M之取代的組合的SEQ ID NO: 2中所示之胺基酸序列之組成。In an embodiment, the esterase comprises at least a combination of substitutions selected from M208A + N211M or M208N + N211M or consists of the amino acid sequence shown in SEQ ID NO: 2 having a combination of substitutions selected from M208A + N211M or M208N + N211M.

在實施例中，酯酶至少包含取代M208N，且進一步包含至少一種選自N211M、H183Q、F187I、N204G、S193E、V200I、T17F、L90Q及H183N之取代。特定言之，酯酶包含至少一種選自以下之取代的組合或由具有選自以下之取代的組合的SEQ ID NO: 2中所示之胺基酸序列組成：M208N + N211M、H183Q + F187I + N204G + M208N + N211M  + S193E + V200I及T17F + L90Q + H183N + S193E + N204G + M208N + N211M + V200I。In an embodiment, the esterase comprises at least the substitution M208N, and further comprises at least one substitution selected from N211M, H183Q, F187I, N204G, S193E, V200I, T17F, L90Q and H183N. Specifically, the esterase comprises at least one combination of substitutions selected from or consists of the amino acid sequence shown in SEQ ID NO: 2 having a combination of substitutions selected from: M208N + N211M, H183Q + F187I + N204G + M208N + N211M + S193E + V200I and T17F + L90Q + H183N + S193E + N204G + M208N + N211M + V200I.

在另一實施例中，酯酶係由具有選自L90W、M208A、M208N、M208S、N122D、Q237E、Q258E、S212T、M208I + N211M之取代的組合的SEQ ID NO: 2中所示之胺基酸序列組成。In another embodiment, the esterase consists of the amino acid sequence shown in SEQ ID NO: 2 having a combination of substitutions selected from L90W, M208A, M208N, M208S, N122D, Q237E, Q258E, S212T, M208I + N211M.

較佳地，酯酶與SEQ ID NO: 2之親本酯酶一樣展現至少一種選自S130、D175、H207、C240或C275之胺基酸殘基，亦即，本發明之酯酶在此等位置中之一者、兩者、三者等或全部處未經修飾。Preferably, the esterase exhibits at least one amino acid residue selected from S130, D175, H207, C240 or C275 like the parent esterase of SEQ ID NO: 2, i.e., the esterase of the present invention is not modified at one, two, three, etc. or all of these positions.

特定言之，酯酶與親本酯酶一樣至少可展現形成酯酶催化位點之胺基酸S130、D175及H207，及/或形成二硫鍵之胺基酸C240及C275。較佳地，酯酶與親本酯酶一樣至少包含選自S130 + D175 + H207、C240 + C275及S130 + D175 + H207 + C240 + C275之胺基酸殘基的組合，更佳與親本酯酶一樣包含組合S130 + D175 + H207 + C240 + C275。Specifically, the esterase can at least exhibit the amino acids S130, D175 and H207 that form the esterase catalytic site, and/or the amino acids C240 and C275 that form disulfide bonds, like the parent esterase. Preferably, the esterase at least comprises a combination of amino acid residues selected from S130 + D175 + H207, C240 + C275, and S130 + D175 + H207 + C240 + C275, like the parent esterase, and more preferably comprises the combination S130 + D175 + H207 + C240 + C275, like the parent esterase.

根據本發明，酯酶與SEQ ID NO: 2之親本酯酶一樣可進一步包含至少一種選自C203及C248之胺基酸殘基。舉例而言，酯酶與親本酯酶一樣可包含胺基酸殘基C203 + C248之組合。替代地，酯酶與親本酯酶一樣至少包含取代C203K/R，較佳為C203K及胺基酸殘基C248。According to the present invention, the esterase may further comprise at least one amino acid residue selected from C203 and C248, like the parent esterase of SEQ ID NO: 2. For example, the esterase may comprise the combination of amino acid residues C203 + C248, like the parent esterase. Alternatively, the esterase may comprise at least the substitution C203K/R, preferably C203K and amino acid residue C248, like the parent esterase.

根據本發明，與SEQ ID NO: 1之酯酶相比，酯酶進一步展現增加之熱穩定性及/或增加之聚酯降解活性。According to the present invention, the esterase further exhibits increased thermostability and/or increased polyester degradation activity compared to the esterase of SEQ ID NO: 1.

變異體之聚酯降解活性本發明之一目標為提供在鹼性條件下具有酯酶活性之新的酶。在特定實施例中，本發明之酶展現角質酶活性。 Polyester degradation activity of variants One object of the present invention is to provide novel enzymes having esterase activity under alkaline conditions. In a specific embodiment, the enzymes of the present invention exhibit cutinase activity.

在特定實施例中，本發明之酯酶具有聚酯降解活性，較佳為聚對苯二甲酸伸乙酯(PET)降解活性及/或聚己二酸對苯二甲酸伸丁酯(PBAT)降解活性及/或聚己內酯(PCL)降解活性及/或聚丁二酸伸丁酯(PBS)活性，更佳為聚對苯二甲酸伸乙酯(PET)降解活性及/或聚己二酸對苯二甲酸伸丁酯(PBAT)降解活性。甚至更佳地，本發明之酯酶具有聚對苯二甲酸伸乙酯(PET)降解活性。In a specific embodiment, the esterase of the present invention has polyester degradation activity, preferably polyethylene terephthalate (PET) degradation activity and/or polyethylene adipate terephthalate (PBAT) degradation activity and/or polycaprolactone (PCL) degradation activity and/or polybutylene succinate (PBS) activity, more preferably polyethylene terephthalate (PET) degradation activity and/or polyethylene adipate terephthalate (PBAT) degradation activity. Even more preferably, the esterase of the present invention has polyethylene terephthalate (PET) degradation activity.

有利的是，本發明之酯酶在20℃至90℃、較佳30℃至90℃、更佳40℃至90℃、更佳50℃至90℃、甚至更佳60℃至90℃、68℃至90℃之溫度範圍內展現聚酯降解活性。特定言之，本發明之酯酶在65℃及90℃、65℃及85℃、65℃及80℃、70℃及90℃、70℃及85℃、70℃及80℃之溫度範圍內展現聚酯降解活性。特定言之，本發明之酯酶在40℃與80℃之間、較佳在50℃與72℃之間、更佳在50℃與65℃之間的溫度下展現聚酯降解活性。在實施例中，本發明之酯酶在55℃與60℃之間、50℃與55℃之間、55℃與65℃之間、60℃與72℃之間、60℃與70℃之間的溫度下展現聚酯降解活性。在特定實施例中，酯酶在至少50℃下、在54℃下、在55℃下、在60℃下、在65℃下、在68℃下或在70℃下展現聚酯降解活性。有利的是，仍可在55℃與70℃之間的溫度下量測聚酯降解活性。在本發明之上下文中，以+/-1℃給定溫度。Advantageously, the esterase of the present invention exhibits polyester degradation activity in a temperature range of 20°C to 90°C, preferably 30°C to 90°C, more preferably 40°C to 90°C, more preferably 50°C to 90°C, even more preferably 60°C to 90°C, 68°C to 90°C. Specifically, the esterase of the present invention exhibits polyester degradation activity in a temperature range of 65°C and 90°C, 65°C and 85°C, 65°C and 80°C, 70°C and 90°C, 70°C and 85°C, 70°C and 80°C. Specifically, the esterase of the present invention exhibits polyester degradation activity at a temperature between 40°C and 80°C, preferably between 50°C and 72°C, more preferably between 50°C and 65°C. In embodiments, the esterases of the invention exhibit polyester degradation activity at temperatures between 55°C and 60°C, between 50°C and 55°C, between 55°C and 65°C, between 60°C and 72°C, between 60°C and 70°C. In particular embodiments, the esterases exhibit polyester degradation activity at least at 50°C, at 54°C, at 55°C, at 60°C, at 65°C, at 68°C or at 70°C. Advantageously, the polyester degradation activity can still be measured at temperatures between 55°C and 70°C. In the context of the present invention, the temperature is given as +/-1°C.

根據本發明，與親本酯酶相比，本發明之酯酶在給定溫度下，且更尤其在40℃與90℃之間、更佳在50℃與90℃之間的溫度下具有增加之聚酯降解活性。有利的是，與SEQ ID NO: 1及/或SEQ ID NO: 2之酯酶相比，本發明之酯酶在40℃與90℃之間、40℃與80℃之間、40℃與70℃之間、50℃與70℃之間、54℃與70℃之間、55℃與70℃之間、60℃與70℃之間、65℃與75℃之間、65℃與80℃之間、65℃與90℃之間的整個溫度範圍內具有增加之聚酯降解活性。特定言之，本發明之酯酶在40℃與80℃之間、較佳在50℃與72℃之間、更佳在50℃與65℃之間的溫度下展現增加之聚酯降解活性。在實施例中，本發明之酯酶在55℃與60℃之間、50℃與55℃之間、55℃與65℃之間、60℃與72℃之間、60℃與70℃之間的溫度下展現增加之聚酯降解活性。更特定言之，本發明之酯酶至少在50℃、54℃、55℃、60℃、65℃或68℃下，較佳在55℃或60℃或65℃下展現增加之聚酯降解活性。有利的是，酯酶之聚酯降解活性比親本酯酶之聚酯降解活性高至少5%，較佳至少10%、20%、50%、100%或更多。According to the present invention, the esterase of the present invention has increased polyester degradation activity compared to the parent esterase at a given temperature, and more particularly at a temperature between 40°C and 90°C, more preferably between 50°C and 90°C. Advantageously, compared to the esterase of SEQ ID NO: 1 and/or SEQ ID NO: 2, the esterase of the present invention has increased polyester degradation activity over the entire temperature range of between 40°C and 90°C, between 40°C and 80°C, between 40°C and 70°C, between 50°C and 70°C, between 54°C and 70°C, between 55°C and 70°C, between 60°C and 70°C, between 65°C and 75°C, between 65°C and 80°C, between 65°C and 90°C. In particular, the esterase of the present invention exhibits increased polyester degradation activity at a temperature between 40°C and 80°C, preferably between 50°C and 72°C, more preferably between 50°C and 65°C. In an embodiment, the esterase of the present invention exhibits increased polyester degradation activity at a temperature between 55°C and 60°C, between 50°C and 55°C, between 55°C and 65°C, between 60°C and 72°C, between 60°C and 70°C. More particularly, the esterase of the present invention exhibits increased polyester degradation activity at least at 50°C, 54°C, 55°C, 60°C, 65°C or 68°C, preferably at 55°C or 60°C or 65°C. Advantageously, the polyester degradation activity of the esterase is at least 5%, preferably at least 10%, 20%, 50%, 100% or more higher than the polyester degradation activity of the parent esterase.

較佳地，酯酶在65℃下之聚酯降解活性比親本酯酶之聚酯降解活性高至少5%，較佳至少10%、20%、30%、50%、100%或更多。Preferably, the polyester degradation activity of the esterase at 65°C is at least 5% higher than the polyester degradation activity of the parent esterase, more preferably at least 10%, 20%, 30%, 50%, 100% or more.

在另一較佳實施例中，酯酶在60℃下之聚酯降解活性比親本酯酶之聚酯降解活性高至少5%，較佳至少10%、20%、50%、100%或更多。In another preferred embodiment, the polyester degradation activity of the esterase at 60°C is at least 5% higher than the polyester degradation activity of the parent esterase, preferably at least 10%, 20%, 50%, 100% or more.

有利的是，本發明之酯酶至少在5至11、較佳在6至9的pH範圍內，更佳在6.5至9之pH範圍內，甚至更佳在6.5至8之pH範圍內，甚至更佳在包含於7與9之間的pH下，尤其在pH 8下展現可量測之酯酶活性。Advantageously, the esterase of the present invention exhibits measurable esterase activity at least in the pH range of 5 to 11, preferably in the pH range of 6 to 9, more preferably in the pH range of 6.5 to 9, even more preferably in the pH range of 6.5 to 8, even more preferably at a pH comprised between 7 and 9, in particular at pH 8.

核酸、表現卡匣、載體、宿主細胞本發明之另一目標為提供編碼上文所定義之酯酶的核酸。 Nucleic Acids, Expression Cassettes, Vectors, Host Cells Another object of the present invention is to provide a nucleic acid encoding an esterase as defined above.

依本文所用，術語「 核酸」、「 核酸序列」、「 聚核苷酸」、「 寡核苷酸」及「 核苷酸序列」係指去氧核糖核苷酸及/或核糖核苷酸之序列。核酸可為DNA (cDNA或gDNA)、RNA或其混合物。核酸可呈單股形式或呈雙螺旋體形式或為其混合物。其可具有重組、人工及/或合成來源，且可包含經修飾之核苷酸，包含例如經修飾之鍵、經修飾之嘌呤或嘧啶鹼基或經修飾之糖。本發明之核酸可呈經分離或純化形式，且藉由此項技術中本身已知的技術來製備、分離及/或操縱，該等技術例如為cDNA文庫之選殖及表現、擴增、酶促合成或重組技術。核酸亦可藉由熟知化學合成技術在活體外合成，例如Belousov (1997) Nucleic Acids Res. 25:3440-3444中所描述。 As used herein, the terms " nucleic acid ", " nucleic acid sequence ", " polynucleotide ", " oligonucleotide " and " nucleotide sequence " refer to a sequence of deoxyribonucleotides and/or ribonucleotides. The nucleic acid may be DNA (cDNA or gDNA), RNA or a mixture thereof. The nucleic acid may be in single-stranded form or in double-helical form or a mixture thereof. It may be of recombinant, artificial and/or synthetic origin and may comprise modified nucleotides, including, for example, modified bonds, modified purine or pyrimidine bases or modified sugars. The nucleic acids of the invention may be in isolated or purified form and prepared, isolated and/or manipulated by techniques known per se in the art, such as cloning and expression of cDNA libraries, amplification, enzymatic synthesis or recombinant techniques. Nucleic acids can also be synthesized in vitro by well-known chemical synthesis techniques, such as described in Belousov (1997) Nucleic Acids Res. 25:3440-3444.

本發明亦涵蓋在嚴格條件下與編碼上文所定義之酯酶的核酸雜交的核酸。較佳地，此類嚴格條件包括於2 X SSC/0.1% SDS中將雜交濾紙在約42℃下培育約2.5小時，接著在65℃下於1 X SSC/0.1% SDS中洗滌濾紙四次，每次15分鐘。所使用方案描述於諸如Sambrook等人(Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor N.Y. (1988))及Ausubel (Current Protocols in Molecular Biology (1989))之參考文獻中。The invention also encompasses nucleic acids hybridized to nucleic acids encoding an esterase as defined above under stringent conditions. Preferably, such stringent conditions include incubating the hybridized filter paper in 2X SSC/0.1% SDS at about 42°C for about 2.5 hours, followed by washing the filter paper four times for 15 minutes each at 65°C in 1X SSC/0.1% SDS. The protocols used are described in references such as Sambrook et al. (Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor N.Y. (1988)) and Ausubel (Current Protocols in Molecular Biology (1989)).

本發明亦涵蓋編碼本發明之酯酶的核酸，其中該核酸之序列或至少該序列之一部分已使用經最佳化之密碼子使用進行工程改造。The present invention also encompasses nucleic acids encoding the esterases of the present invention, wherein the sequence of the nucleic acid, or at least a portion of the sequence, has been engineered with optimized codon usage.

替代地，根據本發明之核酸可由根據本發明之酯酶的序列推斷，且密碼子使用可根據核酸將在其中轉錄之宿主細胞調適。此等步驟可根據熟習此項技術者熟知的方法進行，且其中一些描述於參考手冊Sambrook等人(Sambrook等人, 2001)中。Alternatively, the nucleic acid according to the invention can be deduced from the sequence of the esterase according to the invention, and the codon usage can be adapted according to the host cell in which the nucleic acid is to be transcribed. These steps can be carried out according to methods well known to those skilled in the art, and some of them are described in the reference manual Sambrook et al. (Sambrook et al., 2001).

本發明之核酸可進一步包含其他核苷酸序列，諸如調節區，亦即啟動子、強化子、沉默子、終止子、信號肽及可用於引起或調節多肽在所選擇宿主細胞或系統中之表現的類似者。The nucleic acids of the present invention may further comprise other nucleotide sequences, such as regulatory regions, i.e., promoters, enhancers, silencers, terminators, signal peptides and the like that can be used to induce or regulate the expression of the polypeptide in a selected host cell or system.

本發明進一步係關於包含可操作地連接至一或多個控制序列的根據本發明之核酸的表現卡匣，該一或多個控制序列引導該核酸在適合之宿主細胞中之表現。The invention further relates to an expression cassette comprising a nucleic acid according to the invention operably linked to one or more control sequences, which direct the expression of the nucleic acid in a suitable host cell.

依本文所用，術語「 表現」係指涉及多肽之產生的任何步驟，包括但不限於轉錄、轉錄後修飾、轉譯、轉譯後修飾及分泌。 As used herein, the term " expression " refers to any step involved in the production of a polypeptide, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

術語「表現卡匣」表示包含編碼區(亦即，本發明之核酸)及可操作地連接之調節區(亦即，包含一或多個控制序列之區)的核酸構築體。The term "expression cassette" refers to a nucleic acid construct comprising a coding region (ie, a nucleic acid of the invention) and an operably linked regulatory region (ie, a region comprising one or more control sequences).

通常，表現卡匣包含以下或由以下組成：可操作地連接至控制序列的根據本發明之核酸，該控制序列諸如為轉錄啟動子及/或轉錄終止子。控制序列可包括啟動子，其由宿主細胞或活體外表現系統識別以用於表現編碼本發明之酯酶的核酸。啟動子含有介導酶之表現的轉錄控制序列。啟動子可為在宿主細胞中展示轉錄活性之任何聚核苷酸，包括突變、截短及雜合啟動子，且可自將同源或異源之胞外或胞內多肽編碼成宿主細胞之基因獲得。控制序列亦可為轉錄終止子，其由宿主細胞識別以終止轉錄。終止子可操作地連接至編碼酯酶之核酸的3'端。在宿主細胞中具有功能之任何終止子可用於本發明中。通常，表現卡匣包含以下或由以下組成：可操作地連接至轉錄啟動子及轉錄終止子的根據本發明之核酸。Typically, the expression cassette comprises or consists of a nucleic acid according to the present invention operably linked to a control sequence, such as a transcriptional promoter and/or a transcriptional terminator. The control sequence may include a promoter, which is recognized by a host cell or an in vitro expression system for expressing a nucleic acid encoding an esterase of the present invention. The promoter contains a transcriptional control sequence that mediates the expression of the enzyme. The promoter may be any polynucleotide that exhibits transcriptional activity in a host cell, including mutant, truncated and hybrid promoters, and may be obtained from a gene that encodes a homologous or heterologous extracellular or intracellular polypeptide into a host cell. The control sequence may also be a transcriptional terminator, which is recognized by a host cell to terminate transcription. The terminator is operably linked to the 3' end of the nucleic acid encoding the esterase. Any terminator that is functional in the host cell can be used in the present invention. Typically, the expression cassette comprises or consists of a nucleic acid according to the present invention operably linked to a transcriptional promoter and a transcriptional terminator.

本發明亦係關於包含上文所定義之核酸或表現卡匣的載體。The invention also relates to a vector comprising a nucleic acid or an expression cassette as defined above.

依本文所用，術語「 載體」或「 表現載體」係指包含本發明之表現卡匣的DNA或RNA分子，其用作將重組遺傳物質轉移至宿主細胞中之載體。主要類型之載體為質體、噬菌體、病毒、黏質體及人工染色體。載體本身通常為由插入序列(異源核酸序列，轉殖基因)及充當載體之「主鏈」的較大序列組成的DNA序列。將遺傳資訊轉移至宿主的載體之目的通常為在目標細胞中分離、倍增或表現插入序列。被稱為表現載體(表現構築體)之載體經特定調適以用於在目標細胞中表現異源序列，且通常具有驅動編碼多肽之異源序列之表現的啟動子序列。通常，存在於表現載體中之調節元件包括轉錄啟動子、核糖體結合位點、終止子，且視情況存在操縱子。較佳地，表現載體亦含有用於在宿主細胞中之自主複製的複製起點、可選標記物、有限數目個有效限制酶位點及高複本數之潛能。表現載體之實例為選殖載體、經修飾之選殖載體、經特定設計之質體及病毒。在不同宿主中提供適合水準之多肽表現的表現載體為此項技術中所熟知。載體之選擇將通常視載體與待引入載體之宿主細胞的相容性而定。較佳地，表現載體為線性或環狀雙股DNA分子。 As used herein, the term " vector " or " expression vector " refers to a DNA or RNA molecule comprising an expression cassette of the present invention, which is used as a vector for transferring recombinant genetic material into a host cell. The main types of vectors are plasmids, bacteriophages, viruses, cosmids and artificial chromosomes. The vector itself is usually a DNA sequence composed of an inserted sequence (heterologous nucleic acid sequence, transgene) and a larger sequence that serves as the "backbone" of the vector. The purpose of the vector for transferring genetic information to a host is usually to isolate, multiply or express the inserted sequence in the target cell. Vectors, called expression vectors (expression constructs), are specifically adapted for expressing heterologous sequences in target cells and usually have a promoter sequence that drives the expression of the heterologous sequence encoding a polypeptide. Typically, regulatory elements present in an expression vector include a transcriptional promoter, a ribosome binding site, a terminator, and, if appropriate, an operator. Preferably, the expression vector also contains an origin of replication for autonomous replication in a host cell, a selectable marker, a limited number of effective restriction enzyme sites, and the potential for a high copy number. Examples of expression vectors are cloning vectors, modified cloning vectors, specifically designed plasmids, and viruses. Expression vectors that provide appropriate levels of polypeptide expression in different hosts are well known in the art. The choice of vector will generally depend on the compatibility of the vector with the host cell into which the vector is to be introduced. Preferably, the expression vector is a linear or circular double-stranded DNA molecule.

本發明之另一目標為提供包含上文所描述之核酸、表現卡匣或載體的宿主細胞。由此，本發明係關於根據本發明之核酸、表現卡匣或載體用於轉化、轉染或轉導宿主細胞之用途。載體之選擇將通常視載體與必須引入載體之宿主細胞的相容性而定。Another object of the present invention is to provide a host cell comprising a nucleic acid, expression cassette or vector as described above. Thus, the present invention relates to the use of a nucleic acid, expression cassette or vector according to the present invention for transforming, transfecting or transducing a host cell. The choice of vector will generally depend on the compatibility of the vector with the host cell into which the vector must be introduced.

根據本發明，宿主細胞可以暫時或穩定方式經轉化、轉染或轉導。將本發明之表現卡匣或載體引入宿主細胞中，使得該卡匣或載體作為染色體組成部分或作為自我複製的染色體外載體維持。術語「 宿主細胞」亦涵蓋因複製期間發生之突變而與親本宿主細胞不一致之親本宿主細胞的任何後代。宿主細胞可為適用於產生本發明之變異體的任何細胞，例如原核細胞或真核細胞。原核宿主細胞可為任何革蘭氏陽性(Gram-positive)或革蘭氏陰性(Gram-negative)細菌。宿主細胞亦可為真核細胞，諸如酵母菌、真菌、哺乳動物、昆蟲或植物細胞。在特定實施例中，宿主細胞係選自以下之群：大腸桿菌(Escherichia coli)、芽孢桿菌(Bacillus)、鏈黴菌(Streptomyces)、木黴菌(Trichoderma)、麴黴菌(Aspergillus)、酵母菌(Saccharomyces)、畢赤酵母菌(Pichia)、弧菌(Vibrio)或耶氏酵母菌(Yarrowia)。 According to the present invention, host cells may be transformed, transfected or transduced in a transient or stable manner. An expression cassette or vector of the present invention is introduced into a host cell such that the cassette or vector is maintained as a chromosomal component or as a self-replicating extrachromosomal vector. The term " host cell " also encompasses any progeny of a parent host cell that is not identical to the parent host cell due to mutations that occur during replication. The host cell may be any cell suitable for generating variants of the present invention, such as a prokaryotic cell or a eukaryotic cell. A prokaryotic host cell may be any Gram-positive or Gram-negative bacterium. The host cell may also be a eukaryotic cell, such as a yeast, fungus, mammal, insect or plant cell. In a specific embodiment, the host cell is selected from the group consisting of Escherichia coli, Bacillus, Streptomyces, Trichoderma, Aspergillus, Saccharomyces, Pichia, Vibrio or Yarrowia.

根據本發明之核酸、表現卡匣或表現載體可藉由熟習此項技術者已知的任何方法而引入宿主細胞中，該方法諸如為電穿孔、結合、轉導、勝任細胞轉化、原生質體轉化、原生質體融合、基因槍(biolistic)「基因槍(gene gun)」轉化、PEG介導之轉化、脂質輔助之轉化或轉染、以化學方式介導之轉染、乙酸鋰介導之轉化、脂質體介導之轉化。Nucleic acids, expression cassettes or expression vectors according to the present invention can be introduced into host cells by any method known to those skilled in the art, such as electroporation, conjugation, transduction, competent cell transformation, protoplast transformation, protoplast fusion, biolistic "gene gun" transformation, PEG-mediated transformation, lipid-assisted transformation or transfection, chemically mediated transfection, lithium acetate-mediated transformation, liposome-mediated transformation.

視情況，可將本發明之核酸、卡匣或載體之超過一個複本插入宿主細胞中以增加變異體之產生。Optionally, more than one copy of a nucleic acid, cassette or vector of the invention may be inserted into a host cell to increase the production of variants.

在特定實施例中，宿主細胞為重組微生物。實際上，本發明允許將微生物工程改造成具有改良的使含聚酯材料降解之能力。舉例而言，本發明之序列可用於補充已知能夠使聚酯降解的真菌或細菌之野生型菌株，以改良及/或增加菌株能力。In certain embodiments, the host cell is a recombinant microorganism. In fact, the present invention allows the engineering of microorganisms with improved ability to degrade polyester-containing materials. For example, the sequences of the present invention can be used to supplement wild-type strains of fungi or bacteria known to be able to degrade polyester to improve and/or increase the strain's ability.

酯酶之產生本發明之另一目標為提供一種產生本發明酯酶之方法，其包含表現編碼該酯酶之核酸以及視情況回收該酯酶。 Production of esterases Another object of the present invention is to provide a method for producing the esterase of the present invention, comprising expressing a nucleic acid encoding the esterase and optionally recovering the esterase.

特定言之，本發明係關於產生本發明酯酶之活體外方法，其包含：(a)使本發明之核酸、卡匣或載體與活體外表現系統接觸；及(b)回收所產生的酯酶。活體外表現系統為熟習此項技術者所熟知，且為可商購的。Specifically, the present invention relates to an in vitro method for producing an esterase of the present invention, comprising: (a) contacting a nucleic acid, cassette or vector of the present invention with an in vitro expression system; and (b) recovering the produced esterase. In vitro expression systems are well known to those skilled in the art and are commercially available.

較佳地，該產生方法包含 (a) 在適合於表現核酸之條件下培養包含編碼本發明之酯酶的核酸的宿主細胞；及視情況 (b) 自細胞培養物回收該酯酶。 Preferably, the production method comprises (a) culturing a host cell comprising a nucleic acid encoding an esterase of the present invention under conditions suitable for expression of the nucleic acid; and optionally (b) recovering the esterase from the cell culture.

有利的是，宿主細胞為重組芽孢桿菌、重組大腸桿菌、重組麴黴菌、重組木黴菌、重組鏈黴菌、重組酵母菌、重組畢赤酵母菌、重組弧菌或重組耶氏酵母菌。Advantageously, the host cell is a recombinant Bacillus, a recombinant Escherichia coli, a recombinant Aspergillus, a recombinant Trichoderma, a recombinant Streptococcus, a recombinant Saccharomyces, a recombinant Pichia pastoris, a recombinant Vibrio or a recombinant Yarrowia.

使用此項技術中已知之方法，在適合於產生多肽之營養物培養基中培養宿主細胞。舉例而言，可藉由在適合之培養基中且在允許表現及/或分離酶的條件下進行的搖瓶培養或者實驗室或工業醱酵器中之小規模或大規模醱酵(包括連續、分批、分批補料或固體狀態醱酵)來培養細胞。培養在適合之營養物培養基中進行，該營養物培養基來自商業供應商或根據公佈之組成(例如於美國菌種保藏中心(American Type Culture Collection)之目錄中)製備。The host cells are cultured in a nutrient medium suitable for production of the polypeptide using methods known in the art. For example, the cells may be cultured by shaking flask culture or small-scale or large-scale fermentation (including continuous, batch, fed-batch or solid-state fermentation) in a suitable medium and under conditions that permit expression and/or isolation of the enzyme in a laboratory or industrial fermentor. The culture is carried out in a suitable nutrient medium, which is obtained from a commercial supplier or prepared according to a published composition (e.g., in the catalog of the American Type Culture Collection).

若酯酶分泌至營養物培養基中，則可直接自培養物上清液回收酯酶。相反，可自細胞溶解物或在滲透之後回收酯酶。可使用此項技術中已知之任何方法來回收酯酶。舉例而言，可藉由習知程序自營養物培養基回收酯酶，該等程序包括但不限於收集、離心、過濾、萃取、噴霧乾燥、蒸發或沉澱。視情況，酯酶可藉由此項技術中已知之各種程序部分或完全純化以獲得實質上純的多肽，該等程序包括但不限於層析(例如，離子交換、親和性、疏水性、層析聚焦及尺寸排阻)、電泳程序(例如，製備型等電聚焦)、差分溶解度(例如，硫酸銨沉澱)、SDS-PAGE或萃取。If the esterase is secreted into the nutrient medium, the esterase can be recovered directly from the culture supernatant. Conversely, the esterase can be recovered from a cell lysate or after permeabilization. The esterase can be recovered using any method known in the art. For example, the esterase can be recovered from the nutrient medium by known procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. Optionally, the esterase may be partially or completely purified by various procedures known in the art, including but not limited to chromatography (e.g., ion exchange, affinity, hydrophobic, chromatography focusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction to obtain a substantially pure polypeptide.

酯酶本身可以純化形式單獨或與其他酶組合使用，以催化與使聚酯及/或含聚酯材料(諸如，含有聚酯之塑膠產品)降解及/或再循環有關的酶促反應。酯酶可呈可溶形式或呈固相。特定言之，酯酶可結合至細胞膜或脂質囊泡，或結合至合成性支撐物，諸如玻璃、塑膠、聚合物、濾紙、例如呈珠粒形式之膜、管柱、培養盤及其類似物。The esterase itself can be used in purified form alone or in combination with other enzymes to catalyze enzymatic reactions associated with degradation and/or recycling of polyesters and/or polyester-containing materials (e.g., plastic products containing polyesters). The esterase can be in soluble form or in a solid phase. In particular, the esterase can be bound to a cell membrane or a lipid vesicle, or to a synthetic support such as glass, plastic, polymer, filter paper, a membrane, for example in the form of beads, a column, a culture dish, and the like.

組合物本發明之另一目標為提供一種組合物，其包含本發明之酯酶或宿主細胞或其含有酯酶之萃取物。在本發明之上下文中，術語「組合物」涵蓋任何種類之組合物，其包含本發明之酯酶或宿主細胞或其含有酯酶之萃取物。 Composition Another object of the present invention is to provide a composition comprising the esterase of the present invention or the host cell or an extract thereof containing the esterase. In the context of the present invention, the term "composition" covers any type of composition comprising the esterase of the present invention or the host cell or an extract thereof containing the esterase.

按組合物之總重量計，本發明之組合物可包含0.1重量%至99.9重量%、較佳0.1重量%至50重量%、更佳0.1重量%至30重量%、甚至更佳0.1重量%至5重量%之酯酶。替代地，組合物可包含5重量%至10重量%之本發明之酯酶。Based on the total weight of the composition, the composition of the present invention can include 0.1 wt % to 99.9 wt %, preferably 0.1 wt % to 50 wt %, more preferably 0.1 wt % to 30 wt %, even more preferably 0.1 wt % to 5 wt % of esterase. Alternatively, the composition can include 5 wt % to 10 wt % of the esterase of the present invention.

組合物可呈液體或乾燥形式，例如呈粉末形式。在一些實施例中，組合物為凍乾物。The composition can be in liquid or dry form, for example in powder form. In some embodiments, the composition is a lyophilisate.

組合物可進一步包含賦形劑及/或反應劑等。適當的賦形劑涵蓋：通常用於生物化學中之緩衝液；用於調整pH之試劑；防腐劑，諸如苯甲酸鈉、山梨酸鈉或抗壞血酸鈉；保守劑、保護劑或穩定劑，諸如澱粉、糊精、阿拉伯膠、鹽、糖(例如，山梨糖醇、海藻糖或乳糖)、甘油、聚乙二醇、聚丙二醇、丙二醇；螯合劑，諸如EDTA；還原劑；胺基酸；載劑，諸如溶劑或水溶液；及其類似賦形劑。本發明之組合物可藉由將酯酶與一種或若干種賦形劑混合而獲得。The composition may further comprise excipients and/or reactants, etc. Suitable excipients include: buffers commonly used in biochemistry; reagents for adjusting pH; preservatives, such as sodium benzoate, sodium sorbate or sodium ascorbate; conserving agents, protective agents or stabilizers, such as starch, dextrin, gum arabic, salts, sugars (e.g., sorbitol, trehalose or lactose), glycerol, polyethylene glycol, polypropylene glycol, propylene glycol; chelating agents, such as EDTA; reducing agents; amino acids; carriers, such as solvents or aqueous solutions; and similar excipients. The composition of the present invention can be obtained by mixing esterase with one or more excipients.

舉例而言，按組合物之總重量計，組合物包含0.1重量%至99.9重量%、較佳50重量%至99.9重量%、更佳70重量%至99.9重量%、甚至更佳95重量%至99.9重量%之賦形劑。替代地，組合物可包含90重量%至95重量%之賦形劑。For example, the composition comprises 0.1 wt % to 99.9 wt %, preferably 50 wt % to 99.9 wt %, more preferably 70 wt % to 99.9 wt %, even more preferably 95 wt % to 99.9 wt % of the shaping agent based on the total weight of the composition. Alternatively, the composition may comprise 90 wt % to 95 wt % of the shaping agent.

組合物可進一步包含展現酶活性之其他多肽。本發明之酯酶之量將容易地由熟習此項技術者例如取決於需降解之聚酯及/或組合物中所含有之其他酶/多肽的性質進行調適。The composition may further comprise other polypeptides exhibiting enzymatic activity. The amount of the esterase of the present invention will be easily adapted by those skilled in the art, for example depending on the nature of the polyester to be degraded and/or other enzymes/polypeptides contained in the composition.

本發明之酯酶可與一種或若干種賦形劑(尤其能夠使多肽穩定或保護多肽不降解的賦形劑)一起溶解於水性介質中。舉例而言，本發明之酯酶可最終與其他組分一起溶解於水中，該等其他組分諸如為甘油、山梨糖醇、糊精、澱粉、二醇(諸如丙二醇)、鹽等。所得混合物隨後可經乾燥以獲得粉末。用於乾燥此類混合物之方法為熟習此項技術者所熟知，且包括但不限於凍乾、冷凍乾燥、噴霧乾燥、超臨界乾燥、下吸式蒸發、薄層蒸發、離心蒸發、帶式乾燥、流體化床乾燥、滾筒乾燥或其任何組合。The esterase of the present invention can be dissolved in an aqueous medium together with one or more excipients (especially excipients that can stabilize the polypeptide or protect the polypeptide from degradation). For example, the esterase of the present invention can be finally dissolved in water together with other components, such as glycerol, sorbitol, dextrin, starch, glycols (such as propylene glycol), salts, etc. The resulting mixture can then be dried to obtain a powder. Methods for drying such mixtures are well known to those skilled in the art and include, but are not limited to, freeze drying, freeze drying, spray drying, supercritical drying, downdraft evaporation, thin layer evaporation, centrifugal evaporation, belt drying, fluidized bed drying, drum drying or any combination thereof.

組合物可呈粉末形式，且可包含酯酶及穩定/增溶量之甘油、山梨糖醇或糊精(諸如，麥芽糖糊精及/或環糊精)、澱粉、二醇(諸如，丙二醇)及/或鹽。The composition may be in powder form and may comprise an esterase and a stabilizing/solubilizing amount of glycerol, sorbitol or a dextrin (e.g., maltodextrin and/or cyclodextrin), starch, a glycol (e.g., propylene glycol) and/or a salt.

本發明之組合物可包含至少一種表現本發明之酯酶的重組細胞或其萃取物。「細胞萃取物」表示藉由化學、物理及/或酶促處理而自細胞獲得之任何部分，諸如細胞上清液、細胞碎片、細胞壁、DNA萃取物、酶或酶製備物或衍生自細胞之任何製備物，其基本上不含活細胞。較佳的萃取物為具有酶促活性之萃取物。本發明之組合物可包含本發明之一種或若干種重組細胞或其萃取物以及視情況存在之一種或若干種其他細胞。The composition of the present invention may comprise at least one recombinant cell expressing the esterase of the present invention or an extract thereof. "Cell extract" means any part obtained from cells by chemical, physical and/or enzymatic treatment, such as cell supernatant, cell fragments, cell wall, DNA extract, enzyme or enzyme preparation or any preparation derived from cells, which is essentially free of living cells. Preferred extracts are extracts with enzymatic activity. The composition of the present invention may comprise one or more recombinant cells or extracts thereof of the present invention and one or more other cells as appropriate.

舉例而言，組合物由以下組成或包含以下：表現且分泌本發明之酯酶的重組微生物之培養基。在特定實施例中，組合物包含此類凍乾的培養基。For example, the composition consists of or comprises the following: a culture medium of a recombinant microorganism that expresses and secretes the esterase of the present invention. In a specific embodiment, the composition comprises such a lyophilized culture medium.

酯酶之用途本發明之另一目標為提供使用本發明之酯酶以在好氧或厭氧條件中使聚酯或含聚酯材料降解及/或再循環的方法。本發明之酯酶尤其適用於使PET及含有PET之材料降解，尤其在6與10之間的pH下。 Another object of the present invention is to provide a method for degrading and/or recycling polyester or polyester-containing materials using the esterase of the present invention under aerobic or anaerobic conditions. The esterase of the present invention is particularly suitable for degrading PET and PET-containing materials, especially at a pH between 6 and 10.

因此，本發明之目標為使用本發明之酯酶或具有酯酶活性之對應的重組細胞或其萃取物或者組合物來酶促降解聚酯。Therefore, the object of the present invention is to use the esterase of the present invention or the corresponding recombinant cells or extracts or compositions thereof having esterase activity to enzymatically degrade polyester.

有利的是，酯酶所靶向之聚酯係選自聚對苯二甲酸伸乙酯(PET)、聚對苯二甲酸伸丙酯(PTT)、聚對苯二甲酸伸丁酯(PBT)、聚異山梨醇對苯二甲酸伸乙酯(PEIT)、聚乳酸(PLA)、聚羥基烷酸酯(PHA)、聚丁二酸伸丁酯(PBS)、聚丁二酸己二酸伸丁酯(PBSA)、聚己二酸對苯二甲酸伸丁酯(PBAT)、聚呋喃酸伸乙酯(PEF)、聚己內酯(PCL)、聚(己二酸伸乙酯) (PEA)、聚萘二甲酸伸乙酯(PEN)、「聚烯烴類」聚酯及此等材料之摻合物/混合物，較佳為聚對苯二甲酸伸乙酯。Advantageously, the polyester targeted by the esterase is selected from polyethylene terephthalate (PET), polypropylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), polycaprolactone (PCL), poly(ethylene adipate) (PEA), polyethylene naphthalate (PEN), "polyolefin" polyesters and blends/mixtures of these materials, preferably polyethylene terephthalate.

在較佳實施例中，聚酯為PET，且視情況回收至少單體(例如，單乙二醇或對苯二甲酸)及/或寡聚物(例如，對苯二甲酸甲基-2-羥基乙酯(MHET)、對苯二甲酸雙(2-羥基乙酯) (BHET)、對苯二甲酸1-(2-羥基乙酯) 4-甲酯(HEMT)及對苯二甲酸二甲酯(DMT))。In a preferred embodiment, the polyester is PET, and at least monomers (e.g., monoethylene glycol or terephthalic acid) and/or oligomers (e.g., methyl-2-hydroxyethyl terephthalate (MHET), bis(2-hydroxyethyl) terephthalate (BHET), 1-(2-hydroxyethyl)-4-methyl terephthalate (HEMT) and dimethyl terephthalate (DMT)) are recovered as appropriate.

本發明之又一目標為使用本發明之酯酶或對應的重組細胞或其萃取物或者組合物來酶促降解含聚酯材料中之至少一種聚酯，尤其在6與10之間的pH下。Another object of the present invention is to use the esterase of the present invention or the corresponding recombinant cells or extracts or compositions thereof for enzymatic degradation of at least one polyester in a polyester-containing material, in particular at a pH between 6 and 10.

本發明之另一目標為提供一種用於使含聚酯材料中之至少一種聚酯降解的方法，其中使該含聚酯材料與本發明之酯酶或宿主細胞或其萃取物或者組合物接觸，尤其在6與10之間的pH下，從而使含聚酯材料中之該至少一種聚酯降解。Another object of the present invention is to provide a method for degrading at least one polyester in a polyester-containing material, wherein the polyester-containing material is contacted with an esterase or a host cell or an extract or a composition thereof according to the present invention, in particular at a pH between 6 and 10, thereby degrading the at least one polyester in the polyester-containing material.

有利的是，聚酯經解聚合為單體及/或寡聚物。Advantageously, the polyester is depolymerized into monomers and/or oligomers.

特定言之，本發明提供一種用於使含有PET之材料中之PET降解的方法，其中使該含有PET之材料與本發明之酯酶或宿主細胞或組合物接觸，尤其在6與10之間的pH下，從而使該PET降解。In particular, the present invention provides a method for degrading PET in a PET-containing material, wherein the PET-containing material is contacted with an esterase or a host cell or a composition of the present invention, in particular at a pH between 6 and 10, thereby degrading the PET.

有利的是，至少一種聚酯降解成可再聚合的單體及/或寡聚物，可有利地重新得到該等單體及/或寡聚物以再使用。重新得到的單體/寡聚物可用於再循環(例如，使聚酯再聚合)或甲烷化。在特定實施例中，至少一種聚酯為PET，且重新得到單乙二醇、對苯二甲酸、對苯二甲酸甲基-2-羥基乙酯(MHET)、對苯二甲酸雙(2-羥基乙酯) (BHET)、對苯二甲酸1-(2-羥基乙酯) 4-甲酯(HEMT)及/或對苯二甲酸二甲酯(DMT)。Advantageously, at least one polyester is degraded into repolymerizable monomers and/or oligomers, which can be advantageously recovered for reuse. The recovered monomers/oligomers can be used for recycling (e.g., repolymerizing the polyester) or methanation. In a particular embodiment, at least one polyester is PET, and monoethylene glycol, terephthalic acid, methyl-2-hydroxyethyl terephthalate (MHET), bis(2-hydroxyethyl) terephthalate (BHET), 1-(2-hydroxyethyl)-4-methyl terephthalate (HEMT) and/or dimethyl terephthalate (DMT) are recovered.

較佳地，含聚酯材料中之聚酯充分降解。Preferably, the polyester in the polyester-containing material is substantially degraded.

使含聚酯材料降解所需的時間可視含聚酯材料本身(亦即，含聚酯材料之性質及來源、其組成、形狀等)、所使用酯酶之類型及量以及各種過程參數(亦即，溫度、pH、其他試劑等)而變化。熟習此項技術者可容易地使過程參數適於含聚酯材料及所設想降解時間。The time required to degrade the polyester-containing material may vary depending on the polyester-containing material itself (i.e., the nature and source of the polyester-containing material, its composition, shape, etc.), the type and amount of esterase used, and various process parameters (i.e., temperature, pH, other reagents, etc.). One skilled in the art can readily adapt the process parameters to the polyester-containing material and the envisioned degradation time.

有利的是，降解過程在包含於20℃與90℃之間、較佳40℃與90℃之間、更佳50℃與70℃之間的溫度下實施。在特定實施例中，降解過程係在60℃下實施。在另一特定實施例中，降解過程係在65℃下實施。在另一特定實施例中，降解過程係在70℃下實施。更一般而言，將溫度維持在不活化溫度以下，該不活化溫度對應於酯酶不活化之溫度(亦即，與酯酶在其最佳溫度下之活性相比酯酶喪失超過80%活性之溫度)及/或重組微生物不再合成酯酶之溫度。特定言之，將溫度維持在所靶向聚酯之玻璃轉化溫度(Tg)以下。Advantageously, the degradation process is carried out at a temperature comprised between 20°C and 90°C, preferably between 40°C and 90°C, more preferably between 50°C and 70°C. In a particular embodiment, the degradation process is carried out at 60°C. In another particular embodiment, the degradation process is carried out at 65°C. In another particular embodiment, the degradation process is carried out at 70°C. More generally, the temperature is maintained below the inactivation temperature, which corresponds to a temperature at which the esterase is inactive (i.e., a temperature at which the esterase loses more than 80% of its activity compared to its activity at its optimal temperature) and/or a temperature at which the recombinant microorganism no longer synthesizes the esterase. In particular, the temperature is maintained below the glass transition temperature (Tg) of the targeted polyester.

有利的是，過程在酯酶可使用若干次及/或再循環之溫度下以連續流過程之形式實施。Advantageously, the process is carried out as a continuous flow process at a temperature at which the esterase can be used several times and/or recycled.

有利的是，降解過程係在包含於5至9之間的pH下、較佳在6至9之pH範圍內、更佳在6.5至9之pH範圍內、甚至更佳在6.5至8之pH範圍內、甚至更佳在包含於7與9之間的pH下、尤其在pH 8下實施。Advantageously, the degradation process is carried out at a pH comprised between 5 and 9, preferably in the pH range of 6 to 9, more preferably in the pH range of 6.5 to 9, even more preferably in the pH range of 6.5 to 8, even more preferably at a pH comprised between 7 and 9, in particular at pH 8.

含聚酯材料可在與酯酶接觸之前經預處理，以便以物理方式改變其結構，從而增加聚酯與酯酶之間的接觸表面。The polyester-containing material may be pretreated prior to contact with the esterase in order to physically alter its structure and thereby increase the contact surface between the polyester and the esterase.

本發明之另一目標為提供一種自含聚酯材料產生單體及/或寡聚物之方法，其包含將含聚酯材料暴露於本發明之酯酶或對應的重組細胞或其萃取物或者組合物，尤其在6與10之間的pH下，以及視情況回收單體及/或寡聚物。Another object of the present invention is to provide a method for producing monomers and/or oligomers from a polyester-containing material, which comprises exposing the polyester-containing material to the esterase of the present invention or corresponding recombinant cells or extracts or compositions thereof, in particular at a pH between 6 and 10, and optionally recovering the monomers and/or oligomers.

由解聚合產生之單體及/或寡聚物可依序或連續地回收。視起始的含聚酯材料而定，可回收單一類型之單體及/或寡聚物或若干不同類型之單體及/或寡聚物。The monomers and/or oligomers produced by depolymerization can be recovered sequentially or continuously. Depending on the starting polyester-containing material, a single type of monomer and/or oligomer or several different types of monomers and/or oligomers can be recovered.

本發明之方法尤其適用於自PET及/或包含PET之塑膠產品產生選自單乙二醇及對苯二甲酸之單體，及/或選自對苯二甲酸甲基-2-羥基乙酯(MHET)、對苯二甲酸雙(2-羥基乙酯) (BHET)、對苯二甲酸1-(2-羥基乙酯) 4-甲酯(HEMT)及對苯二甲酸二甲酯(DMT)之寡聚物。The method of the present invention is particularly suitable for producing monomers selected from monoethylene glycol and terephthalic acid, and/or oligomers selected from methyl-2-hydroxyethyl terephthalate (MHET), bis(2-hydroxyethyl) terephthalate (BHET), 1-(2-hydroxyethyl)-4-methyl terephthalate (HEMT) and dimethyl terephthalate (DMT) from PET and/or plastic products containing PET.

回收的單體及/或寡聚物可使用所有適合之純化方法進一步純化且調節成可再聚合形式。The recovered monomers and/or oligomers can be further purified and adjusted to a repolymerizable form using all suitable purification methods.

回收的可再聚合的單體及/或寡聚物可例如再用於合成聚酯。有利的是，再聚合具有相同性質之聚酯。然而，有可能將回收的單體及/或寡聚物與其他單體及/或寡聚物混合，例如以合成新的共聚物。替代地，回收的單體可用作化學中間物以便產生所關注的新化合物。The recovered repolymerizable monomers and/or oligomers can, for example, be reused in the synthesis of polyesters. Advantageously, polyesters having the same properties are repolymerized. However, it is possible to mix the recovered monomers and/or oligomers with other monomers and/or oligomers, for example to synthesize new copolymers. Alternatively, the recovered monomers can be used as chemical intermediates in order to produce new compounds of interest.

作為實例，使包括本發明之酯酶的此類含聚酯材料降解之方法揭示於專利申請案WO 2014/079844、WO 2015/173265、WO 2017/198786、WO 2020/094661、WO 2020/094646、WO 2021/123299、WO 2021/123301及WO 2021/123328中。本發明亦係關於一種使含聚酯材料表面水解或表面官能化之方法，其包含將含聚酯材料暴露於本發明之酯酶或對應的重組細胞或其萃取物或者組合物，尤其在6與10之間的pH下。本發明之方法尤其適用於增加聚酯材料之親水性或吸水性。此類增加之親水性可在織物生產、電子裝置及生物醫學應用中受到特別關注。As an example, methods for degrading such polyester-containing materials comprising the esterase of the present invention are disclosed in patent applications WO 2014/079844, WO 2015/173265, WO 2017/198786, WO 2020/094661, WO 2020/094646, WO 2021/123299, WO 2021/123301 and WO 2021/123328. The present invention also relates to a method for surface hydrolysis or surface functionalization of a polyester-containing material, comprising exposing the polyester-containing material to the esterase of the present invention or a corresponding recombinant cell or an extract or composition thereof, in particular at a pH between 6 and 10. The method of the present invention is particularly suitable for increasing the hydrophilicity or water absorption of a polyester material. Such increased hydrophilicity may be of particular interest in textile production, electronic devices, and biomedical applications.

本發明亦係關於一種用於處理水、廢水或污水之方法，尤其在6與10之間的pH下。在廢水或污水處理應用中，根據本發明之酯酶可用於使由聚酯(較佳為PET)組成之微塑膠粒子降解，如聚合物長絲、纖維或其他類型之基於聚酯之產物碎片及片段，較佳為基於PET之產物碎片及片段。本發明之另一目標為提供一種含聚酯材料，其中包括本發明之酯酶及/或表現且分泌該酯酶之重組微生物。作為實例，用於製備包括本發明之酯酶的此類含聚酯材料的製程揭示於專利申請案WO2013/093355、WO 2016/198650、WO 2016/198652、WO 2019/043145及WO 2019/043134中。The present invention also relates to a method for treating water, wastewater or sewage, in particular at a pH between 6 and 10. In wastewater or sewage treatment applications, the esterase according to the present invention can be used to degrade microplastic particles composed of polyester, preferably PET, such as polymer filaments, fibers or other types of polyester-based product fragments and fragments, preferably PET-based product fragments and fragments. Another object of the present invention is to provide a polyester-containing material, comprising the esterase of the present invention and/or a recombinant microorganism expressing and secreting the esterase. As an example, processes for preparing such polyester-containing materials including the esterase of the present invention are disclosed in patent applications WO 2013/093355, WO 2016/198650, WO 2016/198652, WO 2019/043145 and WO 2019/043134.

由此，本發明之一目標為提供一種含聚酯材料，其含有本發明之酯酶及/或重組細胞及/或組合物或其萃取物以及至少PET。根據實施例，本發明提供一種塑膠產品，其包含PET及具有PET降解活性之本發明之酯酶。Thus, one object of the present invention is to provide a polyester-containing material, which contains the esterase and/or recombinant cells and/or composition of the present invention or its extract and at least PET. According to an embodiment, the present invention provides a plastic product, which contains PET and the esterase of the present invention having PET degradation activity.

由此，本發明之另一目標為提供一種含聚酯材料，其含有本發明之酯酶及/或重組細胞及/或組合物或其萃取物以及至少PBAT。根據實施例，本發明提供一種塑膠產品，其包含PBAT及具有PBAT降解活性之本發明之酯酶。Thus, another object of the present invention is to provide a polyester-containing material, which contains the esterase and/or reorganized cells and/or composition or extract thereof of the present invention and at least PBAT. According to an embodiment, the present invention provides a plastic product, which contains PBAT and the esterase of the present invention having PBAT degradation activity.

由此，本發明之另一目標為提供一種含聚酯材料，其含有本發明之酯酶及/或重組細胞及/或組合物或其萃取物以及至少PBS。根據實施例，本發明提供一種塑膠產品，其包含PBS及具有PBS降解活性之本發明之酯酶。Thus, another object of the present invention is to provide a polyester-containing material, which contains the esterase and/or reorganized cells and/or composition or extract thereof of the present invention and at least PBS. According to an embodiment, the present invention provides a plastic product, which contains PBS and the esterase of the present invention having PBS degradation activity.

由此，本發明之另一目標為提供一種含聚酯材料，其含有本發明之酯酶及/或重組細胞及/或組合物或其萃取物以及至少PCL。根據實施例，本發明提供一種塑膠產品，其包含PCL及具有PCL降解活性之本發明之酯酶。Thus, another object of the present invention is to provide a polyester-containing material, which contains the esterase and/or reorganized cells and/or composition or extract thereof of the present invention and at least PCL. According to an embodiment, the present invention provides a plastic product, which contains PCL and the esterase of the present invention having PCL degradation activity.

傳統地，本發明之酯酶可用於清潔劑、食物、動物飼料、造紙、織物及醫藥應用中。更特定言之，本發明之酯酶可用作清潔劑組合物之組分。清潔劑組合物包括但不限於手洗或機器洗衣清潔劑組合物，諸如適合於預處理染色織品之洗衣添加劑組合物及沖洗添加的織品軟化劑組合物、用於一般家庭硬表面清洗操作之清潔劑組合物、用於手洗或機器洗碗操作之清潔劑組合物。舉例而言，本發明之酯酶可用作清潔劑添加劑。由此，本發明提供包含本發明之酯酶之清潔劑組合物。特定言之，本發明之酯酶可用作清潔劑添加劑以減少織物清洗期間的起球及發灰效應。Traditionally, the esterase of the present invention can be used in detergent, food, animal feed, papermaking, textile and medical applications. More specifically, the esterase of the present invention can be used as a component of a detergent composition. Detergent compositions include but are not limited to hand-washing or machine laundry detergent compositions, such as laundry additive compositions suitable for pre-treatment of dyed fabrics and fabric softener compositions added by rinsing, detergent compositions for general household hard surface cleaning operations, detergent compositions for hand-washing or machine dishwashing operations. For example, the esterase of the present invention can be used as a detergent additive. Thus, the present invention provides a detergent composition comprising the esterase of the present invention. In particular, the esterases of the present invention can be used as detergent additives to reduce pilling and graying effects during fabric washing.

本發明亦係關於在動物飼料中使用本發明之酯酶之方法，以及包含本發明之酯酶之飼料組合物及飼料添加劑。術語「飼料」及「飼料組合物」係指適合於或既定用於由動物攝入的任何化合物、製備物、混合物或組合物。本發明之酯酶亦可用於使蛋白質水解，且用於產生包含肽之水解產物。此類水解產物可用作飼料組合物或飼料添加劑。The present invention also relates to methods of using the esterase of the present invention in animal feeds, and feed compositions and feed additives comprising the esterase of the present invention. The terms "feed" and "feed composition" refer to any compound, preparation, mixture or composition suitable for or intended for ingestion by animals. The esterase of the present invention can also be used to hydrolyze proteins and to produce hydrolyzates comprising peptides. Such hydrolyzates can be used as feed compositions or feed additives.

本發明之另一目標為提供一種在造紙工業中使用本發明之酯酶的方法。更特定言之，本發明之酯酶可用於自造紙機之紙漿及水管線移除黏性物質。Another object of the present invention is to provide a method of using the esterase of the present invention in the papermaking industry. More specifically, the esterase of the present invention can be used to remove sticky substances from the pulp and water pipelines of a paper machine.

實例 實例 1- 酯酶之構築、表現及純化 構築使用質體構築pET26b-LCC-His來產生根據本發明之酯酶。此質體在於選殖編碼SEQ ID NO: 1之酯酶的基因，其針對 NdeI與 XhoI限制位點之間的大腸桿菌表現最佳化。已根據供應商之建議使用兩個定點突變誘發套組來產生酯酶變異體：來自Agilent (Santa Clara, California, USA)之QuikChange II定點突變誘發套組及QuikChange Lightning多定點。 EXAMPLES Example 1 - Construction, Expression and Purification of Esterases The plasmid construct pET26b-LCC-His was used to produce the esterases according to the present invention. This plasmid consists in cloning the gene encoding the esterase of SEQ ID NO: 1, which is optimized for expression in E. coli between the NdeI and XhoI restriction sites. Two site-directed mutagenesis induction kits have been used to generate esterase variants according to the supplier's recommendations: the QuikChange II site-directed mutagenesis induction kit and the QuikChange Lightning Multisite from Agilent (Santa Clara, California, USA).

酯酶之表現及純化在50 mL LB-Miller培養基或ZYM自動誘導培養基(Studier等人, 2005- Prot. Exp. Pur. 41, 207-234)中，依次採用菌株Stellar™ (Clontech, California, USA)及大腸桿菌One Shot® BL21 DE3 (Life technologies, Carlsbad, California, USA)來進行選殖及重組表現。在16℃下利用0.5 mM異丙基β-D-1-硫代哌喃半乳糖苷(IPTG, Euromedex, Souffelweyersheim, France)來進行LB-Miller培養基中之誘導。藉由在Avanti J-26 XP離心機(Beckman Coulter, Brea, USA)中離心(8000 rpm，10℃下20分鐘)來終止培養物。將細胞懸浮於20 mL Talon緩衝液(Tris-HCl 20 mM，NaCl 300 mM，pH 8)中。隨後藉由FB 705音波處理器(Fisherbrand, Illkirch, France)在2分鐘期間利用30%振幅(2秒開啟及1秒斷開循環)對細胞懸浮液進行音波處理。隨後，實現離心步驟：在Eppendorf離心機中，10℃，在11000 rpm下，30分鐘。收集可溶性溶離份且經受親和性層析。此純化步驟已藉由Talon®金屬親和樹脂(Clontech, CA, USA)完成。藉由補充有咪唑之Talon緩衝液步驟來進行蛋白質溶離。純化的蛋白質針對Talon緩衝液經透析，隨後使用Bio-Rad蛋白質分析根據製造商說明(Lifescience Bio-Rad, France)經量化，且儲存在+4℃下。 Expression and purification of esterases The strains Stellar™ (Clontech, California, USA) and Escherichia coli One Shot® BL21 DE3 (Life technologies, Carlsbad, California, USA) were selected and expressed recombinantly in 50 mL LB-Miller medium or ZYM autoinduction medium (Studier et al., 2005- Prot. Exp. Pur. 41, 207-234). Induction in LB-Miller medium was performed with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG, Euromedex, Souffelweyersheim, France) at 16°C. The cultures were terminated by centrifugation (8000 rpm, 20 min at 10 °C) in an Avanti J-26 XP centrifuge (Beckman Coulter, Brea, USA). The cells were suspended in 20 mL Talon buffer (Tris-HCl 20 mM, NaCl 300 mM, pH 8). The cell suspension was then sonicated by an FB 705 sonicator (Fisherbrand, Illkirch, France) during 2 min with 30% amplitude (2 s on and 1 s off cycle). Subsequently, a centrifugation step was achieved: 10 °C, 11000 rpm, 30 min in an Eppendorf centrifuge. The soluble fractions were collected and subjected to affinity chromatography. This purification step has been done with Talon® metal affinity resin (Clontech, CA, USA). Protein elution was performed with a Talon buffer step supplemented with imidazole. The purified protein was dialyzed against Talon buffer and subsequently quantified using the Bio-Rad protein assay according to the manufacturer's instructions (Lifescience Bio-Rad, France) and stored at +4°C.

實例 2- 評估酯酶之降解活性已確定酯酶之降解活性且與SEQ ID NO: 1之酯酶之活性進行比較。 Example 2 - Evaluation of the Degradation Activity of Esterases The degradation activity of the esterases was determined and compared with the activity of the esterase of SEQ ID NO: 1.

使用用於估計比活性之多種方法： (1) 基於PET水解之比活性 (2) 基於呈固態形式之聚酯之降解的降解活性 (3) 基於超過100 mL之反應器中之PET水解的降解活性 (4) 基於PET水解及紫外光吸光度(UV分析)分析之比活性 Several methods for estimating specific activity were used: (1) Specific activity based on PET hydrolysis (2) Degradation activity based on degradation of polyester in solid form (3) Degradation activity based on PET hydrolysis in reactors larger than 100 mL (4) Specific activity based on PET hydrolysis and ultraviolet absorbance (UV analysis) analysis

下文給出關於此類方法之方案的細節。Details on the scheme of such an approach are given below.

2.1. 基於PET水解之比活性 稱取100 mg呈粉末形式之非晶形PET (根據WO 2017/198786製備以達到低於20%之結晶度)，且將其引入100 mL玻璃瓶中。在玻璃瓶(0.2 mg 酶/g PET或1.0 mg 酶/g PET)中引入1 mL酯酶製劑，其包含SEQ ID NO: 1之酯酶(作為參考對照)或本發明之酯酶，其以0.69 µM或3.45 µM在Talon緩衝液(Tris-HCl 20 mM，NaCl 0.3 M，pH 8)中製備。最後，添加pH為8的49 mL 0.1 M磷酸鉀緩衝液。藉由在Max Q 4450培育箱(Thermo Fisher Scientific, Inc. Waltham, MA, USA)中在40℃、45℃、50℃、55℃、60℃、65℃、70℃或72℃及150 rpm下培育各玻璃瓶而開始解聚合。藉由在前24小時期間的不同時間處進行且藉由超高效液相層析(UHPLC)分析的取樣來確定解聚合反應之初始速率，其以每小時產生的當量TA之毫克數為單位。視需要，將樣品稀釋於pH為8的0.1 M磷酸鉀緩衝液中。隨後，向150 µL樣品或稀釋液中添加150 µL甲醇及6.5 µL之6 N HCl。在混合且經由0.45 µm針筒過濾器過濾之後，將樣品加載在UHPLC上以監測對苯二甲酸(TA)、MHET及BHET之釋放。所使用層析系統為Ultimate 3000 UHPLC系統(Thermo Fisher Scientific, Inc. Waltham, MA, USA)，其包括泵模組、自動取樣器、恆溫在25℃的管柱烘箱及240 nm下之UV偵測器。所使用管柱為Discovery® HS C18 HPLC管柱(150×4.6 mm，5 µm，配備有前置管柱，Supelco，Bellefonte，USA)。使用MeOH (30%至90%)於1 mM H 2SO 4中之梯度以1 mL/min分離TA、MHET及BHET。注射液為20 µL樣品。根據在與樣品相同的條件下由市售TA及BHET以及內部合成的MHET所製備之標準曲線來量測TA、MHET及BHET。在反應之水解曲線之線性部分中(亦即在反應開始時)確定PET水解之比活性(當量TA之毫克數/小時/酶之毫克數)，該曲線係由在前24、48、72、96小時期間之不同時間進行之取樣建立。當量TA對應於所量測TA與所量測MHET及BHET中含有之TA的總和。當量TA之該量測亦可用於計算在給定時間及/或在規定時段(例如24 h、48 h、72 h或96 h)之後PET解聚合分析之產率。 2.1. Specific activity based on PET hydrolysis 100 mg of amorphous PET in powder form (prepared according to WO 2017/198786 to achieve a crystallinity of less than 20%) was weighed and introduced into a 100 mL glass bottle. 1 mL of an esterase preparation was introduced into a glass bottle (0.2 mg enzyme /g PET or 1.0 mg enzyme /g PET ), which contained the esterase of SEQ ID NO: 1 (as a reference control) or the esterase of the present invention, which was prepared at 0.69 µM or 3.45 µM in Talon buffer (Tris-HCl 20 mM, NaCl 0.3 M, pH 8). Finally, 49 mL of 0.1 M potassium phosphate buffer at pH 8 was added. Depolymerization was initiated by incubating each glass bottle at 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, or 72°C and 150 rpm in a Max Q 4450 incubator (Thermo Fisher Scientific, Inc. Waltham, MA, USA). The initial rate of depolymerization, expressed as mg equivalent TA produced per hour, was determined by sampling at various times during the first 24 hours and analyzed by ultra-high performance liquid chromatography (UHPLC). Samples were diluted as needed in 0.1 M potassium phosphate buffer, pH 8. Subsequently, 150 µL of methanol and 6.5 µL of 6 N HCl were added to 150 µL of sample or dilution. After mixing and filtering through a 0.45 µm syringe filter, the samples were loaded on UHPLC to monitor the release of terephthalic acid (TA), MHET, and BHET. The chromatography system used was an Ultimate 3000 UHPLC system (Thermo Fisher Scientific, Inc. Waltham, MA, USA), which included a pump module, an autosampler, a column oven maintained at 25 °C, and a UV detector at 240 nm. The column used was a Discovery® HS C18 HPLC column (150 × 4.6 mm, 5 µm, equipped with a precolumn, Supelco, Bellefonte, USA). TA, MHET, and BHET were separated using a gradient of MeOH (30% to 90%) in 1 mM H 2 SO 4 at 1 mL/min. The injection was 20 µL of sample. TA, MHET and BHET are measured according to standard curves prepared from commercially available TA and BHET and MHET synthesized in-house under the same conditions as the samples. The specific activity of PET hydrolysis (mg equivalent TA/hour/mg enzyme) is determined in the linear part of the hydrolysis curve of the reaction (i.e. at the beginning of the reaction), which is established from samples taken at different times during the first 24, 48, 72, 96 hours. The equivalent TA corresponds to the sum of the measured TA and the TA contained in the measured MHET and BHET. This measurement of the equivalent TA can also be used to calculate the yield of PET depolymerization analysis at a given time and/or after a specified period of time (e.g. 24 h, 48 h, 72 h or 96 h).

2.2. 基於呈固態形式之聚酯之降解的活性 將20 µL酶製劑沉積於含有PET之瓊脂盤中所形成之孔中。藉由使500 mg PET溶解於六氟-2-丙醇(HFIP)中且將此培養基傾倒在250 mL水溶液中來實現瓊脂盤之製備。在140毫巴下在52℃下進行HFIP蒸發之後，將溶液與pH為8的含有3%瓊脂之0.2 M磷酸鉀緩衝液以v/v混合。使用大約30 mL混合物來製備各培養盤，且儲存在4℃下。量測由於親本酯酶及變異體對聚酯之降解而形成的暈圈之直徑或表面積且在40℃、45℃、50℃、55℃、60℃、65℃或70℃下2至24小時之後進行比較。 2.2. Activity based on the degradation of polyester in solid form 20 µL of the enzyme preparation were deposited in wells formed in agar plates containing PET. Preparation of the agar plates was achieved by dissolving 500 mg of PET in hexafluoro-2-propanol (HFIP) and pouring this medium in 250 mL of aqueous solution. After evaporation of HFIP at 52 °C at 140 mbar, the solution was mixed v/v with 0.2 M potassium phosphate buffer, pH 8, containing 3% agar. Approximately 30 mL of the mixture were used to prepare each plate and stored at 4 °C. The diameter or surface area of the halo formed due to the degradation of polyester by the parent esterase and the variants was measured and compared after 2 to 24 hours at 40°C, 45°C, 50°C, 55°C, 60°C, 65°C or 70°C.

2.3. 基於反應器中之PET水解的活性 將在195 ml磷酸鈉鉀緩衝液(pH 8.0，100 mM)中所製備之49 mg純化的蛋白質與500 mL桌面式F1 0.5 MB生物反應器(AD Biotec, France)中之50 g起泡的PET (98%純度) (根據WO 2021/123299製備，以達到低於20%之結晶度)混合。在加水套的生物反應器中進行40℃、45℃、50℃、55℃、60℃、65℃、68℃或70℃下之溫度調節，且使用雙Rushton葉輪來維持800 rpm下之持續攪拌。藉由使用ROSITA 2.0軟體(AD Biotec, France)添加20% NaOH (w/w)溶液將pH值調節至pH 8.0。考慮到TPA及MEG之排他性生產，基於NaOH消耗追蹤PET解聚合之動力學(滴定1 mol二酸TPA消耗2 mol NaOH)。藉由確定殘餘PET重量或藉由確定所產生的當量TA或者經由鹼消耗來確定PET解聚合分析之最終產率。藉由在反應結束時經由12至15 µm級11無灰濾紙(Dutscher SAS, Brumath, France)來過濾反應體積且對此保留物進行乾燥，之後對其進行稱重，從而估計殘餘PET之重量確定。使用2.1中所描述之UHPLC方法來實現所產生的當量TA之確定，且基於給定時間處之莫耳濃度比率(TA + MHET + BHET)相比於初始樣品中所含有的TA總量來計算水解百分比。PET解聚合產生之酸單體將經鹼中和，此能夠維持反應器中之pH。使用對應的莫耳鹼消耗來計算所產生的當量TA之確定，且基於給定時間處當量TA之莫耳濃度比率相比於初始樣品中含有的TA總量來計算水解百分比。 2.3. Activity based on PET hydrolysis in the reactor 49 mg of purified protein prepared in 195 ml of potassium sodium phosphate buffer (pH 8.0, 100 mM) was mixed with 50 g of foamed PET (98% purity) (prepared according to WO 2021/123299 to achieve a crystallinity of less than 20%) in a 500 mL tabletop F1 0.5 MB bioreactor (AD Biotec, France). The temperature was regulated at 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 68°C or 70°C in a water-jacketed bioreactor and continuous stirring was maintained at 800 rpm using a dual Rushton impeller. The pH value was adjusted to pH 8.0 by adding a 20% NaOH (w/w) solution using ROSITA 2.0 software (AD Biotec, France). The kinetics of PET depolymerization were followed based on NaOH consumption (2 mol NaOH consumed for titration of 1 mol of diacid TPA), taking into account the exclusive production of TPA and MEG. The final yield of PET depolymerization analysis was determined by determining the weight of residual PET or by determining the equivalent TA produced or by base consumption. The weight determination of residual PET was estimated by filtering the reaction volume at the end of the reaction through 12 to 15 µm grade 11 ash-free filter paper (Dutscher SAS, Brumath, France) and drying this retentate, which was then weighed. The determination of the equivalent TA produced is achieved using the UHPLC method described in 2.1 and the hydrolysis percentage is calculated based on the ratio of the molar concentrations at a given time (TA + MHET + BHET) compared to the total amount of TA contained in the initial sample. The acid monomers produced by the depolymerization of PET will be neutralized with base, which will maintain the pH in the reactor. The determination of the equivalent TA produced is calculated using the corresponding molar base consumption and the hydrolysis percentage is calculated based on the ratio of the molar concentrations of the equivalent TA at a given time compared to the total amount of TA contained in the initial sample.

2.4. 基於PET水解及紫外光吸光度(UV分析)分析之比活性 稱取100 mg呈粉末形式之非晶形PET (根據WO 2017/198786製備以達到低於20%之結晶度)，且將其引入100 mL玻璃瓶中。在玻璃瓶(0.2 mg 酶/g PET或1.0 mg 酶/g PET)中引入1 mL酯酶製劑，其包含SEQ ID NO: 1之酯酶(作為參考對照)或本發明之酯酶，其以0.69 µM或3.45 µM在Talon緩衝液(Tris-HCl 20 mM，NaCl 0.3 M，pH 8)中製備。最後，添加pH為8.0的49 mL 100mM磷酸鉀緩衝液。藉由在Max Q 4450培育箱(Thermo Fisher Scientific, Inc. Waltham, MA, USA)中在50℃、54℃、60℃或65℃及150 rpm下培育各玻璃瓶而開始解聚合。替代地，反應可以在deepwell (ThermoScientific, Abgene, AB-0661, Illkirch, France)中微型化，稱取22 mg呈粉末形式之非晶形PET (根據WO 2017/198786製備以達到低於20%之結晶度)且將其引入deepwell培養盤之各孔中。在deepwell之各孔中引入0.1 mL酯酶製劑，其包含SEQ ID NO: 1之酯酶(作為參考對照)或本發明之酯酶，其以0.138 µM在Talon緩衝液(Tris-HCl 20 mM，NaCl 0.3 M，pH 8或100 mM磷酸鉀緩衝液，pH 8.0)中製備。最後，添加pH為8.0的0.9 mL 100mM磷酸鉀緩衝液。藉由在Infors HT multitron振盪培育箱(Infors HT, Bottmingen, Suisse)中在50℃、54℃、55℃、60℃或65℃及600 rpm下培育各deepwell而開始解聚合。初始解聚合反應速率(以所產生之可溶性降解產物之微莫耳數/小時為單位)係藉由在前24小時期間不同時間進行之取樣確定，且藉由使用Eon微量培養盤分光光度計(BioTek, USA)在242 nm處讀取吸光度進行分析。在光譜之紫外光區中(在242 nm下)反應混合物之吸光度增加指示可溶TA或其酯(BHET及MHET)自不溶PET受質釋放。此波長下之吸光度值可用於根據朗伯-比爾定律(Lambert-Beer law)計算PET水解產物之整體總和，且酶比活性經確定為所產生的總當量TA。視需要，將樣品稀釋於pH為8.0的100 mM磷酸鉀緩衝液中。在反應之水解曲線之線性部分中(亦即在反應開始時)確定PET水解之比活性(可溶產物之微莫耳數/小時/酶之毫克數)，該曲線係藉由在前24小時期間不同時間進行之取樣建立。當量TA之該量測亦可用於計算在給定時間及/或在規定時段(例如24 h、48 h、72 h或96 h)之後PET解聚合分析之產率。 2.4. Specific activity based on PET hydrolysis and ultraviolet absorbance (UV analysis) analysis 100 mg of amorphous PET in powder form (prepared according to WO 2017/198786 to achieve a crystallinity of less than 20%) was weighed and introduced into a 100 mL glass bottle. 1 mL of an esterase preparation was introduced into a glass bottle (0.2 mg enzyme /g PET or 1.0 mg enzyme /g PET ), which contained the esterase of SEQ ID NO: 1 (as a reference control) or the esterase of the present invention, which was prepared at 0.69 µM or 3.45 µM in Talon buffer (Tris-HCl 20 mM, NaCl 0.3 M, pH 8). Finally, 49 mL of 100 mM potassium phosphate buffer at pH 8.0 was added. Depolymerization was initiated by incubating each glass bottle at 50° C., 54° C., 60° C. or 65° C. and 150 rpm in a Max Q 4450 incubator (Thermo Fisher Scientific, Inc. Waltham, MA, USA). Alternatively, the reaction can be miniaturized in a deepwell (ThermoScientific, Abgene, AB-0661, Illkirch, France), weighing 22 mg of amorphous PET in powder form (prepared according to WO 2017/198786 to achieve a crystallinity of less than 20%) and introducing it into each well of a deepwell culture plate. 0.1 mL of esterase preparation was introduced into each well of the deepwell, which contained the esterase of SEQ ID NO: 1 (as a reference control) or the esterase of the present invention, which was prepared at 0.138 μM in Talon buffer (Tris-HCl 20 mM, NaCl 0.3 M, pH 8 or 100 mM potassium phosphate buffer, pH 8.0). Finally, 0.9 mL of 100 mM potassium phosphate buffer, pH 8.0 was added. Depolymerization was started by incubating each deepwell at 50°C, 54°C, 55°C, 60°C or 65°C and 600 rpm in an Infors HT multitron shaking incubator (Infors HT, Bottmingen, Suisse). The initial depolymerization reaction rate (in micromoles of soluble degradation products produced/hour) was determined by sampling at different times during the first 24 hours and analyzed by reading the absorbance at 242 nm using an Eon microplate spectrophotometer (BioTek, USA). The increase in absorbance of the reaction mixture in the UV region of the spectrum (at 242 nm) indicates the release of soluble TA or its esters (BHET and MHET) from the insoluble PET substrate. The absorbance value at this wavelength can be used to calculate the overall sum of PET hydrolysis products according to the Lambert-Beer law, and the specific enzyme activity is determined as the total equivalent TA produced. Samples were diluted in 100 mM potassium phosphate buffer, pH 8.0, as needed. The specific activity of PET hydrolysis (μmol of soluble product/hour/mg of enzyme) is determined in the linear part of the hydrolysis curve of the reaction (i.e. at the beginning of the reaction), which is established by sampling at different times during the first 24 hours. This measurement of equivalent TA can also be used to calculate the yield of PET depolymerization analysis at a given time and/or after a specified period of time (e.g. 24 h, 48 h, 72 h or 96 h).

結果與SEQ ID NO: 1之酯酶相比，基於PET水解在鹼性條件下之比降解活性 依實例2.4中所揭露，在反應開始時，在55℃、60℃或65℃、pH 8下量測本發明之酯酶(變異體)之比降解活性。結果展示於下表1中。SEQ ID NO: 1之酯酶之比降解活性用作參考且被視為100%比降解活性。 表1：與SEQ ID NO: 1相比，本發明之酯酶在pH 8下之比降解活性。 變異體 溫度( ℃) 變異體濃度(mg/g PET) 與SEQ ID NO : 1 相比之相對比降解活性 (%) N9D 60 0.2 122% N9E 60 0.2 117% V28I 60 0.2 112% R30G 55 0.2 115% A62T 60 1 115% A64V 65 0.2 129% A68N 60 1 111% F90Y 60 0.2 111% T109S 65 0.2 141% T109S 60 0.2 118% V115I 65 0.2 116% L124G 60 1 116% S145G 65 0.2 123% L152M 60 1 113% T157P 60 0.2 119% E158A 65 0.2 127% S181K 60 1 111% H183Q 60 0.2 123% H183N 60 0.2 121% F187I 60 1 113% S193L 60 1 112% S193Q 60 1 115% M208T 60 0.2 124% W228S 65 0.2 126% V242E 55 0.2 119% V242E 65 0.2 120% N243Y 65 0.2 129% A24Q + H183Q 55 0.2 153% A24Q + H183Q 65 0.2 119% R30G + H183Q 55 0.2 164% R30G + H183Q 65 0.2 177% S66N + M208T 60 1 117% A62T + M208T 60 0.2 139% A64V + N243Y 65 0.2 134% L74V + H183Q 65 0.2 112% L74V + H183Q 60 0.2 141% N162S + S193Q 60 0.2 125% S181K + S193L 60 1 116% H183Q + F187Q 65 0.2 133% H183L + F187I 60 0.2 119% H183A + F187I 60 0.2 125% H183Y + F187I 60 0.2 126% H183Q + F187I 60 0.2 159% H183N + F187I 60 0.2 132% H183D + F187I 60 0.2 128% H183W + F187I 60 0.2 132% H183E + F187I 60 1 130% H183S + F187I 60 1 132% L227N + H183Q 55 0.2 166% L227N + H183Q 65 0.2 142% R251S + H183Q 55 0.2 139% R251S + H183Q 65 0.2 126% Y26H + A64T + H183Q 55 0.2 149% Y26H + A64T + H183Q 65 0.2 118% G35A + A62T + H183Q 55 0.2 157% G46V + R73H + H183Q 55 0.2 151% T50M + I169M + H183Q 55 0.2 153% A62T + S66N + M208T 60 0.2 132% A64V + V115I + N243Y 65 0.2 132% F90A + H183S + F187L 65 0.2 142% F90L + H183E + F187L 65 0.2 144% F90L + H183N + F187L 65 1 129% F90S + H183S + F187L 65 0.2 129% F90Y + H183L + F187L 65 0.2 134% F90Y + H183N 65 1 127% Y106F + I185V + H183Q 55 0.2 144% R108L + V167L + H183Q 55 0.2 153% N162S + S181K + S193Q 60 1 111% H183Q + F187I + N204G 60 1 162% S22R + G39S + P179N + L239M 55 0.2 114% N204G + H183Q + F187I + R30G 65 1 125% N204G + H183Q + F187I + G39S 65 1 121% N204G + H183Q + F187I + A62T 60 1 149% N204G + H183Q + F187I + A64V 65 0.2 144% N204G + H183Q + F187I + A64V 55 1 133% N204G + H183Q + F187I + A64V 65 1 149% N204G + H183Q + F187I + L124G 60 1 148% N204G + H183Q + F187I + N162S 60 1 154% N204G + H183Q + F187I + S193Q 60 1 155% N204G + H183Q + F187I + Q237L 65 1 148% N204G + H183Q + F187I + Q237L 55 1 127% M208T + N204G + H183Q + F187I 60 1 153% T27A + R30G + S145G + W228S + V242E 55 0.2 119% T27A + R30G + S145G + W228S + V242E 65 0.2 131% N204G + H183Q + F187I + N162S + S181K 60 1 138% G7A + N9T + P192S + P213A + W228R + Q237L 55 0.2 146% N204G + H183Q + F187I + N162S + S181K + S193Q 60 1 146% N204G + H183Q + F187I + W69L + A216C + A246C 60 1 131% 變異體具有SEQ ID NO: 1之精確胺基酸序列，表1中所列之取代除外。 Results Compared with the esterase of SEQ ID NO: 1, the specific degradation activity of the esterase (variant) of the present invention based on PET hydrolysis under alkaline conditions was measured at 55°C, 60°C or 65°C, pH 8 at the beginning of the reaction as disclosed in Example 2.4. The results are shown in Table 1 below. The specific degradation activity of the esterase of SEQ ID NO: 1 was used as a reference and was considered as 100% specific degradation activity. Table 1: Specific degradation activity of the esterase of the present invention at pH 8 compared with SEQ ID NO: 1. Mutant Temperature( ℃) Variant concentration (mg/g PET ) Relative degradation activity compared with SEQ ID NO : 1 (%) N9D 60 0.2 122% N9E 60 0.2 117% V28I 60 0.2 112% R30G 55 0.2 115% A62T 60 1 115% A64V 65 0.2 129% A68N 60 1 111% F90Y 60 0.2 111% T109S 65 0.2 141% T109S 60 0.2 118% V115I 65 0.2 116% L124G 60 1 116% S145G 65 0.2 123% L152M 60 1 113% T157P 60 0.2 119% E158A 65 0.2 127% S181K 60 1 111% H183Q 60 0.2 123% H183N 60 0.2 121% F187I 60 1 113% S193L 60 1 112% S193Q 60 1 115% M208T 60 0.2 124% W228S 65 0.2 126% V242E 55 0.2 119% V242E 65 0.2 120% N243Y 65 0.2 129% A24Q + H183Q 55 0.2 153% A24Q + H183Q 65 0.2 119% R30G + H183Q 55 0.2 164% R30G + H183Q 65 0.2 177% S66N + M208T 60 1 117% A62T + M208T 60 0.2 139% A64V + N243Y 65 0.2 134% L74V + H183Q 65 0.2 112% L74V + H183Q 60 0.2 141% N162S + S193Q 60 0.2 125% S181K + S193L 60 1 116% H183Q + F187Q 65 0.2 133% H183L + F187I 60 0.2 119% H183A + F187I 60 0.2 125% H183Y + F187I 60 0.2 126% H183Q + F187I 60 0.2 159% H183N + F187I 60 0.2 132% H183D + F187I 60 0.2 128% H183W + F187I 60 0.2 132% H183E + F187I 60 1 130% H183S + F187I 60 1 132% L227N + H183Q 55 0.2 166% L227N + H183Q 65 0.2 142% R251S + H183Q 55 0.2 139% R251S + H183Q 65 0.2 126% Y26H + A64T + H183Q 55 0.2 149% Y26H + A64T + H183Q 65 0.2 118% G35A + A62T + H183Q 55 0.2 157% G46V + R73H + H183Q 55 0.2 151% T50M + I169M + H183Q 55 0.2 153% A62T + S66N + M208T 60 0.2 132% A64V + V115I + N243Y 65 0.2 132% F90A + H183S + F187L 65 0.2 142% F90L + H183E + F187L 65 0.2 144% F90L + H183N + F187L 65 1 129% F90S + H183S + F187L 65 0.2 129% F90Y + H183L + F187L 65 0.2 134% F90Y + H183N 65 1 127% Y106F + I185V + H183Q 55 0.2 144% R108L + V167L + H183Q 55 0.2 153% N162S + S181K + S193Q 60 1 111% H183Q + F187I + N204G 60 1 162% S22R + G39S + P179N + L239M 55 0.2 114% N204G + H183Q + F187I + R30G 65 1 125% N204G + H183Q + F187I + G39S 65 1 121% N204G + H183Q + F187I + A62T 60 1 149% N204G + H183Q + F187I + A64V 65 0.2 144% N204G + H183Q + F187I + A64V 55 1 133% N204G + H183Q + F187I + A64V 65 1 149% N204G + H183Q + F187I + L124G 60 1 148% N204G + H183Q + F187I + N162S 60 1 154% N204G + H183Q + F187I + S193Q 60 1 155% N204G + H183Q + F187I + Q237L 65 1 148% N204G + H183Q + F187I + Q237L 55 1 127% M208T + N204G + H183Q + F187I 60 1 153% T27A + R30G + S145G + W228S + V242E 55 0.2 119% T27A + R30G + S145G + W228S + V242E 65 0.2 131% N204G + H183Q + F187I + N162S + S181K 60 1 138% G7A + N9T + P192S + P213A + W228R + Q237L 55 0.2 146% N204G + H183Q + F187I + N162S + S181K + S193Q 60 1 146% N204G + H183Q + F187I + W69L + A216C + A246C 60 1 131% The variants have the exact amino acid sequence of SEQ ID NO: 1, except for the substitutions listed in Table 1.

與SEQ ID NO: 1之酯酶相比在鹼性條件下之PET解聚合產率 在55℃、60℃或65℃、pH 8下，在6小時或24小時之後，根據實例2.4量測本發明之酯酶(變異體)的PET解聚合產率。在本發明之上下文中，使用PET解聚合產率來評估降解活性。結果(平均值)分別展示於表2及表3中。在6小時或24小時之後，SEQ ID NO: 1之酯酶之PET解聚合產率在pH 8下被用作參考，且分別被視為100%降解活性。 表2：6小時之後本發明之酯酶之PET解聚合產率 變異體 溫度 ( ℃ ) 變異體濃度 (mg/g PET) 與 SEQ ID NO : 1 相比 24 小時後 PET 解聚合產率 (%) E182D 55 0.2 112% M208G 55 0.2 122% M208N + N211M 55 0.2 116% A17F + F90Q + H183N + N204G + M208L + N211M 55 0.2 115% H183Q + F187I + N204G + M208N + N211M + S193E 55 0.2 182% M208T + N204G + H183Q + F187I + N211M + S193E 55 0.2 147% H183Q + F187I + N204G + N211M + S193E + V200I + I170V 55 0.2 137% A17F + F90Q + N122D + H183N + S193E + N204G + M208L + N211M 55 0.2 121% A17F + F90Q + Q142E + H183N + S193E + N204G + M208L + N211M 55 0.2 115% A17F + F90Q + N143D + H183N + S193E + N204G + M208L + N211M 55 0.2 123% A17F + F90Q + H183N + S193E + N204G + M208L + N211M + Q237E 55 0.2 124% A17F + F90Q + H183N + S193E + N204G + M208L + N211M + N253D 55 0.2 110% A17F + F90Q + H183N + S193E + N204G + M208L + N211M + Q258E 55 0.2 118% H183Q + F187I + N204G + M208N + N211M + S193E + V200I + I170V 55 0.2 166% M208T + N204G + H183Q + F187I + N211M + S193E + V200I + I170V 55 0.2 135% A17F + F90Q + H183N + S193E + N204G + M208L + N211M + V200I + I170V 55 0.2 119% T16E 55 0.2 109% Q142E 55 0.2 114% N143D 55 0.2 119% Q237E 55 0.2 112% M208K 55 0.2 117% M208Q + N211M 55 0.2 110% V200I + I170V 55 0.2 110% H183Q + F187I + N204G + N211M + S193E 55 0.2 118% A17F + N85D + F90Q + H183N + S193E + N204G + M208L + N211M 55 0.2 118% 變異體具有SEQ ID NO: 1之精確胺基酸序列，表2中所列之取代除外。 表3：24小時之後本發明之酯酶之PET解聚合產率。 變異體 溫度 ( ℃ ) 變異體濃度 (mg/g PET) 與 SEQ ID NO : 1 相比 24 小時後 PET 解聚合產率 (%) N9D 60 0.2 126% N9E 60 0.2 132% V28I 60 0.2 113% R30G 55 0.2 121% A64V 65 0.2 128% T109S 65 0.2 117% T109S 60 0.2 121% V115I 65 0.2 114% S145G 55 0.2 110% S145G 65 0.2 130% T157P 60 0.2 115% E158A 65 0.2 115% K159R 65 0.2 111% F187I 65 1 116% M208T 60 0.2 133% W228R 65 0.2 122% W228S 65 0.2 133% V242E 55 0.2 114% V242E 65 0.2 127% N243Y 65 0.2 127% A24Q + H183Q 55 0.2 118% A24Q + H183Q 65 0.2 159% R30G + H183Q 55 0.2 159% R30G + H183Q 65 0.2 151% A62T + M208T 60 0.2 147% A64V + N243Y 65 0.2 131% L74V + H183Q 60 0.2 121% N162S + S193Q 60 0.2 118% H183Q + F187Q 65 0.2 112% H183L + F187I 60 0.2 123% H183A + F187I 60 0.2 117% H183Y + F187I 60 0.2 127% H183Q + F187I 60 0.2 162% H183N + F187I 60 0.2 132% H183W + F187I 60 0.2 118% H183S + F187I 60 1 111% L227N + H183Q 55 0.2 150% R251S + H183Q 55 0.2 113% R251S + H183Q 65 0.2 125% Y26H + A64T + H183Q 55 0.2 119% Y26H + A64T + H183Q 65 0.2 146% G35A + A62T + H183Q 55 0.2 135% G46V + R73H + H183Q 55 0.2 128% T50M + I169M + H183Q 55 0.2 115% A62T + S66N + M208T 60 0.2 139% A64V + V115I + N243Y 65 0.2 131% Y106F + I185V + H183Q 55 0.2 129% R108L + V167L + H183Q 55 0.2 134% N162S + S181K + S193Q 60 1 111% N204G + H183Q + F187I + R30G 65 1 138% N204G + H183Q + F187I + G39S 65 1 139% N204G + H183Q + F187I + A64V 65 0.2 115% N204G + H183Q + F187I + A64V 55 1 126% N204G + H183Q + F187I + A64V 65 1 140% N204G + H183Q + F187I + N162S 60 1 114% N204G + H183Q + F187I + S193Q 60 1 112% N204G + H183Q + F187I + Q237L 65 1 140% N204G + H183Q + F187I + Q237L 55 1 127% T27A + R30G + S145G + W228S + V242E 55 0.2 121% N204G + H183Q + F187I + N162S + S181K + S193Q 60 1 116% T16E 55 0.2 124% N105D 55 0.2 112% N85D 55 0.2 119% Q142E 55 0.2 127% N253D 55 0.2 118% M208A 55 0.2 120% M208G 55 0.2 130% H183Q + F187I + N204G + N211M + S193E 55 0.2 127% H183Q + F187I + N204G + M208N + N211M + S193E 55 0.2 180% M208T + N204G + H183Q + F187I + N211M + S193E 55 0.2 154% H183Q + F187I + N204G + N211M + S193E + V200I + I170V 55 0.2 117% A17F + N85D + F90Q + H183N + S193E + N204G + M208L + N211M 55 0.2 118% H183Q + F187I + N204G + M208N + N211M + S193E + V200I + I170V 55 0.2 168% A17F + F90Q + N105D + H183N + S193E + N204G + M208L + N211E 55 0.2 131% N143D 55 0.2 110% M208D 55 0.2 111% M208K 55 0.2 108% M208N + N211M 55 0.2 110% M208Q + N211M 55 0.2 108% V200I + I170V 55 0.2 111% M208T + N204G + H183Q + F187I + N211M + S193E + V200I + I170V 55 0.2 121% 變異體具有SEQ ID NO: 1之精確胺基酸序列，表3中所列之取代除外。 PET depolymerization yield under alkaline conditions compared to the esterase of SEQ ID NO: 1 The PET depolymerization yield of the esterase (variant) of the present invention was measured according to Example 2.4 at 55°C, 60°C or 65°C, pH 8 after 6 hours or 24 hours. In the context of the present invention, the PET depolymerization yield is used to assess the degradation activity. The results (average values) are shown in Table 2 and Table 3, respectively. After 6 hours or 24 hours, the PET depolymerization yield of the esterase of SEQ ID NO: 1 at pH 8 was used as a reference and was considered as 100% degradation activity, respectively. Table 2: PET depolymerization yield of the esterase of the present invention after 6 hours Mutant Temperature ( ℃ ) Variant concentration (mg/g PET ) Compared with SEQ ID NO : 1, PET depolymerization yield after 24 hours (%) E182D 55 0.2 112% M208G 55 0.2 122% M208N + N211M 55 0.2 116% A17F + F90Q + H183N + N204G + M208L + N211M 55 0.2 115% H183Q + F187I + N204G + M208N + N211M + S193E 55 0.2 182% M208T + N204G + H183Q + F187I + N211M + S193E 55 0.2 147% H183Q + F187I + N204G + N211M + S193E + V200I + I170V 55 0.2 137% A17F + F90Q + N122D + H183N + S193E + N204G + M208L + N211M 55 0.2 121% A17F + F90Q + Q142E + H183N + S193E + N204G + M208L + N211M 55 0.2 115% A17F + F90Q + N143D + H183N + S193E + N204G + M208L + N211M 55 0.2 123% A17F+F90Q+H183N+S193E+N204G+M208L+N211M+Q237E 55 0.2 124% A17F + F90Q + H183N + S193E + N204G + M208L + N211M + N253D 55 0.2 110% A17F + F90Q + H183N + S193E + N204G + M208L + N211M + Q258E 55 0.2 118% H183Q + F187I + N204G + M208N + N211M + S193E + V200I + I170V 55 0.2 166% M208T + N204G + H183Q + F187I + N211M + S193E + V200I + I170V 55 0.2 135% A17F+F90Q+H183N+S193E+N204G+M208L+N211M+V200I+I170V 55 0.2 119% T16E 55 0.2 109% Q142E 55 0.2 114% N143D 55 0.2 119% Q237E 55 0.2 112% M208K 55 0.2 117% M208Q + N211M 55 0.2 110% V200I + I170V 55 0.2 110% H183Q + F187I + N204G + N211M + S193E 55 0.2 118% A17F + N85D + F90Q + H183N + S193E + N204G + M208L + N211M 55 0.2 118% The variants have the exact amino acid sequence of SEQ ID NO: 1 except for the substitutions listed in Table 2. Table 3: PET depolymerization yields of the esterases of the invention after 24 hours. Mutant Temperature ( ℃ ) Variant concentration (mg/g PET ) Compared with SEQ ID NO : 1, PET depolymerization yield after 24 hours (%) N9D 60 0.2 126% N9E 60 0.2 132% V28I 60 0.2 113% R30G 55 0.2 121% A64V 65 0.2 128% T109S 65 0.2 117% T109S 60 0.2 121% V115I 65 0.2 114% S145G 55 0.2 110% S145G 65 0.2 130% T157P 60 0.2 115% E158A 65 0.2 115% K159R 65 0.2 111% F187I 65 1 116% M208T 60 0.2 133% W228R 65 0.2 122% W228S 65 0.2 133% V242E 55 0.2 114% V242E 65 0.2 127% N243Y 65 0.2 127% A24Q + H183Q 55 0.2 118% A24Q + H183Q 65 0.2 159% R30G + H183Q 55 0.2 159% R30G + H183Q 65 0.2 151% A62T + M208T 60 0.2 147% A64V + N243Y 65 0.2 131% L74V + H183Q 60 0.2 121% N162S + S193Q 60 0.2 118% H183Q + F187Q 65 0.2 112% H183L + F187I 60 0.2 123% H183A + F187I 60 0.2 117% H183Y + F187I 60 0.2 127% H183Q + F187I 60 0.2 162% H183N + F187I 60 0.2 132% H183W + F187I 60 0.2 118% H183S + F187I 60 1 111% L227N + H183Q 55 0.2 150% R251S + H183Q 55 0.2 113% R251S + H183Q 65 0.2 125% Y26H + A64T + H183Q 55 0.2 119% Y26H + A64T + H183Q 65 0.2 146% G35A + A62T + H183Q 55 0.2 135% G46V + R73H + H183Q 55 0.2 128% T50M + I169M + H183Q 55 0.2 115% A62T + S66N + M208T 60 0.2 139% A64V + V115I + N243Y 65 0.2 131% Y106F + I185V + H183Q 55 0.2 129% R108L + V167L + H183Q 55 0.2 134% N162S + S181K + S193Q 60 1 111% N204G + H183Q + F187I + R30G 65 1 138% N204G + H183Q + F187I + G39S 65 1 139% N204G + H183Q + F187I + A64V 65 0.2 115% N204G + H183Q + F187I + A64V 55 1 126% N204G + H183Q + F187I + A64V 65 1 140% N204G + H183Q + F187I + N162S 60 1 114% N204G + H183Q + F187I + S193Q 60 1 112% N204G + H183Q + F187I + Q237L 65 1 140% N204G + H183Q + F187I + Q237L 55 1 127% T27A + R30G + S145G + W228S + V242E 55 0.2 121% N204G + H183Q + F187I + N162S + S181K + S193Q 60 1 116% T16E 55 0.2 124% N105D 55 0.2 112% N85D 55 0.2 119% Q142E 55 0.2 127% N253D 55 0.2 118% M208A 55 0.2 120% M208G 55 0.2 130% H183Q + F187I + N204G + N211M + S193E 55 0.2 127% H183Q + F187I + N204G + M208N + N211M + S193E 55 0.2 180% M208T + N204G + H183Q + F187I + N211M + S193E 55 0.2 154% H183Q + F187I + N204G + N211M + S193E + V200I + I170V 55 0.2 117% A17F + N85D + F90Q + H183N + S193E + N204G + M208L + N211M 55 0.2 118% H183Q + F187I + N204G + M208N + N211M + S193E + V200I + I170V 55 0.2 168% A17F + F90Q + N105D + H183N + S193E + N204G + M208L + N211E 55 0.2 131% N143D 55 0.2 110% M208D 55 0.2 111% M208K 55 0.2 108% M208N + N211M 55 0.2 110% M208Q + N211M 55 0.2 108% V200I + I170V 55 0.2 111% M208T + N204G + H183Q + F187I + N211M + S193E + V200I + I170V 55 0.2 121% The variants have the exact amino acid sequence of SEQ ID NO: 1, except for the substitutions listed in Table 3.

與SEQ ID NO: 1之酯酶相比在鹼性條件下反應器中之PET解聚合產率 在68℃、pH 8下，根據實例2.3在反應器中量測本發明之酯酶(變異體)之PET解聚合產率。在本發明之上下文中，使用PET解聚合產率來評估降解活性。結果展示於下表4中。在pH 8下用SEQ ID NO: 11之酯酶達到90%之PET解聚合產率的時間為22.2小時。使用1.0 mg 酶/g PET進行PET解聚合。 表4：用本發明之酯酶達到90%之PET解聚合產率的時間。 變異體 與SEQ ID NO: 1 相比達到90% PET 解聚合產率的時間(h) A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + H183Q + F187I + C203K + N204G + S206T + M208T + C248S + T252S 12.8 A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + H183Q + F187I + C203K + N204G + S206T + C248S + T252S 17.4 A17T + T27S + S48T + L82I + F90L + G92Y + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + H183Q + F187I + C203K + N204G + M208T + S206T + C248S + T252S 20.8 A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + H183Q + F187I + N204G + S206T + M208T + T252S 11.3 A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + H183Q + F187I + N204G + S206T + T252S 12.1 變異體具有SEQ ID NO: 1之精確胺基酸序列，表4中所列之取代除外。 PET depolymerization yield in reactor under alkaline conditions compared to esterase of SEQ ID NO: 1 The PET depolymerization yield of the esterase of the invention (variant) was measured in a reactor at 68°C, pH 8 according to Example 2.3. In the context of the present invention, the PET depolymerization yield was used to assess the degradation activity. The results are shown in Table 4 below. The time to reach a PET depolymerization yield of 90% with the esterase of SEQ ID NO: 11 at pH 8 was 22.2 hours. PET depolymerization was performed using 1.0 mg enzyme /g PET . Table 4: Time to reach a PET depolymerization yield of 90% with the esterase of the invention. Mutant Time to reach 90% PET depolymerization yield compared to SEQ ID NO: 1 (h) A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + H183Q + F187I + C203K + N204G + S206T + M208T + C248S + T252S 12.8 A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + H183Q + F187I + C203K + N204G + S206T + C248S + T252S 17.4 A17T + T27S + S48T + L82I + F90L + G92Y + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + H183Q + F187I + C203K + N204G + M208T + S206T + C248S + T252S 20.8 A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + H183Q + F187I + N204G + S206T + M208T + T252S 11.3 A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + H183Q + F187I + N204G + S206T + T252S 12.1 The variants have the exact amino acid sequence of SEQ ID NO: 1, except for the substitutions listed in Table 4.

在68℃、pH 8下，根據實例2.3在反應器中量測本發明之酯酶(變異體)之PET解聚合產率，唯一差異在於除20%外，PET之量為14% w/w。在本發明之上下文中，使用PET解聚合產率來評估降解活性。結果展示於下表5中。在pH 8下用SEQ ID NO: 1之酯酶達到90%之PET解聚合產率的時間為20小時。使用1.0 mg 酶/g PET進行PET解聚合。 表5：用本發明之酯酶達到90%之PET解聚合產率的時間 變異體 與SEQ ID NO: 1 相比達到90% PET 解聚合產率的時間(h) H183Q + F187I + N204G 13 h M208T + N204G + H183Q + F187I 12 h S13L + D158E + H183Q + F187I + N204G 14 h S13L + D158E + M208T + N204G + H183Q + F187I 11 h A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I 10 h A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + H183Q + F187I + N204G 11 h A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I + L13S + E158D 11 h 變異體具有SEQ ID NO: 1之精確胺基酸序列，表5中所列之取代除外。 The PET depolymerization yield of the esterase (variant) of the present invention was measured in a reactor according to Example 2.3 at 68°C, pH 8, the only difference being that the amount of PET was 14% w/w in addition to 20%. In the context of the present invention, the PET depolymerization yield was used to assess the degradation activity. The results are shown in Table 5 below. The time to reach a PET depolymerization yield of 90% with the esterase of SEQ ID NO: 1 at pH 8 was 20 hours. PET depolymerization was performed using 1.0 mg enzyme /g PET . Table 5: Time to reach a PET depolymerization yield of 90% with the esterase of the present invention Mutant Time to reach 90% PET depolymerization yield compared to SEQ ID NO: 1 (h) H183Q + F187I + N204G 13h M208T + N204G + H183Q + F187I 12h S13L + D158E + H183Q + F187I + N204G 14h S13L + D158E + M208T + N204G + H183Q + F187I 11h A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I 10h A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + H183Q + F187I + N204G 11h A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I + L13S+E158D 11h The variants have the exact amino acid sequence of SEQ ID NO: 1, except for the substitutions listed in Table 5.

與 SEQ ID NO : 2 之酯酶相比在鹼性條件下之 PET 解聚合產率在55℃、pH 8下，在6小時或24小時之後，根據實例2.4量測本發明之酯酶(變異體)的PET解聚合產率。在本發明之上下文中，使用PET解聚合產率來評估降解活性。結果(平均值)分別在6小時或24小時之後展示於表6及表7中。在6小時或24小時之後，SEQ ID NO: 2之酯酶之PET解聚合產率在pH 8下被用作參考，且分別被視為100%降解活性。 表6：6小時之後本發明之酯酶之PET解聚合產率 變異體 與SEQ ID NO: 2 相比6 小時之後的PET 解聚合產率(%) H183N 167% Q237E 127% M208N 126% M208S 130% H183Q + F187I + N204G + M208N + N211M  + S193E + V200I 115% M208T + N204G + H183Q + F187I + N211M  + S193E + V200I 120% N122D 121% I143D 118% L90W 113% Q258E 134% M208I + N211M 116% M208K + N211M 113% S212T 137% S66N 140% M208P + N211M 113% H183E + F187I 195% N253D 120% H183N + F187I 164% M208A + N211M 131% M208N + N211M 143% M208T + N204G + H183Q + F187I + N211M + S193E 134% T17F + F90Q + H183N + S193E + N204G + M208L + N211M 120% T17F + F90Q + H183N + S193E + N204G + M208L + N211M + N253D 166% T17F + F90Q + H183N + S193E + N204G + M208L + N211M + Q237E 154% T17F + F90Q + H183N + S193E + N204G + M208L + N211M + Q258E 176% T17F + F90Q + H183N + S193E + N204G + M208L + N211M + V200I 140% T17F + F90Q + I145D + H183N + S193E + N204G + M208L + N211M 155% T17F + F90Q + N122D + H183N + S193E + N204G + M208L + N211M 116% T17F + F90Q + Q142E + H183N + S193E + N204G + M208L + N211M 124% T17F + F90Q + S98R + N105D + H183N + S193E + N204G + N211M 133% T17F + N85D + F90Q + H183N + S193E + N204G + M208L + N211M 160% A64V 111% M208A 108% M208G + N211M 111% M208H + N211M 120% M208Q + N211M 112% H183D + F187I 136% M208T + N211M 106% T17F + F90Q + H183N + N204G + M208L + N211M 110% T17F + L90Q + H183N + S193E + N204G + M208M + N211M + V200I 119% T17F + L90Q + H183N + S193E + N204G + M208N + N211M + V200I 112% 表6中所列之變異體分別具有SEQ ID NO: 2之精確胺基酸序列，表6中所列之取代除外。 表7：24小時之後本發明之酯酶之PET解聚合產率。 變異體 與SEQ ID NO: 2 相比24 小時之後的PET 解聚合產率(%) T17F + N85D + F90Q + H183N + S193E + N204G + M208L + N211M 165% T17F + F90Q + I145D + H183N + S193E + N204G + M208L + N211M 135% T17F + F90Q + H183N + S193E + N204G + M208L + N211M + Q237E 142% T17F + F90Q + H183N + S193E + N204G + M208L + N211M + N253D 170% T17F + F90Q + H183N + S193E + N204G + M208L + N211M + Q258E 158% T17F + F90Q + H183N + S193E + N204G + M208L + N211M + V200I 128% T17F + F90Q + S98R + N105D + H183N + S193E + N204G + N211M 119% F92G 134% S212T 127% A64V 109% S66N 128% L90W 120% H183N 161% N122D 117% I143D 117% Q237E 129% N253D 110% Q258E 126% H183D + F187I 147% H183E + F187I 178% H183N + F187I 156% T17F + L90Q + H183N + S193E + N204G + M208N + N211M + V200I 112% M208T + N204G + H183Q + F187I + N211M + S193E 127% M208A 119% M208N 114% M208A + N211M 115% M208G + N211M 119% M208N + N211M 152% T17F + F90Q + H183N + S193E + N204G + M208L + N211M 120% T17F + F90Q + N122D + H183N + S193E + N204G + M208L + N211M 116% T17F + F90Q + Q142E + H183N + S193E + N204G + M208L + N211M 124% N105D 115% S193E 114% M208S 122% M208H + N211M 116% M208I + N211M 112% M208K + N211M 109% M208Q + N211M 108% M208T + N211M 113% 變異體具有SEQ ID NO: 2之精確胺基酸序列，表7中所列之取代除外。 PET depolymerization yield under alkaline conditions compared to the esterase of SEQ ID NO : 2 The PET depolymerization yield of the esterase (variant) of the present invention was measured according to Example 2.4 at 55° C., pH 8 after 6 hours or 24 hours. In the context of the present invention, the PET depolymerization yield is used to assess the degradation activity. The results (mean values) are shown in Table 6 and Table 7 after 6 hours or 24 hours, respectively. After 6 hours or 24 hours, the PET depolymerization yield of the esterase of SEQ ID NO: 2 at pH 8 was used as a reference and was considered as 100% degradation activity, respectively. Table 6: PET depolymerization yield of the esterase of the present invention after 6 hours Mutant Compared with SEQ ID NO: 2, PET depolymerization yield after 6 hours (%) H183N 167% Q237E 127% M208N 126% M208S 130% H183Q + F187I + N204G + M208N + N211M + S193E + V200I 115% M208T + N204G + H183Q + F187I + N211M + S193E + V200I 120% N122D 121% I143D 118% L90W 113% Q258E 134% M208I + N211M 116% M208K + N211M 113% S212T 137% S66N 140% M208P + N211M 113% H183E + F187I 195% N253D 120% H183N + F187I 164% M208A + N211M 131% M208N + N211M 143% M208T + N204G + H183Q + F187I + N211M + S193E 134% T17F + F90Q + H183N + S193E + N204G + M208L + N211M 120% T17F+F90Q+H183N+S193E+N204G+M208L+N211M+N253D 166% T17F+F90Q+H183N+S193E+N204G+M208L+N211M+Q237E 154% T17F+F90Q+H183N+S193E+N204G+M208L+N211M+Q258E 176% T17F + F90Q + H183N + S193E + N204G + M208L + N211M + V200I 140% T17F + F90Q + I145D + H183N + S193E + N204G + M208L + N211M 155% T17F + F90Q + N122D + H183N + S193E + N204G + M208L + N211M 116% T17F + F90Q + Q142E + H183N + S193E + N204G + M208L + N211M 124% T17F + F90Q + S98R + N105D + H183N + S193E + N204G + N211M 133% T17F + N85D + F90Q + H183N + S193E + N204G + M208L + N211M 160% A64V 111% M208A 108% M208G + N211M 111% M208H + N211M 120% M208Q + N211M 112% H183D + F187I 136% M208T + N211M 106% T17F + F90Q + H183N + N204G + M208L + N211M 110% T17F + L90Q + H183N + S193E + N204G + M208M + N211M + V200I 119% T17F + L90Q + H183N + S193E + N204G + M208N + N211M + V200I 112% The variants listed in Table 6 each have the exact amino acid sequence of SEQ ID NO: 2, except for the substitutions listed in Table 6. Table 7: PET depolymerization yield of the esterase of the present invention after 24 hours. Mutant PET depolymerization yield (%) after 24 hours compared to SEQ ID NO: 2 T17F + N85D + F90Q + H183N + S193E + N204G + M208L + N211M 165% T17F + F90Q + I145D + H183N + S193E + N204G + M208L + N211M 135% T17F+F90Q+H183N+S193E+N204G+M208L+N211M+Q237E 142% T17F+F90Q+H183N+S193E+N204G+M208L+N211M+N253D 170% T17F+F90Q+H183N+S193E+N204G+M208L+N211M+Q258E 158% T17F + F90Q + H183N + S193E + N204G + M208L + N211M + V200I 128% T17F + F90Q + S98R + N105D + H183N + S193E + N204G + N211M 119% F92G 134% S212T 127% A64V 109% S66N 128% L90W 120% H183N 161% N122D 117% I143D 117% Q237E 129% N253D 110% Q258E 126% H183D + F187I 147% H183E + F187I 178% H183N + F187I 156% T17F + L90Q + H183N + S193E + N204G + M208N + N211M + V200I 112% M208T + N204G + H183Q + F187I + N211M + S193E 127% M208A 119% M208N 114% M208A + N211M 115% M208G + N211M 119% M208N + N211M 152% T17F + F90Q + H183N + S193E + N204G + M208L + N211M 120% T17F + F90Q + N122D + H183N + S193E + N204G + M208L + N211M 116% T17F + F90Q + Q142E + H183N + S193E + N204G + M208L + N211M 124% N105D 115% S193E 114% M208S 122% M208H + N211M 116% M208I + N211M 112% M208K + N211M 109% M208Q + N211M 108% M208T + N211M 113% The variants have the exact amino acid sequence of SEQ ID NO: 2, except for the substitutions listed in Table 7.

實例 3- 評估本發明之酯酶之熱穩定性已確定本發明之酯酶之熱穩定性，且與SEQ ID NO: 1或SEQ ID NO: 2之酯酶之熱穩定性進行比較。 Example 3 - Evaluation of the Thermostability of the Esterases of the Present Invention The thermostability of the esterases of the present invention was determined and compared with the thermostability of the esterases of SEQ ID NO: 1 or SEQ ID NO: 2.

使用不同方法來評估熱穩定性： (1) 呈溶解狀態之蛋白質的圓二色性； (2) 在給定溫度、時間及緩衝液條件中進行蛋白質培育之後的殘餘酯酶活性； (3) 在給定溫度、時間及緩衝液條件中進行蛋白質培育之後的殘餘聚酯解聚合活性； (4) 在給定溫度、時間及緩衝液條件中進行蛋白質培育之後使分散於瓊脂培養盤中之固體聚酯化合物(諸如PET或PBAT或類似物)降解的能力； (5) 在給定溫度、緩衝液、蛋白質濃度及聚酯濃度條件中進行多輪聚酯解聚合分析之能力； (6) 差示掃描螢光測定法(DSF)； 下文給出關於此類方法之方案的細節。 Thermal stability is assessed using different methods: (1) Circular dichroism of the protein in solution; (2) Residual esterase activity after incubation of the protein at a given temperature, time and buffer; (3) Residual polyester depolymerization activity after incubation of the protein at a given temperature, time and buffer; (4) The ability to degrade solid polyester compounds (such as PET or PBAT or similar) dispersed in agar plates after incubation of the protein at a given temperature, time and buffer; (5) The ability to perform multiple rounds of polyester depolymerization analysis at a given temperature, buffer, protein concentration and polyester concentration; (6) Differential Scanning Fluorescence (DSF); Details of the protocol for this method are given below.

3.1. 圓二色性 已用Jasco 815裝置(Easton, USA)進行圓二色性(CD)以將SEQ ID NO: 1之酯酶之解鏈溫度(Tm)與本發明之酯酶之Tm進行比較。在技術上，在Talon緩衝液中以0.5 mg/mL製備400 µL蛋白質樣品，且將其用於CD。實現280 nm至190 nm之第一掃描，以確定對應於蛋白質之正確摺疊的CD之兩個最大強度。隨後在對應於此類最大強度之波長下進行25℃至110℃之第二掃描，且得到特定曲線(S型3參數y=a/(1+e^((x-x0)/b)))，藉由Sigmaplot版本11.0軟體分析該等曲線，確定x=x0時之Tm。所獲得之 T m 反映給定蛋白質之熱穩定性。 T m 愈高，變異體在高溫下愈穩定。 3.1. Circular Dichroism Circular dichroism (CD) has been performed using a Jasco 815 apparatus (Easton, USA) to compare the melting temperature (Tm) of the esterase of SEQ ID NO: 1 with the Tm of the esterase of the present invention. Technically, 400 µL of protein sample was prepared at 0.5 mg/mL in Talon buffer and used for CD. A first scan from 280 nm to 190 nm was performed to determine the two maximum intensities of the CD corresponding to the correct folding of the protein. A second scan from 25°C to 110°C was then performed at a wavelength corresponding to such maximum intensity, and specific curves (S-type 3 parameters y=a/(1+e^((x-x0)/b))) were obtained, which were analyzed by Sigmaplot version 11.0 software to determine the Tm when x=x0. The obtained Tm reflects the thermal stability of a given protein. The higher the Tm , the more stable the variant is at high temperatures.

3.2. 殘餘酯酶活性 在不同溫度(40℃、50℃、60℃、65℃、70℃、75℃、80℃及90℃)下培育1 mL具有40 mg/L (在Talon緩衝液中) SEQ ID NO: 1之酯酶或本發明之酯酶的溶液長達10天。有規律地獲取樣品，在pH 8.0的0.1 M磷酸鉀緩衝液中稀釋1至500倍，且實現對硝基苯酚丁酸酯(pNP-B)分析。將20 µL樣品與175 µL pH 8.0的0.1 M磷酸鉀緩衝液及5 µL含pNP-B溶液之2-甲基-2丁醇(40 mM)混合。在攪拌下在30℃下進行酶促反應持續15分鐘，且藉由微板分光光度計(Versamax, Molecular Devices, Sunnyvale, CA, USA)獲取405 nm下之吸光度。使用水解曲線之線性部分中針對所釋放的對硝基苯酚之標準曲線來確定pNP-B水解活性(以每分鐘pNPB微莫耳數表現之初始速度)。 3.2. Residual esterase activity 1 mL of a solution with 40 mg/L (in Talon buffer) of the esterase of SEQ ID NO: 1 or the esterase of the present invention was incubated at different temperatures (40°C, 50°C, 60°C, 65°C, 70°C, 75°C, 80°C and 90°C) for up to 10 days. Samples were taken regularly, diluted 1 to 500 times in 0.1 M potassium phosphate buffer, pH 8.0, and subjected to p-nitrophenol butyrate (pNP-B) analysis. 20 µL of sample was mixed with 175 µL of 0.1 M potassium phosphate buffer, pH 8.0 and 5 µL of 2-methyl-2-butanol (40 mM) containing pNP-B solution. The enzymatic reaction was carried out at 30°C for 15 min with stirring, and the absorbance at 405 nm was obtained by a microplate spectrophotometer (Versamax, Molecular Devices, Sunnyvale, CA, USA). The pNP-B hydrolysis activity (initial velocity expressed as micromoles of pNPB per minute) was determined using a standard curve for the released p-nitrophenol in the linear part of the hydrolysis curve.

3.3. 殘餘聚酯解聚合活性 分別在不同溫度(40℃、50℃、60℃、65℃、70℃、75℃、80℃及90℃)下培育10 mL具有40 mg/L (在Talon緩衝液中) SEQ ID NO: 1之酯酶及本發明之酯酶的溶液長達30天。有規律地獲取1 mL樣品，且轉移至含有100 mg以250-500 µm微粉化之非晶形PET (根據WO 2017/198786製備以達到低於20%之結晶度)及49 mL pH 8.0的0.1 M磷酸鉀緩衝液之瓶中，且在50℃、55℃、60℃、65℃或70℃下培育。有規律地對150 µL緩衝液進行取樣。需要時，將樣品稀釋於pH 8的0.1 M磷酸鉀緩衝液中。隨後，向150 µL樣品或稀釋液中添加150 µL甲醇及6.5 µL之6 N HCl。在混合且經由0.45 µm針筒過濾器過濾之後，將樣品加載在UHPLC上以監測對苯二甲酸(TA)、MHET及BHET之釋放。所使用層析系統為Ultimate 3000 UHPLC系統(Thermo Fisher Scientific, Inc. Waltham, MA, USA)，其包括泵模組、自動取樣器、恆溫在25℃的管柱烘箱及240 nm下之UV偵測器。所使用管柱為Discovery® HS C18 HPLC管柱(150×4.6 mm，5 µm，配備有前置管柱，Supelco，Bellefonte，USA)。使用MeOH (30%至90%)於1 mM H 2SO 4中之梯度以1 mL/min分離TA、MHET及BHET。注射液為20 µL樣品。根據在與樣品相同的條件下由市售TA及BHET以及內部合成的MHET所製備之標準曲線來量測TA、MHET及BHET。在水解曲線之線性部分中確定PET水解之活性(每分鐘水解的PET之微莫耳數或每小時產生的當量TA之毫克數)，該曲線係藉由在前24小時期間之不同時間處進行的取樣建立。當量TA對應於所量測TA與所量測MHET及BHET中含有之TA的總和。 3.3. Residual polyester depolymerization activity 10 mL of a solution with 40 mg/L (in Talon buffer) of SEQ ID NO: 1 and the esterase of the present invention was incubated at different temperatures (40°C, 50°C, 60°C, 65°C, 70°C, 75°C, 80°C and 90°C) for up to 30 days. 1 mL samples were taken regularly and transferred to bottles containing 100 mg of amorphous PET micronized at 250-500 µm (prepared according to WO 2017/198786 to achieve a crystallinity of less than 20%) and 49 mL of 0.1 M potassium phosphate buffer at pH 8.0 and incubated at 50°C, 55°C, 60°C, 65°C or 70°C. 150 µL of buffer was sampled regularly. When necessary, the samples were diluted in 0.1 M potassium phosphate buffer, pH 8. Subsequently, 150 µL of methanol and 6.5 µL of 6 N HCl were added to the 150 µL sample or dilution. After mixing and filtering through a 0.45 µm syringe filter, the samples were loaded on UHPLC to monitor the release of terephthalic acid (TA), MHET, and BHET. The chromatography system used was an Ultimate 3000 UHPLC system (Thermo Fisher Scientific, Inc. Waltham, MA, USA), which included a pump module, an autosampler, a column oven maintained at 25 °C, and a UV detector at 240 nm. The column used was a Discovery® HS C18 HPLC column (150×4.6 mm, 5 µm, equipped with a precolumn, Supelco, Bellefonte, USA). TA, MHET and BHET were separated using a gradient of MeOH (30% to 90%) in 1 mM H 2 SO 4 at 1 mL/min. The injection was 20 µL of sample. TA, MHET and BHET were measured according to standard curves prepared from commercially available TA and BHET and MHET synthesized in-house under the same conditions as the samples. The activity of PET hydrolysis (micromoles of PET hydrolyzed per minute or milligrams of equivalent TA produced per hour) was determined in the linear part of the hydrolysis curve, which was established by sampling at different times during the first 24 hours. The equivalent TA corresponds to the sum of the measured TA and the TA contained in the measured MHET and BHET.

3.4. 呈固態形式之聚酯之降解 分別在不同溫度(40℃、50℃、60℃、65℃、70℃、75℃、80℃及90℃)下培育1 mL具有40 mg/L (在Talon緩衝液中) SEQ ID NO: 1之酯酶及本發明之酯酶的溶液長達30天。有規律地，將20 µL酶製劑沉積於含有PET之瓊脂盤中所形成之孔中。藉由使500 mg PET溶解於六氟-2-丙醇(HFIP)中且將此培養基傾倒在250 mL水溶液中來實現含有PET之瓊脂盤之製備。在140毫巴下在52℃下進行HFIP蒸發之後，將溶液與pH為8的含有3%瓊脂之0.2 M磷酸鉀緩衝液以v/v混合。使用大約30 mL混合物來製備各omnitray，且儲存在4℃下。量測由於本發明之親本酯酶及變異體對聚酯之降解而形成的暈圈之直徑或表面積且在50℃、55℃、60℃、65℃或70℃下2至24小時之後進行比較。給定溫度下酶之半衰期對應於暈圈之直徑減小2倍係數所需之時間。 3.4. Degradation of polyester in solid form 1 mL of a solution with 40 mg/L (in Talon buffer) of the esterase of SEQ ID NO: 1 and the esterase of the present invention was incubated at different temperatures (40°C, 50°C, 60°C, 65°C, 70°C, 75°C, 80°C and 90°C) for up to 30 days. Regularly, 20 µL of the enzyme preparation was deposited in the wells formed in the agar plates containing PET. The preparation of the agar plates containing PET was achieved by dissolving 500 mg of PET in hexafluoro-2-propanol (HFIP) and pouring this medium in 250 mL of aqueous solution. After evaporation of HFIP at 52°C at 140 mbar, the solution was mixed v/v with 0.2 M potassium phosphate buffer, pH 8, containing 3% agar. Approximately 30 mL of the mixture was used to prepare each omnitray and stored at 4°C. The diameter or surface area of the halo formed by the degradation of polyester by the parent esterase and variants of the invention was measured and compared after 2 to 24 hours at 50°C, 55°C, 60°C, 65°C or 70°C. The half-life of the enzyme at a given temperature corresponds to the time required for the diameter of the halo to decrease by a factor of 2.

3.5. 多輪聚酯解聚合 在酶促反應器中評估酯酶進行連續輪次之聚酯解聚合分析之能力。藉由3 g非晶形PET (根據WO 2017/198786製備以達到低於20%之結晶度)及100 mL的含有3 mg酯酶的pH為8的10 mM磷酸鉀緩衝液來起始Minibio 500生物反應器(Applikon Biotechnology B.V., Delft, The Netherlands)。使用船用式葉輪將攪拌設定在250 rpm。生物反應器藉由浸沒於外部水浴中而恆溫在50℃、55℃、60℃、65℃或70℃。藉由添加3 M KOH將pH調節在8。藉由BioXpert軟體V2.95監測不同參數(pH、溫度、攪拌、鹼之添加)。每20 h添加1.8 g非晶形PET (根據WO 2017/198786製備以達到低於20%之結晶度)。有規律地對500 µL反應介質進行取樣。藉由HPLC測定TA、MHET及BHET之量，如實例2.3中所描述。使用恆溫在65℃之Aminex HPX-87K管柱(Bio-Rad Laboratories, Inc, Hercules, California, United States)來確定EG之量。溶離劑為5 mM K 2HPO 4，0.6 mL.min - 1。注射液為20 µL。使用折射器來監測乙二醇。基於給定時間處之莫耳濃度比率(TA + MHET + BHET)相比於初始樣品中所含有的TA總量，或基於給定時間處之莫耳濃度比率(EG +MHET + 2×BHET)相比於初始樣品中所含有的EG總量來計算水解百分比。以每小時釋放的TA總毫克數或以每小時EG的總毫克數來計算降解速率。將酶半衰期評估為獲得50%降解速率損失所需的培育時間。 3.5. Multiple rounds of polyester depolymerization The ability of esterases to perform consecutive rounds of polyester depolymerization analysis was evaluated in an enzymatic reactor. A Minibio 500 bioreactor (Applikon Biotechnology BV, Delft, The Netherlands) was initiated with 3 g of amorphous PET (prepared according to WO 2017/198786 to achieve a crystallinity of less than 20%) and 100 mL of 10 mM potassium phosphate buffer, pH 8, containing 3 mg of esterase. Agitation was set at 250 rpm using a marine impeller. The bioreactor was thermostated at 50°C, 55°C, 60°C, 65°C, or 70°C by immersion in an external water bath. The pH was adjusted to 8 by adding 3 M KOH. Different parameters (pH, temperature, stirring, addition of base) were monitored by BioXpert software V2.95. 1.8 g of amorphous PET (prepared according to WO 2017/198786 to achieve a crystallinity of less than 20%) was added every 20 h. 500 µL of the reaction medium was sampled regularly. The amounts of TA, MHET and BHET were determined by HPLC as described in Example 2.3. The amount of EG was determined using an Aminex HPX-87K column (Bio-Rad Laboratories, Inc, Hercules, California, United States) thermostated at 65 °C. The solvent was 5 mM K 2 HPO 4 , 0.6 mL.min - 1 . The injection was 20 µL. A refractometer was used to monitor ethylene glycol. The percentage hydrolysis was calculated based on the ratio of the molar concentrations at a given time (TA + MHET + BHET) compared to the total amount of TA contained in the initial sample, or based on the ratio of the molar concentrations at a given time (EG + MHET + 2×BHET) compared to the total amount of EG contained in the initial sample. The degradation rate was calculated as the total mg TA released per hour or as the total mg EG per hour. The enzyme half-life was estimated as the incubation time required to obtain a 50% loss in degradation rate.

3.6. 差示掃描螢光測定法(DSF) DSF用於藉由確定親本蛋白質(SEQ ID NO: 1)及其變異體的解鏈溫度(Tm)，亦即一半的蛋白質群體展開之溫度來評估他們的熱穩定性。以6.25 µM之濃度製備蛋白質樣品且儲存於由100 mM磷酸鉀緩衝液(pH 8)組成之緩衝液A中。用水將於DMSO中之SYPRO橙色染料5000×儲備溶液首次稀釋至250×。將蛋白質樣品加載至白色透明96孔PCR培養盤(Bio-Rad目錄號HSP9601)上，其中各孔含有25 µL最終體積。各孔中蛋白質及SYPRO橙色染料之最終濃度分別為6 µM (0.17 mg/ml)及10×。每孔所加載的體積如下：24 μL之6.25 µM蛋白質溶液及1 μL之250×Sypro Orange稀釋溶液。隨後用光學品質密封膠帶密封PCR培養盤，且使其在室溫下以1000 rpm旋轉1 min。隨後使用設定成使用450/490激勵及560/580發射濾光器之CFX96即時PCR系統來進行DSF實驗。以0.3℃/秒之速率將樣品自25℃加熱至100℃。每0.03秒進行單次螢光量測。使用Bio-Rad CFX Manager軟體自解鏈曲線之一階導數之峰值確定解鏈溫度。可使用緩衝液類型或緩衝液濃度之變化，而不影響本發明之酯酶與親本酯酶之間的∆Tm，只要相同的緩衝液用於親本酯酶即可。隨後基於Tm值將SEQ ID NO: 1或SEQ ID NO: 2之酯酶與本發明之酯酶進行比較。歸因於來自不同產生批次之相同蛋白質之實驗之間的高再現性，將0.8℃之∆Tm視為比較變異體之有效值。Tm值對應於至少3次量測之平均值。在84.7℃下評估SEQ ID NO: 1之酯酶之Tm。 3.6. Differential Scanning Fluorescence (DSF) DSF was used to assess the thermal stability of the parent protein (SEQ ID NO: 1) and its variants by determining their melting temperature (Tm), i.e., the temperature at which half of the protein population is unfolded. Protein samples were prepared at a concentration of 6.25 µM and stored in buffer A consisting of 100 mM potassium phosphate buffer (pH 8). A 5000× stock solution of SYPRO Orange dye in DMSO was first diluted to 250× with water. Protein samples were loaded onto white clear 96-well PCR plates (Bio-Rad catalog number HSP9601) with each well containing 25 µL final volume. The final concentrations of protein and SYPRO Orange dye in each well were 6 µM (0.17 mg/ml) and 10×, respectively. The volumes loaded per well were as follows: 24 μL of 6.25 µM protein solution and 1 μL of 250× Sypro Orange dilution. The PCR plates were then sealed with optical quality sealing tape and rotated at 1000 rpm for 1 min at room temperature. DSF experiments were then performed using a CFX96 Real-Time PCR System set to use 450/490 excitation and 560/580 emission filters. Samples were heated from 25°C to 100°C at a rate of 0.3°C/sec. Single fluorescence measurements were taken every 0.03 sec. The melting temperature was determined from the peak of one derivative of the melting curve using Bio-Rad CFX Manager software. Changes in buffer type or buffer concentration can be used without affecting the ∆Tm between the esterase of the present invention and the parent esterase, as long as the same buffer is used for the parent esterase. The esterase of SEQ ID NO: 1 or SEQ ID NO: 2 was then compared with the esterase of the present invention based on the Tm value. Due to the high reproducibility between experiments of the same protein from different production batches, a ∆Tm of 0.8°C was considered a valid value for comparing variants. The Tm value corresponds to the average of at least 3 measurements. The Tm of the esterase of SEQ ID NO: 1 was evaluated at 84.7°C.

TW202511481A_113117991_SEQL.xmlTW202511481A_113117991_SEQL.xml

Claims (38)

一種酯酶，其：(i)與SEQ ID NO: 1中所示之全長胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%一致性；(ii)與SEQ ID NO: 1相比，具有至少一種選自以下之胺基酸取代：H183Q/L/A/Y/N/D/E/W、R30G、A64V、A68N、T109S、L124G、S145G、L152M、E158A、K159R、S193L/Q、W228R/S、V242E、G7A、N9T、A24Q、Y26H、T27A、G39S、G46V、T50M、A64T、R73H、L74V、Y106F、R108L、V167L、I169M、P179N、I185V、F187L/Q、P192S、P213A、L227N、Q237L、L239M、R251S、S66T、F90C/V、P151V、A246R、A215E/D、D230E、L15K/R、N143D、V200I及N253D，或至少一種選自以下之取代的組合：N204G + M208L + N211E、F90E + N204G + N211E、F90N + N204G + N211E、F90Q + N204G + N211E、F90R + N204G + N211E、F90W + N204G + N211E、F187I + N204G + N211E，及S98R + E173Q、M208Q + N211M及M208N + N211M，其中參考SEQ ID NO: 1中所示之胺基酸序列對位置進行編號；(iii)具有聚酯降解活性；及(iv)與SEQ ID NO: 1之酯酶相比，在包含於6與10之間的pH下展現增加之熱穩定性及/或增加之聚酯降解活性。An esterase, which: (i) has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the full-length amino acid sequence shown in SEQ ID NO: 1; (ii) compared to SEQ ID NO: 1, has at least one amino acid substitution selected from the following: H183Q/L/A/Y/N/D/E/W, R30G, A64V, A68N, T109S, L124G, S145G, L152M, E158A, K159R, S193L/Q, W228R/S, V242E, G7A, N9T, A24Q, Y26H, T27A, G39S, G46V, T50M, A64T, R73H, L74 V, Y106F, R108L, V167L, I169M, P179N, I185V, F187L/Q, P192S, P213A, L227N, Q237L, L239M, R251S, S66T, F90C/V, P151V, A246R, A215E/D, D230E, L15K/R, N143D, V200I, and N253D, or a combination of at least one substitution selected from the following: N204G + M208L + N211E, F90E + N204G + N211E, F90N + N204G + N211E, F90Q + N204G + N211E, F90R + N204G + N211E, F90W + N204G + N211E, F187I + N204G + N211E, and S98R + E173Q, M208Q + N211M and M208N + N211M, wherein the positions are numbered with reference to the amino acid sequence shown in SEQ ID NO: 1; (iii) have polyester degradation activity; and (iv) exhibit increased thermostability and/or increased polyester degradation activity at a pH comprised between 6 and 10 compared to the esterase of SEQ ID NO: 1. 如請求項1之酯酶，其中該酯酶包含選自以下之至少一種胺基酸取代或至少一種取代之組合：H183Q/L/A/Y/N/D/E/W、R30G、A64V、A68N、T109S、L124G、S145G、L152M、E158A、K159R、S193L/Q、W228R/S、V242E、G7A、N9T、A24Q、Y26H、T27A、G39S、G46V、T50M、A64T、R73H、L74V、Y106F、R108L、V167L、I169M、P179N、I185V、F187L/Q、P192S、P213A、L227N、Q237L、L239M、R251S、S66T、F90C/V、P151V、A246R、A215E/D、D230E、L15K/R、N253D、N204G + M208L + N211E、F90E + N204G + N211E、F90N + N204G + N211E、F90Q + N204G + N211E、F90R + N204G + N211E、F90W + N204G + N211E、F187I + N204G + N211E、S98R + E173Q及M208N + N211M。The esterase of claim 1, wherein the esterase comprises at least one amino acid substitution or a combination of at least one substitution selected from the group consisting of H183Q/L/A/Y/N/D/E/W, R30G, A64V, A68N, T109S, L124G, S145G, L152M, E158A, K159R, S193L/Q, W228R/S, V242E, G7A, N9T, A24Q, Y26H, T27A, G39S , G46V, T50M, A64T, R73H, L74V, Y106F, R108L, V167L, I169M, P179N, I185V, F187L/Q, P192S, P213A , L227N, Q237L, L239M, R251S, S66T, F90C/V, P151V, A246R, A215E/D, D230E, L15K/R, N253D, N204G + M208L + N211E, F90E + N204G + N211E, F90N + N204G + N211E, F90Q + N204G + N211E, F90R + N204G + N211E, F90W + N204G + N211E, F187I + N204G + N211E, S98R + E173Q and M208N + N211M. 如前述請求項中任一項之酯酶，其中該酯酶進一步包含至少一種選自以下之取代：G35A、W69L、F90L/Y、N162S、S181K、N204G、A216C、N243Y、A246C、V28I、T157P、T16K/R、Y4K/R、V219E/D、M208N、N211M、A17F、M208L、M208T、V200I、I170V、N122D、Q142E、Q237E、Q258E、N85D、N105D、L13S、E158D。The esterase of any of the preceding claims, wherein the esterase further comprises at least one substitution selected from the group consisting of G35A, W69L, F90L/Y, N162S, S181K, N204G, A216C, N243Y, A246C, V28I, T157P, T16K/R, Y4K/R, V219E/D, M208N, N211M, A17F, M208L, M208T, V200I, I170V, N122D, Q142E, Q237E, Q258E, N85D, N105D, L13S, E158D. 如前述請求項中任一項之酯酶，其中該酯酶包含至少一種選自以下之取代：R30G、A64V、L124G、S145G、H183Q/N、S193L/Q、W228S及V242E，且進一步包含至少一種選自以下之取代：G35A、W69L、F90L/Y、V115I、N162S、S181K、N204G、A216C、N243Y、A246C、V28I、T157P、A17F、F90Q、M208L、N211M、N122D、S193E、Q142E、N143D、Q237E、N253D、Q258E、V200I、I170V、N85D、N105D、N211E、F187I、M208N、M208T、L13S、E158D，較佳選自G35A、W69L、F90L/Y、V115I、N162S、S181K、N204G、A216C、N243Y、A246C、V28I及T157P。The esterase of any of the preceding claims, wherein the esterase comprises at least one substitution selected from the group consisting of R30G, A64V, L124G, S145G, H183Q/N, S193L/Q, W228S, and V242E, and further comprises at least one substitution selected from the group consisting of G35A, W69L, F90L/Y, V115I, N162S, S181K, N204G, A216C, N243Y, A246C, V28I, T157P, A17F, F90Q, M2 08L, N211M, N122D, S193E, Q142E, N143D, Q237E, N253D, Q258E, V200I, I170V, N85D, N105D, N211E, F187I, M208N, M208T, L13S, E158D, preferably G35A, W69L, F90L/Y, V115I, N162S, S181K, N204G, A216C, N243Y, A246C, V28I and T157P. 如前述請求項中任一項之酯酶，其中該酯酶包含至少一種選自H183Q/L/A/Y/N/D/E/W之取代，且進一步包含至少一種選自以下之取代：F187I、F187L、A24Q、R30G、L74V、F187Q、F187I、L227N、R251S、Y26H、A64T、G35A、A62T、G46V、R73H、T50M、I169M、Y106F、I185V、R108L、V167L、N204G、G39S、A64V、L124G、N162S、S193Q、Q237L、M208T、S181K、W69L、A216C、A246C、F90I、F90Y、A17F、F90Q、M208L、N211M、N122D、S193E、Q142E、N143D、Q237E、N253D、Q258E、V200I、I170V、N85D、N105D、N211E、M208N、M208T、L13S及E158D。The esterase of any of the preceding claims, wherein the esterase comprises at least one substitution selected from H183Q/L/A/Y/N/D/E/W, and further comprises at least one substitution selected from F187I, F187L, A24Q, R30G, L74V, F187Q, F187I, L227N, R251S, Y26H, A64T, G35A, A62T, G46V, R73H, T50M, I169M, Y106F, I185V, R108L, V167L, N204G , G39S, A64V, L124G, N162S, S193Q, Q237L, M208T, S181K, W69L, A216C, A246C, F90I, F90Y, A17F, F90Q, M208L, N211M, N122D, S193E, Q142E, N143D, Q237E, N253D, Q258E, V200I, I170V, N85D, N105D, N211E, M208N, M208T, L13S, and E158D. 如請求項5之酯酶，其中該酯酶包含至少一種選自以下之取代的組合或由具有一種選自以下之取代的組合的SEQ ID NO: 1中所示之胺基酸序列組成：H183L + F187I、H183A + F187I、H183Y + F187I、H183D + F187I、H183W + F187I、H183E + F187I、F90L + H183E + F187L、F90Y + H183L + F187L、A24Q + H183Q、R30G + H183Q、L74V + H183Q、H183Q + F187Q、H183Q + F187I、L227N + H183Q、R251S + H183Q、Y26H + A64T + H183Q、G35A + A62T + H183Q、G46V + R73H + H183Q、T50M + I169M + H183Q、Y106F + I185V + H183Q、R108L + V167L + H183Q、H183Q + F187I + N204G、N204G + H183Q + F187I + R30G、N204G + H183Q + F187I + G39S、N204G + H183Q + F187I + A62T、N204G + H183Q + F187I + A64V、N204G + H183Q + F187I + L124G、N204G + H183Q + F187I + N162S、N204G + H183Q + F187I + S193Q、N204G + H183Q + F187I + Q237L、M208T + N204G + H183Q + F187I、N204G + H183Q + F187I + N162S + S181K、N204G + H183Q + F187I + N162S + S181K + S193Q及N204G + H183Q + F187I + W69L + A216C + A246C、A17F + F90Q + H183N + N204G + M208L + N211M、H183Q + F187I + N204G + M208N + N211M + S193E、M208T + N204G + H183Q + F187I + N211M + S193E、H183Q + F187I + N204G + N211M + S193E + V200I + I170V、A17F + F90Q + N122D + H183N + S193E + N204G + M208L + N211M、A17F + F90Q + Q142E + H183N + S193E + N204G + M208L + N211M、A17F + F90Q + N143D + H183N + S193E + N204G + M208L + N211M、A17F + F90Q + H183N + S193E + N204G + M208L + N211M + Q237E、A17F + F90Q + H183N + S193E + N204G + M208L + N211M + N253D、A17F + F90Q + H183N + S193E + N204G + M208L + N211M + Q258E、H183Q + F187I + N204G + M208N + N211M + S193E + V200I + I170V、M208T + N204G + H183Q + F187I + N211M + S193E + V200I + I170V、A17F + N85D + F90Q + H183N + S193E + N204G + M208L + N211M、A17F + F90Q + N105D + H183N + S193E + N204G + M208L + N211E、H183Q + F187I + N204G + L13S + E158D、M208T + N204G + H183Q + F187I + L13S + E158D、A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I、A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + H183Q + F187I + N204G、A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I + L13S + E158D。The esterase of claim 5, wherein the esterase comprises at least one combination of substitutions selected from the following or consists of the amino acid sequence shown in SEQ ID NO: 1 having a combination of substitutions selected from the following: H183L + F187I, H183A + F187I, H183Y + F187I, H183D + F187I, H183W + F187I, H183E + F187I, F90L + H183E + F187L, F90Y + H183L + F187L, A24Q + H183Q, R30G + H183Q, L74V + H183Q, H183Q + F187Q, H183Q + F187I, L227N + H183Q, R251S + H183Q, Y26H + A64T + H183Q, G35A + A62T + H183Q, G46V + R73H + H183Q, T50M + I169M + H183Q, Y106F + I185V + H183Q, R108L + V167L + H183Q, H183Q + F187I + N204G, N204G + H183Q + F187I + R30G, N204G + H183Q + F187I + G39S, N204G + H183Q + F187I + A62T, N204G + H183Q + F187I + A64V, N204G + H183Q + F187I + L124G, N204G + H183Q + F187I + N162S, N204G + H183Q + F187I + S193Q, N204G + H183Q + F187I + Q237L, M208T + N204G + H183Q + F187I, N204G + H183Q + F187I + N162S + S181K, N204G + H183Q + F187I + N162S + S181K + S193Q and N204G + H183Q + F187I + W69L + A216C + A246C, A17F + F90Q + H183N + N204G + M208L + N211M, H183Q + F187I + N204G + M208N + N211M + S193E, M208T + N204G + H183Q + F187I + N211M + S193E, H183Q + F187I + N204G + N211M + S193E + V200I + I170V, A17F + F90Q + N122D + H183N + S193E + N204G + M208L + N211M, A17F + F90Q + Q142E + H183N + S193E + N204G + M208L + N211M, A17F + F90Q + N143D + H183N + S193E + N204G + M208L + N211M, A17F + F90Q+H183N+ S193E + N204G + M208L + N211M + Q237E, A17F + F90Q + H183N + S193E + N204G + M208L + N211M + N253D, A17F + F90Q + H183N + S193E + N204G + M208L + N211M + Q258E, H183Q + F187I + N204G + M208N + N211M + S193E + V200I + I170V, M208T + N204G + H183Q + F187I + N211M + S193E + V200I + I170V, A17F + N85D + F90Q + H183N + S193E + N204G + M208L + N211M, A17F + F90Q + N105D + H183N + S193E + N204G + M208L + N211E, H183Q + F187I + N204G + L13S + E158D, M208T + N204G + H183Q + F187I + L13S + E158D, A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I, A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + H183Q + F187I + N204G、A17T + T27S + S48T+L82I+F90L+G92F+G135A+A140S+N143I+S145T+A149G+S164P+V167Q+S206T+T252S+I170V+M208T+N204G+H183Q+F187I+L13S+E158D. 如請求項5之酯酶，其中該酯酶包含取代H183Q，且進一步包含選自以下之至少一種取代、至少兩種取代、至少三種取代：A24Q、R30G、L74V、F187Q、F187I、L227N、R251S、Y26H、A64T、G35A、A62T、G46V、R73H、T50M、I169M、Y106F、I185V、R108L、V167L、N204G、G39S、A64V、L124G、N162S、S193Q、Q237L、M208T、S181K、W69L、A216C、A246C、N204G、M208N、N211M、S193E、V200I、I170V、L13S、E158D。The esterase of claim 5, wherein the esterase comprises substitution H183Q and further comprises at least one substitution, at least two substitutions, or at least three substitutions selected from the group consisting of A24Q, R30G, L74V, F187Q, F187I, L227N, R251S, Y26H, A64T, G35A, A62T, G46V, R73H, T50M, I169M, Y1 06F, I185V, R108L, V167L, N204G, G39S, A64V, L124G, N162S, S193Q, Q237L, M208T, S 181K, W69L, A216C, A246C, N204G, M208N, N211M, S193E, V200I, I170V, L13S, E158D. 如請求項7之酯酶，其中該酯酶包含至少一種選自以下之取代的組合或由具有一種選自以下之取代的組合的SEQ ID NO: 1中所示之胺基酸序列組成：A24Q + H183Q、R30G + H183Q、L74V + H183Q、H183Q + F187Q、H183Q + F187I、L227N + H183Q、R251S + H183Q、Y26H + A64T + H183Q、G35A + A62T + H183Q、G46V + R73H + H183Q、T50M + I169M + H183Q、Y106F + I185V + H183Q、R108L + V167L + H183Q、H183Q + F187I + N204G、N204G + H183Q + F187I + R30G、N204G + H183Q + F187I + G39S、N204G + H183Q + F187I + A62T、N204G + H183Q + F187I + A64V、N204G + H183Q + F187I + L124G、N204G + H183Q + F187I + N162S、N204G + H183Q + F187I + S193Q、N204G + H183Q + F187I + Q237L、M208T + N204G + H183Q + F187I、N204G + H183Q + F187I + N162S + S181K、N204G + H183Q + F187I + N162S + S181K + S193Q、N204G + H183Q + F187I + W69L + A216C + A246C、H183Q + F187I + N204G + M208N + N211M + S193E、M208T + N204G + H183Q + F187I + N211M + S193E、H183Q + F187I + N204G + N211M + S193E + V200I + I170V、H183Q + F187I + N204G + M208N + N211M + S193E + V200I + I170V、M208T + N204G + H183Q + F187I + N211M + S193E + V200I + I170V、H183Q + F187I + N204G + N211M + S193E 、N204G + H183Q + F187I + L13S + E158D、N204G + H183Q + F187I、M208T + N204G + H183Q + F187I + L13S + E158D、H183Q + F187I + N204G + L13S + E158D、M208T + N204G + H183Q + F187I + L13S + E158D、A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I、A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + H183Q + F187I + N204G、A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I + L13S + E158D。The esterase of claim 7, wherein the esterase comprises at least one combination of substitutions selected from the following or consists of the amino acid sequence shown in SEQ ID NO: 1 having a combination of substitutions selected from the following: A24Q + H183Q, R30G + H183Q, L74V + H183Q, H183Q + F187Q, H183Q + F187I, L227N + H183Q, R251S + H183Q, Y26H + A64T + H183Q, G35A + A62T + H183Q, G46V + R73H + H183Q, T50M + I169M + H183Q, Y106F + I185V + H183Q, R108L + V167L + H183Q, H183Q + F187I + N204G, N204G + H183Q + F187I + R30G, N204G + H183Q + F187I + G39S, N204G + H183Q + F187I + A62T, N204G + H183Q + F187I + A64V, N204G + H183Q + F187I + L124G, N204G + H183Q + F187I + N162S, N204G + H183Q + F187I + S193Q, N204G + H183Q + F187I + Q237L, M208T + N204G + H183Q + F187I, N204G + H183Q + F187I + N162S + S181K, N204G + H183Q + F187I + N162S + S181K + S193Q, N204G + H183Q + F187I + W69L + A216C + A246C, H183Q + F187I + N204G + M208N + N211M + S193E, M208T + N204G + H183Q + F187I + N211M + S193E, H183Q + F187I + N204G + N211M + S193E + V200I + I170V, H183Q + F187I + N204G + M208N + N211M + S193E+V200I+ I170V, M208T + N204G + H183Q + F187I + N211M + S193E + V200I + I170V, H183Q + F187I + N204G + N211M + S193E, N204G + H183Q + F187I + L13S + E158D, N204G + H183Q + F187I, M208T + N204G + H183Q + F187I + L13S + E158D, H183Q + F187I + N204G + L13S + E158D, M208T + N204G + H183Q + F187I + L13S + E158D, A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I, A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + H183Q + F187I + N204G, A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S+N143I+S145T+A149G+S164P+V167Q+S206T+T252S+I170V+M208T+N204G+H183Q+F187I+L13S+E158D. 如請求項7之酯酶，其中該酯酶至少包含N204G + H183Q + F187I之取代的組合，且視情況進一步包含至少一種選自以下之取代：R30G、G39S、A62T、A64V、W69L、L124G、N162S、S181K、S193Q、M208T、A216C、Q237L、A246C、M208N、N211M、S193E、V200I、I170V、L13S、E158D。An esterase as claimed in claim 7, wherein the esterase comprises at least a combination of substitutions N204G + H183Q + F187I, and optionally further comprises at least one substitution selected from the following: R30G, G39S, A62T, A64V, W69L, L124G, N162S, S181K, S193Q, M208T, A216C, Q237L, A246C, M208N, N211M, S193E, V200I, I170V, L13S, E158D. 如請求項9之酯酶，其中該酯酶包含至少一種選自以下之取代的組合或由具有一種選自以下之取代的組合的SEQ ID NO: 1中所示之胺基酸序列組成：N204G + H183Q + F187I + R30G、N204G + H183Q + F187I + G39S、N204G + H183Q + F187I + A62T、N204G + H183Q + F187I + A64V、N204G + H183Q + F187I + L124G、N204G + H183Q + F187I + N162S、N204G + H183Q + F187I + S193Q、N204G + H183Q + F187I + Q237L、M208T + N204G + H183Q + F187I、N204G + H183Q + F187I + N162S + S181K、N204G + H183Q + F187I + N162S + S181K + S193Q、N204G + H183Q + F187I + W69L + A216C + A246C、H183Q + F187I + N204G + M208N + N211M + S193E、H183Q + F187I + N204G + M208N + N211M + S193E + V200I + I170V、M208T + N204G + H183Q + F187I + N211M + S193E、M208T + N204G + H183Q + F187I + N211M + S193E + V200I + I170V、H183Q + F187I + N204G + N211M + S193E、H183Q + F187I + N204G + N211M + S193E + V200I + I170V、H183Q + F187I + N204G + L13S + E158D、M208T + N204G + H183Q + F187I + L13S + E158D、A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I、A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + H183Q + F187I + N204G、A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I + L13S + E158D，較佳選自N204G + H183Q + F187I + R30G、N204G + H183Q + F187I + G39S、N204G + H183Q + F187I + A62T、N204G + H183Q + F187I + A64V、N204G + H183Q + F187I + L124G、N204G + H183Q + F187I + N162S、N204G + H183Q + F187I + S193Q、N204G + H183Q + F187I + Q237L、M208T + N204G + H183Q + F187I、N204G + H183Q + F187I + N162S + S181K、N204G + H183Q + F187I + N162S + S181K + S193Q、N204G + H183Q + F187I + W69L + A216C + A246C、H183Q + F187I + N204G + M208N + N211M + S193E、H183Q + F187I + N204G + M208T + N211M + S193E、H183Q + F187I + N204G + N211M + S193E + V200I + I170V、H183Q + F187I + N204G + M208N + N211M + S193E + V200I + I170V、H183Q + F187I + N204G + M208T + N211M + S193E + V200I + I170V，更佳選自N204G + H183Q + F187I + A62T、N204G + H183Q + F187I + A64V、N204G + H183Q + F187I + L124G、N204G + H183Q + F187I + N162S、N204G + H183Q + F187I + S193Q、N204G + H183Q + F187I + Q237L、M208T + N204G + H183Q + F187I、N204G + H183Q + F187I + N162S + S181K + S193Q、H183Q + F187I + N204G + M208N + N211M + S193E、H183Q + F187I + N204G + M208T + N211M + S193E及H183Q + F187I + N204G + M208N + N211M + S193E + V200I + I170V。The esterase of claim 9, wherein the esterase comprises at least one combination of substitutions selected from the following or consists of the amino acid sequence shown in SEQ ID NO: 1 having a combination of substitutions selected from the following: N204G + H183Q + F187I + R30G, N204G + H183Q + F187I + G39S, N204G + H183Q + F187I + A62T, N204G + H183Q + F187I + A64V, N204G + H183Q + F187I + L124G, N204G + H183Q + F187I + N162S, N204G + H183Q + F187I + S193Q, N204G + H183Q + F187I + Q237L, M208T + N204G + H183Q + F187I, N204G + H183Q + F187I + N162S + S181K, N204G + H183Q + F187I + N162S + S181K + S193Q, N204G + H183Q + F187I + W69L + A216C + A246C, H183Q + F187I + N204G + M208N + N211M + S193E, H183Q + F187I + N204G + M208N + N211M + S193E + V200I + I170V, M208T + N204G + H183Q + F187I + N211M + S193E, M208T + N204G + H183Q + F187I + N211M + S193E + V200I + I170V, H183Q + F187I + N204G + N211M + S193E, H183Q + F187I + N204G + N211M + S193E + V200I + I170V, H183Q + F187I + N204G + L13S + E158D, M208T + N204G + H183Q + F187I + L13S + E158D, A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I, A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + H183Q + F187I + N204G, A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I + L13S + E158D, preferably N204G + H183Q + F187I + R30G, N204G + H183Q + F187I + G39S, N204G + H183Q + F187I + A62T, N204G + H183Q + F187I + A64V, N204G + H183Q + F187I + L124G, N204G + H183Q + F187I + N162S, N204G + H183Q + F187I + S193Q, N204G + H183Q + F187I + Q237L, M208T + N204G + H183Q + F187I, N204G + H183Q + F187I + N162S + S181K, N204G + H183Q + F187I + N162S + S181K + S193Q, N204G + H183Q + F187I + W69L + A216C + A246C, H183Q + F187I + N204G + M208N + N211M + S193E, H183Q + F187I + N204G + M208T + N211M + S193E, H183Q + F187I + N204G + N211M + S193E + V200I + I170V, H183Q + F187I + N204G + M208N + N211M + S193E + V200I + I170V, H183Q + F187I + N204G + M208T + N211M + S193E + V200I + I170V, preferably N204G + H183Q + F187I + A62T, N204G + H183Q + F187I + A64V, N204G + H183Q + F187I + L124G, N204G + H183Q + F187I + N162S, N204G + H183Q + F187I + S193Q, N204G + H183Q + F187I + Q237L, M208T + N204G + H183Q + F187I, N204G + H183Q + F187I + N162S + S181K + S193Q, H183Q + F187I + N204G + M208N + N211M + S193E, H183Q + F187I + N204G + M208T + N211M + S193E and H183Q + F187I + N204G + M208N + N211M + S193E + V200I + I170V. 如請求項9之酯酶，其中該酯酶至少包含選自H183Q + F187I + N204G + N211M + S193E之取代的組合或由具有一種選自H183Q + F187I + N204G + N211M + S193E之取代的組合的SEQ ID NO: 1中所示之胺基酸序列組成，且進一步包含至少一種選自M208N/T、V200I及I170V之取代，較佳地，該酯酶包含至少一種選自N204G + H183Q + F187I + M208N/T + N211M + S193E，較佳選自H183Q + F187I + N204G + M208N + N211M + S193E之取代。An esterase as claimed in claim 9, wherein the esterase comprises at least a combination of substitutions selected from H183Q + F187I + N204G + N211M + S193E or consists of the amino acid sequence shown in SEQ ID NO: 1 having a combination of substitutions selected from H183Q + F187I + N204G + N211M + S193E, and further comprises at least one substitution selected from M208N/T, V200I and I170V, preferably, the esterase comprises at least one substitution selected from N204G + H183Q + F187I + M208N/T + N211M + S193E, preferably selected from H183Q + F187I + N204G + M208N + N211M + S193E. 如請求項5之酯酶，其中該酯酶包含取代H183N，且進一步包含至少一種選自以下之取代：F187I、F90L、F187L、F90Y、A17F、F90Q、N204G、M208L、N211M、N122D、S193E、Q142E、N143D、Q237E、N253D、Q258E、V200I、I170V、N85D、N105D、N211E、L13S、E158D。The esterase of claim 5, wherein the esterase comprises the substitution H183N and further comprises at least one substitution selected from the group consisting of F187I, F90L, F187L, F90Y, A17F, F90Q, N204G, M208L, N211M, N122D, S193E, Q142E, N143D, Q237E, N253D, Q258E, V200I, I170V, N85D, N105D, N211E, L13S, E158D. 如請求項12之酯酶，其中該酯酶包含至少一種選自以下之取代的組合或由具有一種選自以下之取代的組合的SEQ ID NO: 1中所示之胺基酸序列組成：H183N + F187I、F90L + H183N +  F187L、F90Y + H183N、A17F + F90Q + H183N + N204G + M208L + N211M、A17F + F90Q + N122D + H183N + S193E + N204G + M208L + N211M、A17F + F90Q + Q142E + H183N + S193E + N204G + M208L + N211M、A17F + F90Q + N143D + H183N + S193E + N204G + M208L + N211M、A17F + F90Q + H183N + S193E + N204G + M208L + N211M + Q237E、A17F + F90Q + H183N + S193E + N204G + M208L + N211M + N253D、A17F + F90Q + H183N + S193E + N204G + M208L + N211M + Q258E、A17F + F90Q + H183N + S193E + N204G + M208L + N211M + V200I + I170V、A17F + N85D + F90Q + H183N + S193E + N204G + M208L + N211M及A17F + F90Q + N105D + H183N + S193E + N204G + M208L + N211E。The esterase of claim 12, wherein the esterase comprises at least one combination of substitutions selected from the following or consists of the amino acid sequence shown in SEQ ID NO: 1 having a combination of substitutions selected from the following: H183N + F187I, F90L + H183N + F187L, F90Y + H183N, A17F + F90Q + H183N + N204G + M208L + N211M, A17F + F90Q + N122D + H183N + S193E + N204G + M208L + N211M, A17F + F90Q + Q142E + H183N + S193E + N204G + M208L + N211M, A17F + F90Q + N143D + H183N + S193E + N204G + M208L + N211M, A17F + F90Q + H183N + S193E + N204G + M208L + N211M + Q237E, A17F + F90Q + H183N + S193E + N204G + M208L + N211M + N253D, A17F + F90Q + H183N + S193E + N204G + M208L + N211M + Q258E, A17F + F90Q + H183N + S193E + N204G + M208L + N211M + V200I + I170V, A17F + N85D + F90Q + H183N + S193E + N204G + M208L + N211M and A17F + F90Q + N105D + H183N + S193E + N204G + M208L + N211E. 如請求項1或2之酯酶，其中該酯酶至少包含M208N + N211M之取代的組合。The esterase of claim 1 or 2, wherein the esterase comprises at least a substitution combination of M208N + N211M. 如請求項1之酯酶，其中該酯酶包含至少一種選自以下之取代的組合或由具有一種選自以下之取代的組合的SEQ ID NO: 1中所示之胺基酸序列組成：M208N + N211M、H183Q + F187I + N204G + M208N + N211M + S193E、H183Q + F187I + N204G、H183Q + F187I + N204G + L13S + E158D、M208T + N204G + H183Q + F187I、M208T + N204G + H183Q + F187I + L13S + E158D、A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I、A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + H183Q + F187I + N204G、A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I + L13S + E158D。The esterase of claim 1, wherein the esterase comprises at least one combination of substitutions selected from the following or consists of the amino acid sequence shown in SEQ ID NO: 1 having a combination of substitutions selected from the following: M208N + N211M, H183Q + F187I + N204G + M208N + N211M + S193E, H183Q + F187I + N204G, H183Q + F187I + N204G + L13S + E158D, M208T + N204G + H183Q + F187I, M208T + N204G + H183Q + F187I + L13S + E158D, A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I, A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + H183Q + F187I + N204G, A17T + T27S + S48T + L82I + F90L + G92F + G135A + A140S + N143I + S145T + A149G + S164P+V167Q+S206T+T252S+I170V+M208T+N204G+H183Q+F187I+L13S+E158D. 一種酯酶變異體，其：(i)與SEQ ID NO: 1中所示之全長胺基酸序列具有至少97%、98%或99%一致性；(ii)與SEQ ID NO: 1之胺基酸序列相比，具有至少一種選自以下之胺基酸取代：H183Q/L/A/Y/N/D/E/W、R30G、A64V、A68N、T109S、L124G、S145G、L152M、K159R、S193L/Q、W228R/S、V242E、G7A、N9T、A24Q、Y26H、T27A、G39S、G46V、T50M、A64T、R73H、L74V、Y106F、R108L、V167L、I169M、P179N、I185V、F187L/Q、P192S、L227N、Q237L、L239M、R251S、N9E、V28I、A62T、F90Y/R/W/L、V115I、S181K、H183S、F187I、M208T、A246K、S66N、N243Y、S66T、F90C/V、P151V、A246R、A215E/D、D230E、L15K/R、T16K/R、Y4K/R、V219E/D、N143D、N253D、E182D、M208G、T16E、Q142E、Q237E、M208K、N105D、N85D、M208A、M208D，或至少一種選自以下之取代的組合：N204G + M208L + N211E、F90E + N204G + N211E、F90N + N204G + N211E、F90Q + N204G + N211E、F90R + N204G + N211E、F90W + N204G + N211E、F187I + N204G + N211E、S98R + E173Q、M208Q + N211M及M208N + N211M，其中參考SEQ ID NO: 1中所示之胺基酸序列對位置進行編號；(iii)與SEQ ID NO: 1之親本酯酶一樣至少具有胺基酸C240、C275、I170、G92、P213、E182、L13及E158；(iv)具有聚酯降解活性；及(v)與SEQ ID NO: 1之酯酶相比，在包含於6與10之間的pH下展現增加之熱穩定性及/或增加之聚酯降解活性。An esterase variant, which: (i) has at least 97%, 98% or 99% identity to the full-length amino acid sequence shown in SEQ ID NO: 1; (ii) has at least 97%, 98% or 99% identity to the full-length amino acid sequence shown in SEQ ID NO: 1, has at least one amino acid substitution selected from the following: H183Q/L/A/Y/N/D/E/W, R30G, A64V, A68N, T109S, L124G, S145G, L152M, K159R, S193L/Q, W228R/S, V242E, G7A, N9T, A24Q, Y26H, T27A, G39S, G46V, T50M, A64T, R73H, L74V, Y106F, R108L, V167L, I169M, P179N, I185V, F187L/Q, P192S, L227N, Q237L, L239M, R251S, N9E, V28I, A62T, F90Y/R/W/L, V115I, S181K, H183S, F187I, M208T, A246K, S66N, N243Y, S66T, F90C/V, P151V, A246R, A215E/D, D230E, L15K/R, T16K/R, Y4K/R, V219E/D, N143D, N253D, E182D, M208G, T16E, Q142E, Q237E, M208K, N105D, N85D, M208A, M208D, or a combination of at least one substitution selected from the following: N204G + M208L + N211E, F90E + N204G + N211E, F90N + N204G + N211E, F90Q + N204G + N211E, F90R + N204G + N211E, F90W + N204G + N211E, F187I + N204G + N211E, S98R + E173Q, M208Q + N211M and M208N + N211M, wherein the positions are numbered with reference to the amino acid sequence shown in SEQ ID NO: 1; (iii) with SEQ ID NO: (iv) having polyester degradation activity; and (v) exhibiting increased thermostability and/or increased polyester degradation activity at a pH comprised between 6 and 10 compared to the esterase of SEQ ID NO: 1. 如請求項16之酯酶，其中該酯酶包含至少一種選自以下之取代：N9E、V28I、A62T、F90Y、V115I、S181K、H183S、F187I、M208T、S66N、N243Y、E182D、M208G、T16E、Q142E、Q237E、M208K、N105D、N85D、M208A、M208D，較佳選自N9E、V28I、A62T、F90Y、V115I、S181K、H183S、F187I、M208T、S66N、N243Y、E182D、M208G、T16E、Q142E、N105D、N85D及M208A，更佳選自N9E、H183S、M208T、N243Y、M208G、T16E、Q142E、M208A及F187I。The esterase of claim 16, wherein the esterase comprises at least one substitution selected from the group consisting of N9E, V28I, A62T, F90Y, V115I, S181K, H183S, F187I, M208T, S66N, N243Y, E182D, M208G, T16E, Q142E, Q237E, M208K, N105D, N85D, M208A, M208D, preferably N9 E, V28I, A62T, F90Y, V115I, S181K, H183S, F187I, M208T, S66N, N243Y, E182D, M208G, T16E, Q142E, N105D, N85D and M208A, more preferably selected from N9E, H183S, M208T, N243Y, M208G, T16E, Q142E, M208A and F187I. 如請求項16之酯酶，其中該酯酶包含取代F187I，且進一步包含一種、兩種或更多種選自以下之取代：R30G、G39S、A62T、A64V、W69L、L124G、N162S、S181K、H183L/A/Y/Q/N/D/W/E/S、S193Q、N204G、M208T、A216C、Q237L、A246C、M208N、N211M、S193E、V200I及I170V，較佳其中該酯酶包含至少一種選自以下之取代的組合或由具有一種選自以下之取代的組合的SEQ ID NO: 1中所示之胺基酸序列組成：H183L + F187I、H183A + F187I、H183Y + F187I、H183Q + F187I、H183N + F187I、H183D + F187I、H183W + F187I、H183E + F187I、H183S + F187I、H183Q + F187I + N204G、N204G + H183Q + F187I + R30G、N204G + H183Q + F187I + G39S、N204G + H183Q + F187I + A62T、N204G + H183Q + F187I + A64V、N204G + H183Q + F187I + L124G、N204G + H183Q + F187I + N162S、N204G + H183Q + F187I + S193Q、N204G + H183Q + F187I + Q237L、M208T + N204G + H183Q + F187I、N204G + H183Q + F187I + N162S + S181K、N204G + H183Q + F187I + N162S + S181K + S193Q、N204G + H183Q + F187I + W69L + A216C + A246C、H183Q + F187I + N204G + M208N + N211M + S193E、M208T + N204G + H183Q + F187I + N211M + S193E、H183Q + F187I + N204G + N211M + S193E、H183Q + F187I + N204G + N211M + S193E + V200I + I170V、M208T + N204G + H183Q + F187I。The esterase of claim 16, wherein the esterase comprises substitution F187I and further comprises one, two or more substitutions selected from the group consisting of R30G, G39S, A62T, A64V, W69L, L124G, N162S, S181K, H183L/A/Y/Q/N/D/W/E/S, S193Q, N204G, M208T, A216C, Q237L, A246C, M208N, N211M, S193E, V200I and I170V, preferably wherein the esterase comprises at least one combination of substitutions selected from the group consisting of or consists of the amino acid sequence shown in SEQ ID NO: 1 having a combination of substitutions selected from the group consisting of H183L + F187I, H183A + F187I, H183Y + F187I, H183Q + F187I, H183N + F187I, H183D + F187I, H183W + F187I, H183E + F187I, H183S + F187I, H183Q + F187I + N204G, N204G + H183Q + F187I + R30G, N204G + H183Q + F187I + G39S, N204G + H183Q + F187I + A62T, N204G + H183Q + F187I + A64V, N204G + H183Q + F187I + L124G, N204G + H183Q + F187I + N162S, N204G + H183Q + F187I + S193Q, N204G + H183Q + F187I + Q237L, M208T + N204G + H183Q + F187I, N204G + H183Q + F187I + N162S + S181K, N204G + H183Q + F187I + N162S + S181K + S193Q, N204G + H183Q + F187I + W69L + A216C + A246C, H183Q + F187I + N204G + M208N + N211M + S193E, M208T + N204G + H183Q + F187I + N211M + S193E, H183Q + F187I + N204G + N211M + S193E, H183Q + F187I + N204G + N211M + S193E + V200I + I170V, M208T + N204G + H183Q + F187I. 如請求項16之酯酶，其中該酯酶包含取代M208T，且進一步包含至少一種選自A62T、S66N、H183Q、F187I、N204G、N211M、S193E、V200I、I170V之取代，較佳其中該酯酶至少包含選自以下之取代的組合或由具有一種選自以下之取代的組合的SEQ ID NO: 1中所示之胺基酸序列組成：A62T + S66N + M208T、M208T + N204G + H183Q + F187I、M208T + N204G + H183Q + F187I + N211M + S193E及M208T + N204G + H183Q + F187I，較佳選自A62T + S66N + M208T及M208T + N204G + H183Q + F187IM208T + N204G + H183Q + F187I。The esterase of claim 16, wherein the esterase comprises the substitution M208T and further comprises at least one substitution selected from A62T, S66N, H183Q, F187I, N204G, N211M, S193E, V200I, I170V, preferably wherein the esterase comprises at least a combination of substitutions selected from the following or consists of the amino acid sequence shown in SEQ ID NO: 1 having a combination of substitutions selected from the following: A62T + S66N + M208T, M208T + N204G + H183Q + F187I, M208T + N204G + H183Q + F187I + N211M + S193E and M208T + N204G + H183Q + F187I, preferably selected from A62T + S66N + M208T and M208T + N204G + H183Q + F187IM208T + N204G + H183Q + F187I. 如請求項16之酯酶，其中該酯酶至少包含N204G + H183Q + F187I之取代的組合，且視情況進一步包含至少一種選自R30G、G39S、A62T、A64V、W69L、L124G、N162S、S181K、S193Q、M208T、A216C、Q237L、A246C、M208N、N211M、S193E之取代，較佳其中該酯酶包含至少一種選自以下之取代的組合或由具有一種選自以下之取代的組合的SEQ ID NO: 1中所示之胺基酸序列組成：N204G + H183Q + F187I + R30G、N204G + H183Q + F187I + G39S、N204G + H183Q + F187I + A62T、N204G + H183Q + F187I + A64V、N204G + H183Q + F187I + L124G、N204G + H183Q + F187I + N162S、N204G + H183Q + F187I + S193Q、N204G + H183Q + F187I + Q237L、M208T + N204G + H183Q + F187I、N204G + H183Q + F187I + N162S + S181K、N204G + H183Q + F187I + N162S + S181K + S193Q、N204G + H183Q + F187I + W69L + A216C + A246C、H183Q + F187I + N204G + M208N + N211M + S193E、M208T + N204G + H183Q + F187I + N211M + S193E，更佳選自N204G + H183Q + F187I + R30G、N204G + H183Q + F187I + G39S、N204G + H183Q + F187I + A62T、N204G + H183Q + F187I + A64V、N204G + H183Q + F187I + L124G、N204G + H183Q + F187I + N162S、N204G + H183Q + F187I + S193Q、N204G + H183Q + F187I + Q237L、M208T + N204G + H183Q + F187I、N204G + H183Q + F187I + N162S + S181K、N204G + H183Q + F187I + N162S + S181K + S193Q及N204G + H183Q + F187I + W69L + A216C + A246C、H183Q + F187I + N204G + M208N + N211M + S193E、H183Q + F187I + N204G + M208T + N211M + S193E、H183Q + F187I + N204G + N211M + S193E + V200I + I170V，甚至更佳選自N204G + H183Q + F187I + A62T、N204G + H183Q + F187I + A64V、N204G + H183Q + F187I + L124G、N204G + H183Q + F187I + N162S、N204G + H183Q + F187I + S193Q、N204G + H183Q + F187I + Q237L、M208T + N204G + H183Q + F187I、N204G + H183Q + F187I + N162S + S181K + S193Q、H183Q + F187I + N204G + M208N + N211M + S193E及H183Q + F187I + N204G + M208T + N211M + S193E。The esterase of claim 16, wherein the esterase comprises at least a combination of substitutions of N204G + H183Q + F187I, and optionally further comprises at least one substitution selected from R30G, G39S, A62T, A64V, W69L, L124G, N162S, S181K, S193Q, M208T, A216C, Q237L, A246C, M208N, N211M, S193E, preferably wherein the esterase comprises at least one combination of substitutions selected from the following or consists of the amino acid sequence shown in SEQ ID NO: 1 having a combination of substitutions selected from the following: N204G + H183Q + F187I + R30G, N204G + H183Q + F187I + G39S, N204G + H183Q + F187I + A62T, N204G + H183Q + F187I + A64V, N204G + H183Q + F187I + L124G, N204G + H183Q + F187I + N162S, N204G + H183Q + F187I + S193Q, N204G + H183Q + F187I + Q237L, M208T + N204G + H183Q + F187I, N204G + H183Q + F187I + N162S + S181K, N204G + H183Q + F187I + N162S + S181K + S193Q, N204G + H183Q + F187I + W69L + A216C + A246C, H183Q + F187I + N204G + M208N + N211M + S193E, M208T + N204G + H183Q + F187I + N211M + S193E, preferably N204G + H183Q + F187I + R30G, N204G + H183Q + F187I + G39S, N204G + H183Q + F187I + A62T, N204G + H183Q + F187I + A64V, N204G + H183Q + F187I + L124G, N204G + H183Q + F187I + N162S, N204G + H183Q + F187I + S193Q, N204G + H183Q + F187I + Q237L, M208T + N204G + H183Q + F187I, N204G + H183Q + F187I + N162S + S181K, N204G + H183Q + F187I + N162S + S181K + S193Q and N204G + H183Q + F187I + W69L + A216C + A246C, H183Q + F187I + N204G + M208N + N211M + S193E, H183Q + F187I + N204G + M208T + N211M + S193E, H183Q + F187I + N204G + N211M + S193E + V200I + I170V, or even better, choose from N204G + H183Q + F187I + A62T, N204G + H183Q + F187I + A64V, N204G + H183Q + F187I + L124G, N204G + H183Q + F187I + N162S, N204G + H183Q + F187I + S193Q, N204G + H183Q + F187I + Q237L, M208T + N204G + H183Q + F187I, N204G + H183Q + F187I + N162S + S181K + S193Q, H183Q + F187I + N204G + M208N + N211M + S193E and H183Q + F187I + N204G + M208T + N211M + S193E. 如請求項16之酯酶，其中該酯酶至少包含M208N + N211M之取代的組合。The esterase of claim 16, wherein the esterase comprises at least a substitution combination of M208N + N211M. 如請求項1至21中任一項之酯酶，其中該酯酶與SEQ ID NO: 1之親本酯酶一樣展現至少一種選自S130、D175、H207、C240或C275之胺基酸殘基。The esterase of any one of claims 1 to 21, wherein the esterase exhibits at least one amino acid residue selected from S130, D175, H207, C240 or C275 like the parent esterase of SEQ ID NO: 1. 一種酯酶變異體，其：(i)與SEQ ID NO: 2中所示之全長胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%一致性；(ii)與SEQ ID NO: 2之胺基酸序列相比，包含至少一種選自以下之胺基酸取代：H183Q/L/A/Y/N/D/E/W、R30G、A64V、A68N、T109S、L124G、T145G、L152M、E158A、K159R、S193L/Q、W228R/S、V242E、G7A、N9T、A24Q、Y26H、T27S27A、G39S、G46V、T50M、A64T、R73H、L74V、Y106F、R108L、Q167L、I169M、P179N、I185V、F187L/Q、P192S、P213A、L227N、Q237L、L239M、R251S、S66T、L90C/V、F92K、P151V、A246R、A215E/D、D230E、L15K/R、I143D、N253D及V200I，或至少一種選自以下之取代的組合：N204G + M208L + N211E、L90E + N204G + N211E、L90N + N204G + N211E、L90Q + N204G + N211E、L90R + N204G + N211E、L90W + N204G + N211E、F187I + N204G + N211E及S98R + E173Q、M208A + N211M、M208G + N211M、M208K + N211M、M208N + N211M、M208P + N211M、M208Q + N211M、M208H + N211M及M208T + N211M，其中參考SEQ ID NO: 2中所示之胺基酸序列對位置進行編號；(iii)具有聚酯降解活性；及(iv)與SEQ ID NO: 2之酯酶相比，在包含於6與10之間的pH下展現增加之熱穩定性及/或增加之聚酯降解活性。An esterase variant which: (i) has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the full-length amino acid sequence shown in SEQ ID NO: 2; (ii) has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the full-length amino acid sequence shown in SEQ ID NO: 2, comprising at least one amino acid substitution selected from the following: H183Q/L/A/Y/N/D/E/W, R30G, A64V, A68N, T109S, L124G, T145G, L152M, E158A, K159R, S193L/Q, W228R/S, V242E, G7A, N9T, A24Q, Y26H, T27S27A, G39S, G46V, T50M, A64T, R73H, L 74V, Y106F, R108L, Q167L, I169M, P179N, I185V, F187L/Q, P192S, P213A, L227N, Q237L, L239M, R251S, S66T, L90C/V, F92K, P151V, A246R, A215E/D, D230E, L15K/R, I143D, N253D, and V200I, or a combination of at least one substitution selected from the following: N204G + M208L + N211E, L90E + N204G + N211E, L90N + N204G + N211E, L90Q + N204G + N211E, L90R + N204G + N211E, L90W + N204G + N211E, F187I + N204G + N211E and S98R + E173Q, M208A + N211M, M208G + N211M, M208K + N211M, M208N + N211M, M208P + N211M, M208Q + N211M, M208H + N211M and M208T + N211M, with reference to SEQ ID NO: (iii) having polyester degradation activity; and (iv) exhibiting increased thermostability and/or increased polyester degradation activity at a pH comprised between 6 and 10 compared to the esterase of SEQ ID NO: 2. 如請求項23之酯酶，其中該酯酶包含至少一種選自A64V、S66N、H183N、I143D、N253D，較佳選自H183N及S66N之取代。The esterase of claim 23, wherein the esterase comprises at least one substitution selected from A64V, S66N, H183N, I143D, N253D, preferably selected from H183N and S66N. 如請求項23之酯酶，其中該酯酶包含至少一種選自H183Q/L/A/Y/N/D/E/W之取代，且進一步包含至少一種選自以下之取代：F187I、L90Q、I145D、L90Q、M208A、M208L、M208M、M208N、M208T、N105D、N122D、N204G、N211M、N253D、N85D、Q142E、Q237E、Q258E、S193E、S98R、T17F及V200I，較佳其中該酯酶包含至少一種來自以下之取代的組合或由具有一種選自以下之取代的組合的SEQ ID NO: 2中所示之胺基酸序列組成：H183D + F187I、H183E + F187I、H183N + F187I、M208T + N204G + H183Q + F187I + N211M + S193E、H183Q + F187I + N204G + N211M + S193E + V200I、T17F + L90Q + H183N + N204G + M208L + N211M、T17F + L90Q + H183N + S193E + N204G + M208L + N211M、H183Q + F187I + N204G + M208N + N211M  + S193E + V200I、M208T + N204G + H183Q + F187I + N211M + S193E + V200I、T17F + N85D + L90Q + H183N + S193E + N204G + M208L + N211M、T17F + L90Q + N122D + H183N + S193E + N204G + M208L + N211M、T17F + L90Q + Q142E + H183N + S193E + N204G + M208L + N211M、T17F + L90Q + I145D + H183N + S193E + N204G + M208L + N211M、T17F + L90Q + H183N + S193E + N204G + M208L + N211M + Q237E、T17F + L90Q + H183N + S193E + N204G + M208L + N211M + N253D、T17F + L90Q + H183N + S193E + N204G + M208L + N211M + Q258E、T17F + L90Q + H183N + S193E + N204G + M208L + N211M + V200I、T17F + L90Q + S98R + N105D + H183N + S193E + N204G + N211M、T17F + L90Q + H183N + S193E + N204G + M208N + N211M + V200I、T17F + L90Q + H183N + S193E + N204G + M208A + N211M + V200I、T17F + L90Q + H183N + S193E + N204G + M208M + N211M + V200I、H183Q + F187I + N204G + M208N + N211M + S193E、H183Q + F187I + N204G、H183Q + F187I + N204G + M208T、H183Q + F187I + N204G + M208T + L13S + E158D、T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + M208T + L13S + E158D、T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + M208T、T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G、T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + L13S + E158D，較佳選自H183D + F187I、H183E + F187I、H183N + F187I、M208T + N204G + H183Q + F187I + N211M + S193E、H183Q + F187I + N204G + N211M + S193E + V200I、T17F + L90Q + H183N + N204G + M208L + N211M、T17F + L90Q + H183N + S193E + N204G + M208L + N211M、H183Q + F187I + N204G + M208N + N211M + S193E + V200I、M208T + N204G + H183Q + F187I + N211M + S193E + V200I、T17F + N85D + L90Q + H183N + S193E + N204G + M208L + N211M、T17F + L90Q + N122D + H183N + S193E + N204G + M208L + N211M、T17F + L90Q + Q142E + H183N + S193E + N204G + M208L + N211M、T17F + L90Q + I145D + H183N + S193E + N204G + M208L + N211M、T17F + L90Q + H183N + S193E + N204G + M208L + N211M + Q237E、T17F + L90Q + H183N + S193E + N204G + M208L + N211M + N253D、T17F + L90Q + H183N + S193E + N204G + M208L + N211M + Q258E、T17F + L90Q + H183N + S193E + N204G + M208L + N211M + V200I、T17F + L90Q + S98R + N105D + H183N + S193E + N204G + N211M、T17F + L90Q + H183N + S193E + N204G + M208N + N211M + V200I、T17F + L90Q + H183N + S193E + N204G + M208A + N211M + V200I、T17F + L90Q + H183N + S193E + N204G + M208M + N211M + V200I。The esterase of claim 23, wherein the esterase comprises at least one substitution selected from H183Q/L/A/Y/N/D/E/W, and further comprises at least one substitution selected from the following: F187I, L90Q, I145D, L90Q, M208A, M208L, M208M, M208N, M208T, N105D, N122D, N204G, N211M, N253D, N85D, Q142E, Q237E, Q258E, S193E, S98R, T17F and V200I, preferably wherein the esterase comprises at least one combination of substitutions from the following or consists of the amino acid sequence shown in SEQ ID NO: 2 having a combination of substitutions selected from the following: H183D + F187I, H183E + F187I, H183N + F187I, M208T + N204G + H183Q + F187I + N211M + S193E, H183Q + F187I + N204G + N211M + S193E + V200I, T17F + L90Q + H183N + N204G + M208L + N211M, T17F + L90Q + H183N + S193E + N204G + M208L + N211M, H183Q + F187I + N204G + M208N + N211M + S193E + V200I, M208T + N204G + H183Q + F187I + N211M + S193E + V200I, T17F + N85D + L90Q + H183N + S193E + N204G + M208L + N211M, T17F + L90Q + N122D + H183N + S193E + N204G + M208L + N211M, T17F + L90Q + Q142E + H183N + S193E + N204G + M208L + N211M, T17F + L90Q + I145D + H183N + S193E + N204G + M208L + N211M, T17F + L90Q + H183N + S193E + N204G + M208L + N211M + Q237E, T17F + L90Q + H183N + S193E + N204G + M208L + N211M + N253D, T17F + L90Q + H183N + S193E + N204G + M208L + N211M + Q258E, T17F + L90Q + H183N + S193E + N204G + M208L + N211M + V200I, T17F + L90Q + S98R + N105D + H183N + S193E + N204G + N211M, T17F + L90Q + H183N + S193E + N204G + M208N + N211M + V200I, T17F + L90Q + H183N + S193E + N204G + M208A + N211M + V200I, T17F + L90Q + H183N + S193E + N204G + M208M + N211M + V200I, H183Q + F187I + N204G + M208N + N211M + S193E, H183Q + F187I + N204G, H183Q + F187I + N204G + M208T, H183Q + F187I + N204G + M208T + L13S + E158D, T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + M208T + L13S + E158D, T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + M208T, T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G, T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + L13S + E158D, preferably H183D + F187I, H183E + F187I, H183N + F187I, M208T + N204G + H183Q + F187I + N211M + S193E, H183Q + F187I + N204G + N211M + S193E + V200I, T17F + L90Q + H183N + N204G + M208L + N211M, T17F + L90Q + H183N + S193E + N204G + M208L + N211M, H183Q + F187I + N204G + M208N + N211M + S193E + V200I, M208T + N204G + H183Q + F187I + N211M + S193E + V200I, T17F + N85D + L90Q + H183N + S193E + N204G+M208L+ N211M, T17F + L90Q + N122D + H183N + S193E + N204G + M208L + N211M, T17F + L90Q + Q142E + H183N + S193E + N204G + M208L + N211M, T17F + L90Q + I145D + H183N + S193E + N204G + M208L + N211M, T17F + L90Q + H183N + S193E + N204G + M208L + N211M + Q237E, T17F + L90Q + H183N + S193E + N204G + M208L + N211M + N253D, T17F + L90Q + H183N + S193E + N204G + M208L + N211M + Q258E, T17F + L90Q + H183N + S193E + N204G + M208L + N211M + V200I, T17F + L90Q + S98R + N105D + H183N + S193E + N204G + N211M, T17F + L90Q + H183N + S193E + N204G + M208N + N211M + V200I, T17F + L90Q + H183N + S193E + N204G + M208A + N211M + V200I, T17F + L90Q + H183N + S193E + N204G + M208M + N211M + V200I. 如請求項25之酯酶，其中該酯酶包含取代H183Q，且進一步包含至少一種、兩種、三種、四種、五種、六種或更多種選自F187I、M208N、M208T、N204G、N211M、S193E、V200I之取代，較佳其中該酯酶包含至少一種選自以下之取代的組合或由具有一種選自以下之取代的組合的SEQ ID NO: 2中所示之胺基酸序列組成：H183Q + F187I + N204G + M208T + N211M + S193E、H183Q + F187I + N204G + M208N + N211M + S193E + V200I、H183Q + F187I + N204G + M208T + N211M + S193E + V200I、H183Q + F187I + N204G + M208N + N211M + S193E、H183Q + F187I + N204G、H183Q + F187I + N204G + M208T、H183Q + F187I + N204G + M208T + L13S + E158D、T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + M208T + L13S + E158D、T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + M208T、T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G、T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + L13S + E158D，較佳選自H183Q + F187I + N204G + M208T + N211M + S193E、H183Q + F187I + N204G + M208N + N211M + S193E + V200I、H183Q + F187I + N204G + M208T + N211M  + S193E + V200I。The esterase of claim 25, wherein the esterase comprises the substitution H183Q and further comprises at least one, two, three, four, five, six or more substitutions selected from F187I, M208N, M208T, N204G, N211M, S193E, V200I, preferably wherein the esterase comprises at least one combination of substitutions selected from the following or consists of the amino acid sequence shown in SEQ ID NO: 2 having a combination of substitutions selected from the following. N211M + S193E + V200I, H183Q + F187I + N204G + M208N + N211M + S193E, H183Q + F187I + N204G, H183Q + F187I + N204G + M208T, H183Q + F187I + N204G + M208T + L13S + E158D, T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + M208T + L13S + E158D, T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + M208T, T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G, T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + L13S + E158D, better choices are H183Q + F187I + N204G + M208T + N211M + S193E, H183Q + F187I + N204G + M208N + N211M + S193E + V200I, H183Q + F187I + N204G + M208T + N211M + S193E + V200I. 如請求項25或26之酯酶，其中該酯酶至少包含N204G + H183Q + F187I之取代的組合，且視情況進一步包含至少一種選自M208T、N211M、S193E、V200I、M208N、L13S、E158D之取代，較佳其中該酯酶包含至少一種選自以下之取代的組合或由具有一種選自以下之取代的組合的SEQ ID NO: 2中所示之胺基酸序列組成：M208T + N204G + H183Q + F187I + N211M + S193E、H183Q + F187I + N204G + M208N + N211M + S193E + V200I、M208T + N204G + H183Q + F187I + N211M + S193E + V200I、H183Q + F187I + N204G + M208N + N211M + S193E、H183Q + F187I + N204G、H183Q + F187I + N204G + M208T、H183Q + F187I + N204G + M208T + L13S + E158D、T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + M208T + L13S + E158D、T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + M208T、T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G、T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + L13S + E158D，更佳選自M208T + N204G + H183Q + F187I + N211M + S193E、H183Q + F187I + N204G + M208N + N211M + S193E + V200I、M208T + N204G + H183Q + F187I + N211M + S193E + V200I。The esterase of claim 25 or 26, wherein the esterase comprises at least a combination of substitutions of N204G + H183Q + F187I, and optionally further comprises at least one substitution selected from M208T, N211M, S193E, V200I, M208N, L13S, E158D, preferably wherein the esterase comprises at least one combination of substitutions selected from the following or consists of the amino acid sequence shown in SEQ ID NO: 2 having a combination of substitutions selected from the following: M208T + N204G + H183Q + F187I + N211M + S193E, H183Q + F187I + N204G + M208N + N211M + S193E + V200I, M208T + N204G + H183Q + F187I + N211M + S193E + V200I, H183Q + F187I + N204G + M208N + N211M + S193E, H183Q + F187I + N204G, H183Q + F187I + N204G + M208T, H183Q + F187I + N204G + M208T + L13S + E158D, T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + M208T + L13S + E158D, T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + M208T, T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G, T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + L13S + E158D, better choices are M208T + N204G + H183Q + F187I + N211M + S193E, H183Q + F187I + N204G + M208N + N211M + S193E + V200I, M208T + N204G + H183Q + F187I + N211M + S193E + V200I. 如請求項25之酯酶，其中該酯酶包含取代H183N，且進一步包含至少一種、兩種、三種、四種、五種、六種、七種或更多種選自F187I、L90Q、I145D、L90Q、M208A、M208L、M208N、N105D、N122D、N204G、N211M、N253D、N85D、Q142E、Q237E、Q258E、S193E、S98R、T17F、V200I之取代，較佳其中該酯酶包含至少一種選自以下之取代的組合或由具有一種選自以下之取代的組合的SEQ ID NO: 2中所示之胺基酸序列組成：H183N + F187I、T17F + L90Q + H183N + N204G + M208L + N211M、T17F + L90Q + H183N + S193E + N204G + M208L + N211M、T17F + N85D + L90Q + H183N + S193E + N204G + M208L + N211M、T17F + L90Q + N122D + H183N + S193E + N204G + M208L + N211M、T17F + L90Q + Q142E + H183N + S193E + N204G + M208L + N211M、T17F + L90Q + I145D + H183N + S193E + N204G + M208L + N211M、T17F + L90Q + H183N + S193E + N204G + M208L + N211M + Q237E、T17F + L90Q + H183N + S193E + N204G + M208L + N211M + N253D、T17F + L90Q + H183N + S193E + N204G + M208L + N211M + Q258E、T17F + L90Q + H183N + S193E + N204G + M208L + N211M + V200I、T17F + L90Q + S98R + N105D + H183N + S193E + N204G + N211M、T17F + L90Q + H183N + S193E + N204G + M208N + N211M + V200I、T17F + L90Q + H183N + S193E + N204G + M208A + N211M + V200I、T17F + L90Q + H183N + S193E + N204G + M208M + N211M + V200I。The esterase of claim 25, wherein the esterase comprises the substitution H183N and further comprises at least one, two, three, four, five, six, seven or more substitutions selected from F187I, L90Q, I145D, L90Q, M208A, M208L, M208N, N105D, N122D, N204G, N211M, N253D, N85D, Q142E, Q237E, Q258E, S193E, S98R, T17F, V200I, preferably wherein the esterase comprises at least one combination of substitutions selected from the following or consists of the amino acid sequence shown in SEQ ID NO: 2 having a combination of substitutions selected from the following: H183N + F187I, T17F + L90Q + H183N + N204G + M208L + N211M, T17F + L90Q + H183N + S193E + N204G + M208L + N211M, T17F + N85D + L90Q + H183N + S193E + N204G + M208L + N211M, T17F + L90Q + N122D + H183N + S193E + N204G + M208L + N211M, T17F + L90Q + Q142E + H183N + S193E + N204G + M208L + N211M, T17F + L90Q + I145D + H183N + S193E + N204G + M208L + N211M, T17F + L90Q + H183N + S193E + N204G + M208L + N211M + Q237E, T17F + L90Q + H183N + S193E + N204G + M208L + N211M + N253D, T17F + L90Q + H183N + S193E + N204G + M208L + N211M + Q258E, T17F + L90Q + H183N + S193E + N204G + M208L + N211M + V200I, T17F + L90Q + S98R + N105D + H183N + S193E + N204G + N211M, T17F + L90Q + H183N + S193E + N204G + M208N + N211M + V200I, T17F + L90Q + H183N + S193E + N204G + M208A + N211M + V200I, T17F + L90Q + H183N + S193E + N204G + M208M + N211M + V200I. 如請求項25之酯酶，其中該酯酶至少包含M208N + N211M的組合。The esterase of claim 25, wherein the esterase comprises at least the combination of M208N + N211M. 如請求項25之酯酶，其中該酯酶包含至少一種選自以下之取代或取代之組合：M208N + N211M、H183Q + F187I + N204G + M208N + N211M + S193E、H183Q + F187I + N204G、H183Q + F187I + N204G + M208T、H183Q + F187I + N204G + M208T + L13S + E158D、T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + M208T + L13S + E158D、T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + M208T、T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G、T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + L13S + E158D。The esterase of claim 25, wherein the esterase comprises at least one substitution or combination of substitutions selected from the group consisting of M208N + N211M, H183Q + F187I + N204G + M208N + N211M + S193E, H183Q + F187I + N204G, H183Q + F187I + N204G + M208T, H183Q + F187I + N204G + M208T + L13S + E158D, T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + M208T + L13S + E158D, T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + M208T, T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G, T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G+L13S+E158D. 一種酯酶變異體，其：(i)與SEQ ID NO: 2中所示之全長胺基酸序列具有至少97%、98%或99%一致性；(ii)與SEQ ID NO: 2之胺基酸序列相比，具有至少一種選自L90W、M208A、M208N、M208S、N122D、Q237E、Q258E及S212T之胺基酸取代，或至少M208I + N211M之取代的組合，其中參考SEQ ID NO: 2中所示之胺基酸序列對位置進行編號；(iii)與SEQ ID NO: 2之親本酯酶一樣至少具有胺基酸C240、C275、V170、F92、P213、E182、L13及E158；(iv)具有聚酯降解活性；及(v)與SEQ ID NO: 2之酯酶相比，在包含於6與10之間的pH下展現增加之熱穩定性及/或增加之聚酯降解活性。An esterase variant, which: (i) has at least 97%, 98% or 99% identity to the full-length amino acid sequence shown in SEQ ID NO: 2; (ii) compared with the amino acid sequence of SEQ ID NO: 2, has at least one amino acid substitution selected from L90W, M208A, M208N, M208S, N122D, Q237E, Q258E and S212T, or a combination of at least M208I + N211M substitutions, wherein the positions are numbered with reference to the amino acid sequence shown in SEQ ID NO: 2; (iii) has at least amino acids C240, C275, V170, F92, P213, E182, L13 and E158 as the parent esterase of SEQ ID NO: 2; (iv) has polyester degradation activity; and (v) has the same amino acid sequence as SEQ ID NO: 2, exhibiting increased thermostability and/or increased polyester degradation activity at a pH comprised between 6 and 10. 如請求項31之酯酶，其中該酯酶至少包含取代M208N，且進一步包含至少一種選自N211M、H183Q、F187I、N204G、S193E、V200I、T17F、L90Q及H183N之取代，較佳其中該酯酶包含至少一種選自以下之取代的組合或由具有一種選自以下之取代的組合的SEQ ID NO: 2中所示之胺基酸序列組成：M208N + N211M、H183Q + F187I + N204G + M208N + N211M  + S193E + V200I及T17F + L90Q + H183N + S193E + N204G + M208N + N211M + V200I。An esterase as claimed in claim 31, wherein the esterase comprises at least the substitution M208N and further comprises at least one substitution selected from N211M, H183Q, F187I, N204G, S193E, V200I, T17F, L90Q and H183N, preferably wherein the esterase comprises at least one combination of substitutions selected from the following or consists of the amino acid sequence shown in SEQ ID NO: 2 having a combination of substitutions selected from the following: M208N + N211M, H183Q + F187I + N204G + M208N + N211M + S193E + V200I and T17F + L90Q + H183N + S193E + N204G + M208N + N211M + V200I. 如請求項23至32中任一項之酯酶，其中該酯酶與SEQ ID NO: 2之親本酯酶一樣展現至少一種選自S130、D175、H207、C240或C275之胺基酸殘基。The esterase of any one of claims 23 to 32, wherein the esterase exhibits at least one amino acid residue selected from S130, D175, H207, C240 or C275 like the parent esterase of SEQ ID NO: 2. 一種使聚酯降解之方法，其包含 a.  使該聚酯與如請求項1至33中任一項之酯酶或包含且表現編碼如請求項1至33中任一項之酯酶之核酸的宿主細胞或其包含該酯酶之萃取物或包含如請求項1至33中任一項之酯酶之組合物接觸；及視情況 b.  回收單體及/或寡聚物。 A method for degrading polyester, comprising: a. contacting the polyester with an esterase as claimed in any one of claims 1 to 33 or a host cell containing and expressing a nucleic acid encoding an esterase as claimed in any one of claims 1 to 33 or an extract thereof containing the esterase or a composition containing an esterase as claimed in any one of claims 1 to 33; and, as appropriate, b. recovering monomers and/or oligomers. 一種使含聚酯材料中之至少一種聚酯降解之方法，其包含 a.  使該含聚酯材料與如請求項1至33中任一項之酯酶或包含且表現編碼如請求項1至33中任一項之酯酶之核酸的宿主細胞或其包含該酯酶之萃取物或包含如請求項1至33中任一項之酯酶之組合物接觸；及視情況 b.  回收單體及/或寡聚物。 A method for degrading at least one polyester in a polyester-containing material, comprising: a. contacting the polyester-containing material with an esterase as described in any one of claims 1 to 33 or a host cell containing and expressing a nucleic acid encoding an esterase as described in any one of claims 1 to 33 or an extract thereof containing the esterase or a composition containing the esterase as described in any one of claims 1 to 33; and, as appropriate, b. recovering monomers and/or oligomers. 如請求項34或35之方法，其中該聚酯係選自聚對苯二甲酸伸乙酯(PET)、聚對苯二甲酸伸丙酯(PTT)、聚對苯二甲酸伸丁酯(PBT)、聚異山梨醇對苯二甲酸伸乙酯(PEIT)、聚乳酸(PLA)、聚羥基烷酸酯(PHA)、聚丁二酸伸丁酯(PBS)、聚丁二酸己二酸伸丁酯(PBSA)、聚己二酸對苯二甲酸伸丁酯(PBAT)、聚呋喃酸伸乙酯(PEF)、聚己內酯(PCL)、聚(己二酸伸乙酯) (PEA)、聚萘二甲酸伸乙酯(PEN)及此等材料之摻合物/混合物，較佳為聚對苯二甲酸伸乙酯。A method as claimed in claim 34 or 35, wherein the polyester is selected from polyethylene terephthalate (PET), polypropylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), polycaprolactone (PCL), poly(ethylene adipate) (PEA), polyethylene naphthalate (PEN) and blends/mixtures of these materials, preferably polyethylene terephthalate. 一種含聚酯材料，其含有如請求項1至33中任一項之酯酶或包含且表現編碼如請求項1至33中任一項之酯酶之核酸的宿主細胞或其包含該酯酶之萃取物或包含如請求項1至33中任一項之酯酶之組合物。A polyester-containing material, comprising an esterase as claimed in any one of claims 1 to 33, or a host cell comprising and expressing a nucleic acid encoding an esterase as claimed in any one of claims 1 to 33, or an extract thereof comprising the esterase, or a composition comprising the esterase as claimed in any one of claims 1 to 33. 一種清潔劑組合物，其包含如請求項1至33中任一項之酯酶或包含且表現編碼如請求項1至33中任一項之酯酶之核酸的宿主細胞，或其包含該酯酶之萃取物，或包含如請求項1至33中任一項之酯酶之組合物。A cleaning agent composition comprising the esterase of any one of claims 1 to 33 or a host cell comprising and expressing a nucleic acid encoding the esterase of any one of claims 1 to 33, or an extract thereof comprising the esterase, or a composition comprising the esterase of any one of claims 1 to 33.
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