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WO2024236008A2 - Novel esterases and uses thereof - Google Patents

Novel esterases and uses thereof Download PDF

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Publication number
WO2024236008A2
WO2024236008A2 PCT/EP2024/063319 EP2024063319W WO2024236008A2 WO 2024236008 A2 WO2024236008 A2 WO 2024236008A2 EP 2024063319 W EP2024063319 W EP 2024063319W WO 2024236008 A2 WO2024236008 A2 WO 2024236008A2
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WIPO (PCT)
Prior art keywords
esterase
seq
combination
amino acid
substitution
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PCT/EP2024/063319
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French (fr)
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WO2024236008A3 (en
Inventor
Isabelle ANDRÉ
Grégory ARNAL
Nicolas Chabot
Marc GUEROULT
Alain Marty
Alexandra TAUZIN
Vincent TOURNIER
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Carbios SA
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Carbios SA
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Publication of WO2024236008A3 publication Critical patent/WO2024236008A3/en
Anticipated expiration legal-status Critical
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J11/00—Recovery or working-up of waste materials
    • C08J11/04—Recovery or working-up of waste materials of polymers
    • C08J11/10—Recovery or working-up of waste materials of polymers by chemically breaking down the molecular chains of polymers or breaking of crosslinks, e.g. devulcanisation
    • C08J11/105—Recovery or working-up of waste materials of polymers by chemically breaking down the molecular chains of polymers or breaking of crosslinks, e.g. devulcanisation by treatment with enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • 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)
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • 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

Definitions

  • the present invention relates to novel esterases, more particularly to esterases having improved degrading activity and/or improved thermostability compared to a parent esterase at a pH comprised between 6 and 10, preferably at a pH comprised between 7 and 9.
  • the present invention also relates to uses of said novel esterases for degrading polyester containing material, such as plastic products.
  • the esterases of the invention are particularly suited to degrade polyethylene terephthalate, and polyethylene terephthalate containing material.
  • Esterases are able to catalyze the hydrolysis of a variety of polymers, including polyesters.
  • esterases have shown promising effects in a number of industrial applications, including detergents for dishwashing and laundry applications, degrading enzymes for processing biomass and food, biocatalysts in detoxification of environmental pollutants or for the treatment of polyester fabrics in the textile industry.
  • the use of esterases as degrading enzymes for hydrolyzing polyethylene terephthalate (PET) is of particular interest. Indeed, PET is used in a large number of technical fields, such as in the manufacture of clothes, carpets, or in the form of a thermoset resin for the manufacture of packaging or automobile plastics, etc., so that PET accumulation in landfills becomes an increasing ecological problem.
  • polyesters and particularly of PET
  • enzymes may accelerate hydrolysis of polyester containing material, and more particularly of plastic and textile products, even up to the monomer level.
  • hydrolysate i.e., monomers and oligomers
  • esterases have been identified as candidate degrading enzymes for polyesters, and some variants of such esterases have been developed.
  • cutinases also known as cutin hydrolases (EC 3.1.1.74)
  • Cutinases have been identified from various fungi (P.E. Kolattukudy in "Lipases", Ed. B. Borg- strom and H.L. Brockman, Elsevier 1984, 471-504), bacteria and plant pollen.
  • metagenomics approaches have led to identification of additional esterases. For instance, a metagenome- derived cutinase (as described in Sulaiman et al., Appl Environ Microbiol.
  • the present invention provides new variants of esterases exhibiting increased polyester degrading activity and/or increased thermostability at pH between 6 and 10 compared to a parent esterase, having the amino acid sequence as set forth in SEQ ID NO: 1.
  • esterases of the present invention are particularly useful in processes for degrading plastic products under basic conditions, more particularly plastic products containing PET.
  • 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 set forth in SEQ ID NO: 1, (ii) has at least one amino acid substitution selected from 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, H69M, P179N, 1185 V, F187L/Q, P192S, P213A, L227N, Q237L
  • the esterase comprises at least one amino acid substitution or at least one combination of substitutions selected from H183Q/L/A/Y/N/DZE/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, H85V, F187L/Q, P192S, P213A, L227N, Q237L, L239M, R251S, S66T, F90C/V, P151V, A246R, A215E/D, D230E, L15K/R, N253D, N204G + M208L + N211E, F
  • esterase variant which (i) has at least 97%, 98% or 99% identity to the full length amino acid sequence set forth in SEQ ID NO: 1, (ii) has at least one amino acid substitution selected from 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, VI 151,
  • 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 set forth in SEQ ID NO:2, (ii) comprises at least one amino acid substitution selected from 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, L
  • the present invention also relates to an expression cassette or an expression vector comprising said nucleic acid, and to a host cell comprising said nucleic acid, expression cassette or vector.
  • the present invention also provides a composition comprising an esterase of the present invention, a host cell of the present invention, or extract thereof.
  • At least step a) is performed in basic conditions, particularly at a pH between 6 and 10, preferably at a pH between 7 and 9.
  • the invention provides a method of degrading PET, comprising contacting PET with at least one esterase of the invention, and optionally recovering monomers and/or oligomers of PET.
  • at least the step of contacting PET with said esterase of the invention is performed in basic conditions, particularly at a pH between 6 and 10, preferably at a pH between 7 and 9.
  • the invention also relates to the use of an esterase of the invention for degrading PET or a plastic product containing PET.
  • said use is performed in basic conditions, particularly at a pH between 6 and 10, preferably at a pH between 7 and 9.
  • the present invention also relates to a polyester containing material in which an esterase or a host cell or a composition of the invention is included.
  • the present invention also relates to a detergent composition comprising the esterase or host cell according to the invention or a composition comprising an esterase of the present invention.
  • peptide refers to a chain of amino acids linked by peptide bonds, regardless of the number of amino acids forming said chain.
  • the amino acids are herein represented by their one-letter or three-letters code according to the following nomenclature: A: alanine (Ala); C: cysteine (Cys); D: aspartic acid (Asp); E: glutamic acid (Glu); F: phenylalanine (Phe); G: glycine (Gly); H: histidine (His); I: isoleucine (He); K: lysine (Lys); L: leucine (Leu); M: methionine (Met); N: asparagine (Asn); P: proline (Pro); Q: glutamine (Gin); R: arginine (Arg); S: serine (Ser); T: threonine (Thr); V: valine (Vai); W: tryptophan
  • esterase refers to an enzyme which belongs to a class of hydrolases classified as EC 3.1.1 according to Enzyme Nomenclature that catalyzes the hydrolysis of esters into an acid and an alcohol.
  • cutinase or “cutin hydrolase” refers to the esterases classified as EC 3.1.1.74 according to Enzyme Nomenclature that are able to catalyse the chemical reaction of production of cutin monomers from cutin and water.
  • parent protein refers to the esterase having the amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO:2.
  • mutant and variant refer to polypeptides derived from SEQ ID NO: 1 or SEQ ID NO:2 and comprising at least one modification or alteration, i.e., a substitution, insertion, and/or deletion, at one or more (e.g., several) positions as compared to SEQ ID NO: 1 or SEQ ID NO:2, and having a polyester degrading activity.
  • the variants may be obtained by various techniques well known in the art.
  • examples of techniques for altering the DNA sequence encoding the parent protein include, but are not limited to, site-directed mutagenesis, random mutagenesis and synthetic oligonucleotide construction.
  • modification and “alteration” as used herein in relation to a particular position means that the amino acid in this particular position has been modified compared to the amino acid in this particular position in the parent protein.
  • substitution means that an amino acid residue is replaced by another amino acid residue.
  • substitution refers to the replacement of an amino acid residue by another selected from the naturally-occurring standard 20 amino acid residues, rare naturally occurring amino acid residues (e.g. hydroxyproline, hydroxylysine, allohydroxylysine, 6-N-methylysine, N-ethylglycine, N-methylglycine, N-ethylasparagine, allo-isoleucine, N-methylisoleucine, N- methylvaline, pyroglutamine, aminobutyric acid, ornithine, norleucine, norvaline), and non- naturally occurring amino acid residue, often made synthetically, (e.g.
  • substitution refers to the replacement of an amino acid residue by another selected from the naturally-occurring standard 20 amino acid residues (G, P, A, V, L, I, M, C, F, Y, W, H, K, R, Q, N, E, D, S and T).
  • the sign “+” indicates a combination of substitutions.
  • L82A denotes that amino acid residue (Leucine, L) at position 82 of the parent sequence is substituted by an Alanine (A).
  • V/I/M denotes that amino acid residue (Alanine, A) at position 121 of the parent sequence is substituted by one of the following amino acids: Valine (V), Isoleucine (I), or Methionine (M).
  • V Valine
  • I Isoleucine
  • M Methionine
  • conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid 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 small amino acids (glycine, alanine and serine).
  • basic amino acids arginine, lysine and histidine
  • acidic amino acids glutmic acid 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
  • small amino acids glycine, alanine and serine
  • amino acid positions disclosed in the present application are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1.
  • sequence 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 when aligned so as to maximize overlap and identity while minimizing sequence gaps.
  • sequence identity may be determined using any of a number of mathematical global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithm (e.g. Needleman and Wunsch algorithm; Needleman and Wunsch, 1970) which aligns the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g.
  • Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software available on internet web sites 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 needed to achieve maximal alignment over the full length of the sequences being compared.
  • polymer refers to a chemical compound or mixture of compounds whose structure is constituted of multiple monomers (repeat units) linked by covalent chemical bonds.
  • polymer includes natural or synthetic polymers, constituted of a single type of repeat unit (i.e., homopolymers) or of a mixture of different repeat units (i.e., copolymers or heteropolymers).
  • oligomers refer to molecules containing from 2 to about 20 monomers.
  • the polyester containing material refers to textile, fabrics or fibers comprising at least one polyester.
  • the polyester containing material refers to plastic waste or fiber waste comprising at least one polyester.
  • the plastic article is a manufactured product, such as rigid or flexible packaging (bottle, trays, cups, etc.), agricultural films, bags and sacks, disposable items or the like, carpet scrap, fabrics, textiles, etc.
  • the plastic article may contain additional substances or additives, such as plasticizers, minerals, organic fillers or dyes.
  • the plastic article may comprise a mix of semi-crystalline and/or amorphous polymers and/or additives.
  • polyethylene terephthalate PET
  • polytrimethylene terephthalate PTT
  • polybutylene terephthalate PBT
  • polyethylene isosorbide terephthalate PEIT
  • polylactic acid PLA
  • PHA polyhydroxyalkanoate
  • PBS polybutylene succinate
  • PBSA polybutylene succinate adipate
  • PBAT polybutylene adipate terephthalate
  • PCL polyethylene adipate
  • PEA polyethylene naphthalate
  • PEN polyethylene naphthalate
  • Polyesters can also encompasses “polyolefin-like” polyesters, preferably “polyethylene-like” polyesters which correspond to polyolefin (preferably polyethylene) into which ester segments have been introduced (generally achieved by polycondensation of long-chain a, co-difunctional monomers), as defined in Lebarbe et al. Green Chemistry Issue 4 2014.
  • “basic conditions” refer to conditions (e.g., medium, solution, etc.) at a pH comprised between 6 and 10.
  • “basic conditions” refer to the conditions to perform the degradation step of the polyester, i.e., the esterase is contacted with the polyester in a medium having a pH between 6 and 10.
  • the esterases of the present invention exhibit an increased activity and/or an increased thermostability as compared to the parent esterase in basic conditions. Particularly, the esterases of the present invention exhibit an increased activity and/or an increased thermostability as compared to the parent esterase when submitted at a pH between 6 and 10.
  • the increased activity and/or increased thermostability may be observed at specific pH between 6 and 10 and/or in a range of pH between 6 and 10.
  • the increased activity and/or increased thermostability may be observed at least at pH 6, at pH 6.5, at pH 7, at pH 7.5, at pH 8, at pH 8.5, at pH 9, at pH 9.5, and/or at pH 10.
  • the increased activity and/or increased thermostability may also be observed in the whole range of pH 6 to 10, in the whole range of pH 6 to 9, in the whole range of pH 6.5 to 9, in the whole range of pH 7 to 9, in the whole range of pH 7.5 to 8.5, in the whole range of pH 7 to 8.5, in the whole range of pH 7.5 to 9.
  • esterases that exhibit an increased activity at a pH comprised between 6 and 10, compared to the esterase having the amino acid sequence as set forth in the parent esterases at same pH.
  • the parent esterase may be either the esterase of SEQ ID NO: 1 or SEQ ID NO:2.
  • the term “increased activity” or “increased degrading activity” indicates an increased ability of the esterase to degrade a polyester and/or an increased ability to adsorb on a polyester, at given conditions (e.g., temperature, pH, concentration) as compared to the ability of the parent esterase to degrade and/or adsorb on same polyester under same conditions.
  • the esterase of the invention has an increased PET degrading activity.
  • Such an increased activity may be at least 10% greater than the polyester degrading activity of the parent esterase , preferably at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130% or greater.
  • the degrading activity is a depolymerization activity leading to monomers and/or oligomers of the polyester, which can be further retrieved and optionally reused.
  • the esterase exhibits an increased degrading activity at least under basic conditions, in particular at a pH comprised between 6 and 10, as compared to the degrading activity of the parent esterase at same pH.
  • 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 instance at pH 8.
  • the “degrading activity” may be evaluated by measuring, after a defined period of time (for example after 24h, 48h or 72h), the rate and/or yield of oligomers and/or monomers released under suitable conditions of temperature, pH and buffer, when contacting the polymer or the polymer-containing plastic product with a degrading enzyme.
  • the ability of an enzyme to adsorb on a substrate may be evaluated by the one skilled in the art, according to methods known per se in the art. For instance, the ability of an enzyme to adsorb on a substrate can be measured from a solution containing the enzyme and wherein the enzyme has been previously incubated with a substrate under suitable conditions.
  • target amino acid residues in the parent esterase may be advantageously modified to improve the stability of corresponding esterases in basic conditions at elevated temperatures (i.e., improved thermostability), and advantageously at temperature at or above 50°C and at or below 90°C, preferably at or above 60°C and at or below 80°C, more preferably at or above 65°C and at or below 75°C.
  • thermostability under basic conditions as compared to the thermostability of the esterase having the amino acid sequence set forth in the parent esterase at same pH.
  • thermostability of the esterases is improved, in basic conditions, as compared to the thermostability of the parent esterase, at temperature(s) 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.
  • thermostability of the esterases is improved, in acidic conditions, as compared to the thermostability of the parent esterase, at temperature(s) between 40°C and 80°C, between 50°C and 72°C, 55°C and 60°C, between 50°C and 55°C, between 60°C and 72°C.
  • thermostability of the esterases is improved, as compared to the thermostability of the parent esterase, at least at temperatures between 50°C and 65°C. Within the context of the invention, temperatures are given at +/- 1°C.
  • the thermostability may be evaluated through the assessment of the melting temperature (Tm) of the esterase.
  • Tm melting temperature
  • the “melting temperature” refers to the temperature at which half of the enzyme population considered is unfolded or misfolded.
  • esterases of the invention show an increased Tm of about 0.8°C, 1°C, 2°C, 3°C, 4°C, 5°C, 10°C or more, as compared to the Tm of the parent esterase under acidic conditions, in particular at a pH comprised between 6 and 10.
  • esterases of the present invention can have an increased half- life at a temperature between 50°C and 90°C, as compared to the parent esterase.
  • the esterases of the present invention exhibit an increased thermostability as compared to the thermostability of the esterase having the amino acid sequence set forth 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.
  • the esterase exhibits an 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 instance at pH 8.
  • the melting temperature (Tm) of an esterase may be measured by the one skilled in the art, according to methods known per se in the art.
  • the DSF may be used to quantify the change in thermal denaturation temperature of the esterase and thereby to determine its Tm.
  • the Tm can be assessed by analysis of the protein folding using circular dichroism.
  • the Tm is measured using DSF or circular dichroism as exposed in the experimental part.
  • comparisons of Tm are performed with Tm that are measured under same conditions (e.g. pH, nature and amount of polyesters, etc.).
  • thermostability may be evaluated by measuring the esterase activity and/or the polyester depolymerization activity of the esterase after incubation at different temperatures and comparing with the esterase activity and/or polyester depolymerization activity of the parent esterase.
  • the ability to perform multiple rounds of polyester’s depolymerization assays at different temperatures can also be evaluated.
  • a rapid and valuable test may consist of the evaluation, by halo diameter measurement, of the esterase ability to degrade a solid polyester compound dispersed in an agar plate after incubation at different temperatures.
  • the esterase comprises at least one amino acid substitution or at least one combination of substitutions selected from 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,
  • the esterase comprises at least one substitution selected from R30G, A64V, L124G, S145G, H183Q/L/A/Y/N/D/E/W, S193L/Q, W228S and V242E, preferably selected from R30G, A64V, L124G, S145G, H183Q/N, S193L/Q, W228S and V242E.
  • the esterase further comprises at least one substitution selected from G35A, W69L, F90L/Y, N162S, S181K, N204G, A216C, N243Y, A246C, V28I, T157P, T16K/R, Y4K/R, V219E/D, M208N, N21 IM, A17F, M208L, M208T, V200I, I170V, N122D, Q142E, Q237E, Q258E, N85D, N105D, LBS, E158D, preferably selected from selected from G35A, W69L, F90L/Y, V115I, N162S, S181K, N204G, A216C, N243Y, A246C, V28I, T157P, T16K/R, Y4K/R and V219E/D, more preferably selected from G35A, W69L, F90L/Y, VI 151, N162S, S
  • the esterase comprises at least one substitution or at least one combination of substitutions selected from R30G, A64V, A68N, T109S, L124G, S145G, L152M, E158A, K159R, H183Q/N, S193L/Q, W228R/S, V242E, H183L + Fl 871, H183A + F187I, H183Y + Fl 871, H183D + Fl 871, H183W + Fl 871, H183E + Fl 871, A62T + S66N + M208T, F90A + H183S + F187L, F90L + H183E + F187L, F90S + H183S + F187L, F90Y + H183L + F187L and S22R + G39S + P179N + L239M, more preferably selected R30G, A64V, T109S, S145G, E158A, H183Q/N, W228S, V242E, H183A + F187I,
  • the esterase comprises at least one substitution selected from R30G, A64V, L124G, S145G, H183Q/N, S193L/Q, W228S and V242E and further comprises at least one substitution selected from G35A, W69L, F90L/Y, VI 151, N162S, S181K, N204G, A216C, N243Y, A246C, V28I, T157P, A17F, F90Q, M208L, N21 IM, N122D, S193E, Q142E, N143D, Q237E, N253D, Q258E, V200I, Il 70V, N85D, N105D, N21 IE, Fl 871, M208N, M208T, LBS, E158D, preferably selected from G35A, W69L, F90L/Y, VI 151, N162S, S181K, N204G, A216C, N243Y, A246C, V28I
  • the esterase comprises at least the combination of substitutions selected from, or consists of the amino acid sequence as set forth in SEQ ID NO: 1 with the combination of substitutions, V200I + Il 70V.
  • the esterase comprises the substitution A64V and further comprises at least one substitution selected from N243Y, VI 151, N204G, H183Q and Fl 871. Particularly, the esterase comprises at least one combination of substitutions selected from A64V + N243Y, A64V + VI 151 + N243 Y and N204G + Hl 83Q + F 1871 + A64V.
  • the esterase comprises the substitution S145G and further comprises at least one substitution selected from T27A, R30G, W228S and V242E. Particularly, the esterase comprises at least the combination of substitutions T27A + R30G + S145G + W228S + V242E.
  • 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, Fl 871, 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, N
  • the esterase comprises at least one combination of substitutions selected from, or consists of the amino acid sequence as set forth in SEQ ID NO: 1 with the combination of substitutions selected from H183L + F187I, H183A + F187I, H183Y + F187I, H183D + F187I, H183W + F187I, + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I, A17T + T27S + S48T + L82I + F90L + G92F + G135A + AMOS + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + H183Q + F187I + N204G, A17T + T27S + S48T + L82I + F90L + G92F + G135A + AMOS + N143I + S145T + A149G + S164P + V167Q + S206T + T252S +
  • the esterase comprises the substitution H183Q and further comprises at least one substitution, at least two substitutions, at least three substitutions selected from A24Q, R30G, L74V, F187Q, Fl 871, 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, LBS, E158D, preferably selected from A24Q, R30G, L74V, F187Q, F187I, L227N, R251S, Y26H, A64T, G35A, A62T, G46
  • the esterase comprises at least one combination of substitutions selected from, or consists of the amino acid sequence as set forth in SEQ ID NO: 1 with a combination of substitutions selected from A24Q + H183Q, R30G + H183Q, L74V + H183Q, H183Q + F187Q, H183Q + Fl 871, L227N + H183Q, R251S + H183Q, Y26H + A64T + Hl 83Q, G35 A + A62T + Hl 83Q, G46V + R73H + Hl 83Q, T50M + H69M + Hl 83Q, Y106F + I185V + H183Q, R108L + V167L + H183Q, H183Q + F187I + N204G, N204G + H183Q + F187I + R30G, N204G + H183Q + F187I + G39S, N204G + H183Q + F187I + A62T, N204G + H183Q + F187I + A
  • the esterase comprises the substitution H183N and further comprises at least one substitution selected from Fl 871, 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.
  • the esterase comprises at least one combination of substitutions selected from, or consists of the amino acid sequence as set forth in SEQ ID NO: 1 with a combination of substitutions selected from H183N + Fl 871, F90L + H183N + F187L, F90Y + H183N, A17F + F90Q + H183N + N204G + M208L + N211M, A17F + F90Q + N122D + H183N + S193E + N204G + M208L + N21 IM, A17F + F90Q + Q142E + H183N + S193E + N204G + M208L + N211M, A17F + F90Q + N143D + H183N + S193E + N204G + M208L + N21 IM, A17F + F90Q + H183N + S193E + N204G + M208L + N211M, A17F + F90Q + H183N + S193E + N204G + M208L + N211M, A17
  • the esterase comprises the substitution S193L and further comprises at least the substitution S181K. Particularly, the esterase comprises at least the combination of substitutions S181K + S193L.
  • the esterase comprises the substitution S193Q and further comprises at least one substitution selected from N162S, S181K, N204G, H183Q and F187I. Particularly, 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.
  • the esterase comprises the substitution W228S and further comprises at least one substitution selected from T27A, R30G, S145G and V242E. Particularly, the esterase comprises at least the combination of substitutions selected from T27A + R30G + S145G + W228S + V242E.
  • the esterase comprises the substitution V242E and further comprises at least one substitution selected from T27A, R30G, S145G and W228S. Particularly, the esterase comprises at least the combination of substitutions selected from T27A + R30G + S145G + W228S + V242E.
  • the esterase comprises at least one combination of substitutions selected from, or consists of the amino acid sequence as set forth in SEQ ID NO: 1 with a combination of substitutions selected from, 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, Y
  • the esterase comprises at least the combination of substitutions N204G + H183Q + Fl 871 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, V200I, H70V, LBS, E158D, preferably selected from R30G, G39S, A62T, A64V, W69L, L124G, N162S, S181K, S193Q, M208T, A216C, Q237L and A246C.
  • the esterase comprises at least one combination of substitutions selected from, or consists of the amino acid sequence as set forth in SEQ ID NO: 1 with a combination of substitutions selected from 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 + Fl 871, N204G + H183Q + F187I + N162S + S181K, N204G + H183Q + F187I + N162S + S181K + S193Q, N204G + H183Q + F187I + W69L
  • the esterase comprises at least the combination of substitutions N204G + H183Q + F187I and optionally further comprises at least one substitution selected from M208T, L13S and E158D.
  • the esterase comprises at least one combination of substitutions selected from, or consists of the amino acid sequence as set forth in SEQ ID NO:1 with a combination of substitutions selected from N204G + H183Q + Fl 871 + M208T, N204G + H183Q + F187I + M208T + L13S + E158D, N204G + H183Q + F187I + L13S + E158D.
  • the esterase comprises at least the combination of substitutions M208N + N211M.
  • the esterase comprises at least the combination of substitutions selected from H183Q + F187I + N204G + N211M + S193E and further comprises at least one substitution selected from M208N/T, V200I and II 70V.
  • the esterase comprises at least one combination of substitutions selected from N204G + H183Q + F187I + M208N/T + N21 IM + S193E, preferably the combination H183Q + F187I + N204G + M208N + N21 IM + S193E.
  • the esterase comprises at least one substitution or combination of substitutions selected from 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 + N243 Y, L74V + H183Q, N162S + S193Q, S181K + S19
  • the esterase consists of amino acid sequence as set forth in SEQ ID NO: 1 with a substitution or a combination of substitutions selected from 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
  • the esterase comprises at least one combination of substitutions selected from, or consists of the amino acid sequence as set forth in SEQ ID NO: 1 with one combination + S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I, A17T + T27S + S48T + L82I + F90L + G92F + G135A + AMOS + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + H183Q + F187I + N204G, A17T + T27S + S48T + L82I + F90L + G92F + G135A + AMOS + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I + L13S + E158D.
  • the esterase of the invention exhibits an increased polyester degrading activity and/or an increased thermostability as compared to the esterase of SEQ ID NO: 1 in a range of pH between 6 and 10.
  • the esterase of the invention exhibits an increased polyester degrading activity and/or an increased thermostability as compared to the esterase of SEQ ID NO: 1 in the range of pH from 6 to 10, from 6 to 9, from 6.5 to 9, from 7 to 9, from 7.5 to 8.5, from 7 to 8.5, from 7.5 to 9.
  • the designation of a range of pH includes the lower and upper limit of said range.
  • the esterase exhibits an increased polyester degrading activity as compared to the esterase of SEQ ID NO: 1, at least under basic conditions.
  • the esterase exhibits an increased polyester degrading activity at least at pH between 7.5 and 9, such as at pH 8.
  • the esterase may exhibit an increased specific degrading activity and/or an increased PET depolymerization yield after a defined period of time, for example after 24h, 48h or 72h, as compared to the esterase of SEQ ID NO: 1.
  • the esterase comprises at least one substitution or combination of substitutions as described above and exhibits an increased polyester degrading activity as 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.
  • the esterase comprises at least one substitution or combination of substitutions as described above and has an increased PET depolymerization yield after 24 h compared to SEQ ID NO: 1.
  • the esterase comprises at least one substitution or combination of substitutions as described above and exhibits an increased specific degrading activity as compared to the esterase of SEQ ID NO: lat 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.
  • the esterase comprises at least the substitution or combination of substitutions as described above and exhibits an increased PET depolymerization yield after 48h 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.
  • the esterase may exhibit an increased thermostability as compared to the esterase of SEQ ID NO: 1.
  • the esterase may exhibits an increased polyester degrading activity at least at pH between 7 and 9, such as at pH 8.
  • the esterase comprises at least one substitution selected from Fl 871 and S193Q or at least the combination of substitutions S181K + S193L and exhibits an 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 as compared to the esterase of SEQ ID NO: 1.
  • the esterase of the invention further exhibits an increased thermostability and/or an increased polyester degrading activity as 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 comprised between 5 and 5.5, even more preferably at pH 5.2.
  • the esterase comprises at least one substitution or at least one combination of substitutions as described above and exhibits an increased thermostability and/or an increased polyester degrading activity as compared to the esterase of SEQ ID NO: 1 at a pH comprised between 6 and 10, particularly between 7.5 and 9 and further exhibits an increased thermostability and/or an increased polyester degrading activity as compared to the esterase of SEQ ID NO: 1 at a pH between 3 and 6, preferably at a pH comprised between 5 and 6, more preferably comprised between 5 and 5.5, even more preferably at pH 5.2.
  • the esterase may comprise at least one amino acid residue selected from S130, D175, H207, C240 or C275 as in the parent esterase of SEQ ID NO: 1, i.e., the esterase of the invention is not modified at one, two, three, etc., or all of these positions.
  • the esterase may exhibit at least the amino acids S130, D175 and H207 forming the catalytic site of the esterase and/or the amino acids C240 and C275 forming disulphide bond as in the parent esterase.
  • the esterase comprises at least a combination of amino acid residues selected from S130 + D175 + H207, C240 + C275 and SI 30 + DI 75 + H207 + C240 + C275, as in the parent esterase, more preferably the combination S130 + D175 + H207 + C240 + C275 as in the parent esterase.
  • the esterase may further comprise at least one amino acid residue selected from C203 and C248 as in the parent esterase of SEQ ID NO: 1.
  • the esterase may comprise the combination of amino acid residues C203 + C248 as in the parent esterase.
  • the esterase comprises at least the substitution C203K/R, preferably C203K and the amino acid residue C248 as in the parent esterase.
  • the esterase comprises at least one amino acid substitution selected from 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, Il 85V, F187L/Q, P192S, L227N, Q237L, L239M, R251S, N9E, V28I, A62T, F90Y/R/W7L, VI 151, S181K, H183S, F187I, M208T, A246K, S66N, N243Y, S66T, F90C/V, P151V,
  • the esterase comprises at least one substitution selected from N9E, V28I, A62T, F90Y, VI 151, S181K, H183S, F187I, M208T, S66N, N243Y, E182D, M208G, T16E, Q142E, Q237E, M208K, N105D, N85D, M208A, M208D preferably selected from N9E, V28I, A62T, F90Y, VI 151, 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.
  • the esterase comprises one or two substitutions selected from A215E/D, D230E, L15K/R, T16K/R, Y4K/R and V219E/D. Particularly, the esterase comprises a combination of substitutions selected from A215E + T16K/R, A215D + T16R, D230E + Y4K/R and V219E/D + L15K/R.
  • the esterase comprises at least one, preferably at least two substitutions selected from 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, VI 151, S181K, H183S, Fl 871, M208T, S66N and N243Y.
  • the esterase comprises a combination of substitutions selected from S66N + M208T, A62T + M208T.
  • the esterase comprises at least one substitution selected from R30G, A62T, A64V, F90Y, VI 151, L124G, S145G, L152M, K159R, H183Q/N/D/E, F187I, S193L/Q, M208T, W228R/S, V242E and N243Y, preferably selected from R30G, A62T, A64V, F90Y, VI 151, L124G, S145G, L152M, K159R, H183Q/N, F187I, S193L/Q, M208T, W228R/S, V242E and N243 Y.
  • the esterase may further comprise from one to seven substitutions at amino acid positions selected from SI, Y4, Q5, R6, N9, PIO, Ti l, 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, 154, M56, P58, G59, Y60, T61, A62, D63, A64, S65, S66, L67, A68, W69, L70, R72, R73, L74, L82, 184, N85, T86, N87, S88, R89, F90, D91, G92, P93, D94, S95, R96, S98, Q99, A103, L104, N105, L107, R108, SI 13, VI 15, LI 19, A121, N122,
  • the esterase comprises the substitution F90Y and further comprises at least one substitution selected from H183L/N. Particularly, the esterase comprises a combination of substitutions selected from F90Y + H183L + F187L and F90Y + H183N. According to another embodiment, the esterase comprises the substitution VI 151 and further comprises at least one substitution selected from A64V and N243Y. Particularly, the esterase comprises the combination of substitutions A64V + VI 151 + N243Y.
  • the esterase comprises the substitution Fl 871 and further comprises one, two or more substitutions selected from 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 selected from R30G, G39S, A62T, A64V, W69L, L124G, N162S, S181K, H183L/A/Y/Q/N/D/W/E/S, S193Q, N204G, M208T, A216C, Q237L, A246C.
  • the esterase comprises at least one combination of substitutions selected from, or consists of the amino acid sequence as set forth in SEQ ID NO: 1 with a combination of substitutions selected from H183L + F187I, H183A + F187I, H183Y + F187I, H183Q + F187I, H183N + F187I, H183D + F187I, H183W + F187I, H183E + F 1871, Hl 83 S + F 1871, Hl 83Q + F 1871 + N204G, N204G + Hl 83Q + F 1871 + 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
  • the esterase comprises the substitution M208T and further comprises at least one substitution selected from A62T, S66N, H183Q, F187I, N204G, N21 IM, S193E, V200I, I170V, preferably selected from A62T, S66N, H183Q, F187I and N204G.
  • the esterase comprises at least the combination of substitutions selected from, or consists of the amino acid sequence as set forth in SEQ ID NO: 1 with a combination of substitutions selected from 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.
  • the esterase comprises the substitution N243 Y and further comprises at least one substitution selected from A64V and VI 151. Particularly, the esterase comprises at least the combination of substitutions selected from A64V + N243 Y and A64V + V115I + N243Y.
  • the esterase comprises at least one combination of substitutions selected from, or consists of the amino acid sequence as set forth in SEQ ID NO: 1 with the combination of substitutions selected from 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, R
  • the esterase comprises at least the combination of substitutions N204G + H183Q + Fl 871 and optionally further comprises at least one substitution selected from R30G, G39S, A62T, A64V, W69L, L124G, N162S, S181K, S193Q, M208T, A216C, Q237L, A246C, M208N, N21 IM, S193E, preferably selected from R30G, G39S, A62T, A64V, W69L, L124G, N162S, S181K, S193Q, M208T, A216C, Q237L and A246C.
  • the esterase comprises at least one combination of substitutions selected from or consists of the amino acid sequence as set forth in SEQ ID NO: 1 with a combination of substitutions selected from 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 +
  • the esterase comprises at least the combination of substitutions M208N + N211M.
  • the esterase comprises at least one substitution or combination of substitutions selected from or consists of the amino acid sequence as set forth in SEQ ID NO: 1 with a combination of substitutions selected from 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, VI 151, S 18 IK, F 1871, M208T, N243 Y, S66N + M208T, A62T + M208T, F90A + H183S + F187L, F90L + H183E + F187L, F90S + H183S + F187L, S22R + G39S + P179N + L239M, A24Q + H183Q, R30G + H183Q, A64V + N243Y, L74V + H183
  • the esterase comprises at least one substitution selected from Fl 871 and S193Q or at least one combination of substitutions selected from S181K + S193L and exhibits an 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 as compared to the esterase of SEQ ID NO: 1.
  • the esterase may comprise at least one amino acid residue selected from S130, D175, H207, C240 or C275 as in the parent esterase of SEQ ID NO: 1, i.e., the esterase of the invention is not modified at one, two, three, etc., or all of these positions.
  • the esterase may exhibit at least the amino acids S130, D175 and H207 forming the catalytic site of the esterase and/or the amino acids C240 and C275 forming disulphide bond as in the parent esterase.
  • the esterase comprises at least a combination of amino acid residues selected from S130 + D175 + H207, C240 + C275 and SI 30 + DI 75 + H207 + C240 + C275, as in the parent esterase, more preferably the combination S130 + D175 + H207 + C240 + C275 as in the parent esterase.
  • the esterase consists of the amino acid sequence as set forth in SEQ ID NO: 1 with a combination of substitutions selected from A17T + T27S + S48T + L82I + F90L + G92F + G135A + AMOS + N143I + S145T + A149G + S164P + V167Q + H183Q + F187I + C203K + N204G + S206T + M208T + C248S + T252S, A17T + T27S + S48T + L82I + F90L + G92F + G135A + AMOS + N143I + S145T + A149G + S164P + V167Q + H183Q + F187I + C203K + N204G + S206T + C248S + T252S, A17T + T27S + S48T + L82I + F90L + G92Y + G135A + AMOS + N143I + S145T + A149G + S164P + V167Q +
  • the esterases of the invention are derived from SEQ ID NO:2, which corresponds to the amino acid sequence of SEQ ID NO: 1, with the combination of substitutions A17T + T27S + S48T + L82I + F90L + G92F + G135A + AMOS + N143I + S145T + A149G + S164P + V167Q + 1170V + S206T + T252S as compared to SEQ ID NO: 1. That is to say that variants derived from SEQ ID NO:2 comprise at least one of these substitutions as compared to SEQ ID NO: 1 and one or more additional substitutions as described in the present application.
  • 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 set forth in SEQ ID NO:2, (ii) comprises at least one amino acid substitution selected from 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, L
  • the esterase comprises at least one substitution selected from A64V, S66N, H183N, I143D, N253D, preferably selected from H183N and S66N.
  • 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.
  • the esterase may further comprises a substitution at at least one amino acid position selected from SI, Y4, Q5, R6, N9, PIO, Ti l, 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, 154, M56, P58, G59, Y60, T61, A62, D63, A64, S65, S66, L67, A68, W69, L70, R72, R73, L74, 182, 184, N85, T86, N87, S88, R89, L90, D91, F92, P93, D94, S95, R96, S98, Q99, A103, L104, N105, L107, R108, SI 13, VI 15, LI 19, A121, N122, L
  • the esterase further comprises at least one substitution selected from G35A, W69L, L90L/Y, VI 151, N162S, S181K, N204G, A216C, N243Y, A246C, V28I, T157P, T16K/R, Y4K/R, V219E/D, Fl 871, L90Q, H183D, H183E, H183N, H183Q, I145D, L90Q, M208A, M208L, M208N, M208T, N105D, N122D, N211M, N253D, N85D, Q142E, Q237E, Q258E, S193E, S98R, T17F, V200I, L I 3S, E158D, preferably selected from G35A, W69L, L90L/Y, VI 151, N162S, SI 8 IK, N204G, A216C, N243Y, A246C, V28I, T157P, T16
  • 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 G35A, W69L, L90L/Y, VI 151, 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 and V200I, preferably selected from G35
  • 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/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, L90VY, as compared to the amino acid sequence SEQ ID NO:2.
  • the esterase comprises at least one combination of substitutions selected from H183L + Fl 871, Hl 83 A + Fl 871, H183Y + Fl 871, H183D + Fl 871, 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
  • 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, L90Q, I145D, L90Q, M208A, M208L, M208M, M208N, M208T, N105D, N122D, N204G, N211M, N253D, N85D, Q142E, Q237E, Q258E, S193E, S98R, T17F and V200I.
  • the esterase comprises the substitution H183Q and further comprises at least one, two, three or more substitutions selected from 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, as compared to the amino acid sequence SEQ ID NO:2.
  • the esterase comprises at least one combination of substitutions selected from A24Q + H183Q, R30G + H183Q, L74V + H183Q, H183Q + F187Q, H183Q + F187I, L227N + H183Q, R251 S + H183Q, Y26H + A64T + H183Q, G35A + A62T + Hl 83Q, 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 + N
  • the esterase comprises the substitution H183Q and further comprises at least one, two, three, four, five, six or more substitutions selected from Fl 871, M208N, M208T, N204G, N211M, S193E, V200I.
  • the esterase comprises at least one combination of substitutions selected from, or consists of the amino acid sequence as set forth in SEQ ID NO:2 with a combination of substitutions selected from H183Q + F187I + N204G + M208T + N211M + S193E, H183Q + F187I + N204G + M208N + N21 IM + S 193E + V200I, H183Q + F187I + N204G + M208T + N21 IM + S193E + V200I, H183Q + F187I + N204G + M208N + N21 IM + S193E, H183Q + F187I + N204G, H183Q + F187I + N204G + M208T, H183Q + F187I + N204G + M208T + LI 3 S + E158D, T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + I143N + T145S + G149A + P164S,
  • 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.
  • the esterase comprises at least one combination of substitutions selected from, or consists of the amino acid sequence as set forth in SEQ ID NO:2 with a combination of substitutions selected from H183N + F187I, T17F + L90Q + H183N + N204G + M208L + N211M, T17F + L90Q + H183N + S193E + N204G + M208L + N21 IM, T17F + N85D + L90Q + H183N + S193E + N204G + M208L + N211M, T17F + L90Q + N122D + H183N + S193E + N204G + M208L + N21 IM, T17F + L90Q + Q142E + H183N + S193E + N204G + M208L + N211M, T17F + L90Q + I145D + H183N + S193E + N204G + M208L + N21 IM, T17F + L90Q + H183N + S193M,
  • the esterase comprises at least the combination of substitutions N204G + H183Q + Fl 871 and optionally further comprises at least one substitution selected from R30G, G39S, A62T, A64V, W69L, L124G, N162S, S181K, S193Q, M208T, A216C, Q237L and A246C, as compared to the amino acid sequence SEQ ID NO:2.
  • the esterase comprises at least one combination of substitutions selected from 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 + F 1871 + N162S + S 18 IK + S 193Q and N204G + Hl 83Q + F 1871 + W69L + A216C + A246C.
  • the esterase may further comprise at least the substitution C203K
  • the esterase comprises at least the combination of substitutions N204G + H183Q + Fl 871 and optionally further comprises at least one substitution selected from M208T, N211M, S193E, V200I, M208N, LBS, E158D.
  • the esterase comprises at least one combination of substitutions selected from, or consists of the amino acid sequence as set forth in SEQ ID NO:2 with a combination of substitutions selected from M208T + N204G + H183Q + F187I + N211M + S193E, H183Q + F187I + N204G + M208N + N211M + S193E + V200I, M208T + N204G + H183Q + F187I + N21 IM + S193E + V200I, H183Q + F187I + N204G + M208N + N21 IM + S193E, H183Q + F187I + N204G, H183Q + F187I + N204G + M208T, Hl 83Q + F 1871 + N204G + M208T + L13 S + El 58D, T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + H43N + T145S + G149A + P
  • the esterase comprises at least the combination of substitutions M208N + N211M.
  • the esterase comprises at least one substitution or combination of substitutions selected from 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, H183
  • the esterase comprises at least one substitution or combination of substitutions selected from or consists of the amino acid sequence as set forth in SEQ ID NO:2 with a combination of substitutions selected from M208N + N21 IM, H183Q + F187I + N204G + M208N + N21 IM + S 193E, Hl 83Q + F 1871 + N204G, Hl 83Q + F 1871 + N204G + M208T, H183Q + F187I + N204G + M208T + L13S + E158D, T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + H43N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + M208T + L13S + E158D, T17A + S27T + T48S + I82L + L90F + N204G + M208T + L13
  • the esterase comprises at least one substitution selected from Fl 871 and S193Q or at least the combination of substitutions S181K + S193L as compared to the amino acid sequence SEQ ID NO:2 and exhibits an 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 as compared to the esterase of SEQ ID NO:2.
  • the esterase exhibits at least one amino acid residue selected from S130, D175, H207, C240 or C275 as in the parent esterase of SEQ ID NO:2, i.e. the esterase of the invention is not modified at one, two, three, etc., or all of these positions.
  • the esterase may exhibit at least the amino acids S130, D175 and H207 forming the catalytic site of the esterase and/or the amino acids C240 and C275 forming disulphide bond as in the parent esterase.
  • the esterase comprises at least a combination of amino acid residues selected from S130 + D175 + H207, C240 + C275 and SI 30 + DI 75 + H207 + C240 + C275, as in the parent esterase, more preferably the combination S130 + D175 + H207 + C240 + C275 as in the parent esterase.
  • the esterase may further comprise at least one amino acid residue selected from C203 and C248 as in the parent esterase of SEQ ID NO:2.
  • the esterase may comprise the combination of amino acid residues C203 + C248 as in the parent esterase.
  • the esterase comprises at least the substitution C203K/R, preferably C203K and the amino acid residue C248 as in the parent esterase.
  • the esterase further exhibits an increased thermostability and/or an increased polyester degrading activity as compared to the esterase of SEQ ID NO: 1.
  • the esterase comprises at least one substitution selected from M208N, M208S, N122D, Q237E, Q258E, and S212T.
  • the esterase may further comprises at least one substitution at at least one amino acid position selected from SI, Y4, Q5, R6, N9, PIO, Ti l, 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, 154, M56, P58, G59, Y60, T61, A62, D63, A64, S65, S66, L67, A68, W69, L70, R72, R73, L74, 182, 184, N85, T86, N87, S88, R89, L90, D91, F92, P93, D94, S95, R96, S98, Q99, A103, L104, N105, L107, R108, SI 13, VI 15, LI 19, A121, N122,
  • the esterase comprises at least one substitution selected from M208A, M208N, N122D, Q237E and Q258E and further comprises at least one substitution selected from N21 IM, H183Q, Fl 871, N204G, S193E, V200I, T17F, L90Q, H183N and M208L.
  • the esterase comprises at least the combination of substitutions selected from or consists of the amino acid sequence as set forth in SEQ ID NO:2 with the combination of substitutions selected from M208A + N21 IM or M208N + N21 IM.
  • 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.
  • the esterase comprises at least one combination of substitutions selected from, or consists of the amino acid sequence as set forth in SEQ ID NO:2 with a combination of substitutions selected from M208N + N211M, H183Q + F187I + N204G + M208N + N211M + S193E + V200I and T 17F + L90Q + H183N + S193E + N204G + M208N + N211M + V200I.
  • the esterase consists of the amino acid sequence as set forth in SEQ ID NO:2 with a combination of substitutions selected from L90W, M208A, M208N, M208S, N122D, Q237E, Q258E, S212T, M208I + N211M.
  • the esterase exhibits at least one amino acid residue selected from S130, D175, H207, C240 or C275 as in the parent esterase of SEQ ID NO:2, i.e. the esterase of the invention is not modified at one, two, three, etc., or all of these positions.
  • the esterase may exhibit at least the amino acids S130, D175 and H207 forming the catalytic site of the esterase and/or the amino acids C240 and C275 forming disulphide bond as in the parent esterase.
  • the esterase comprises at least a combination of amino acid residues selected from S130 + D175 + H207, C240 + C275 and SI 30 + DI 75 + H207 + C240 + C275, as in the parent esterase, more preferably the combination S130 + D175 + H207 + C240 + C275 as in the parent esterase.
  • the esterase may further comprise at least one amino acid residue selected from C203 and C248 as in the parent esterase of SEQ ID NO:2.
  • the esterase may comprise the combination of amino acid residues C203 + C248 as in the parent esterase.
  • the esterase comprises at least the substitution C203K/R, preferably C203K and the amino acid residue C248 as in the parent esterase.
  • the esterase further exhibits an increased thermostability and/or an increased polyester degrading activity as compared to the esterase of SEQ ID NO: 1.
  • the esterase of the invention has a polyester degrading activity, preferably a polyethylene terephthalate (PET) degrading activity, and/or a polybutylene adipate terephthalate (PBAT) degrading activity and/or a polycaprolactone (PCL) degrading activity and/or a polybutylene succinate (PBS) activity, more preferably a polyethylene terephthalate (PET) degrading activity, and/or a polybutylene adipate terephthalate (PBAT) degrading activity.
  • PBS polybutylene succinate
  • the esterase of the invention has a polyethylene terephthalate (PET) degrading activity.
  • the esterase of the invention exhibits a polyester degrading activity in a range of temperatures from 20°C to 90°C, preferably from 30°C to 90°C, more preferably from 40°C to 90°C, more preferably from 50°C to 90°C, even more preferably from 60°C to 90°C, from 68°C to 90°C.
  • the esterase of the invention exhibits a polyester degrading activity in a range of temperatures from 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.
  • the esterase of the invention exhibits a polyester degrading 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.
  • the esterase of the invention exhibits a polyester degrading 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.
  • the esterase exhibits a polyester degrading 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.
  • a polyester degrading activity is still measurable at a temperature between 55°C and 70°C.
  • temperatures are given at +/- 1°C.
  • the esterase of the invention has an increased polyester degrading activity at a given temperature, compared to the parent esterase, and more particularly at a temperature between 40°C and 90°C, more preferably between 50°C and 90°C.
  • the esterase of the invention has an increased polyester degrading activity compared to the esterase of SEQ ID NO: 1 and/or SEQ ID NO:2 in the whole range of temperatures 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.
  • the esterase of the invention exhibits an increased polyester degrading 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.
  • the esterase of the invention exhibits an increased polyester degrading 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.
  • the esterase of the invention exhibits an increased polyester degrading activity at least at 50°C, 54°C, 55 °C, 60°C, 65 °C or 68°C, preferably at 55 °C or at 60°C or at 65 °C.
  • the esterase has a polyester degrading activity at least 5% higher than the polyester degrading activity of the parent esterase, preferably at least 10%, 20%, 50%, 100% or more.
  • the esterase has a polyester degrading activity at 65°C at least 5% higher than the polyester degrading activity of the parent esterase, preferably at least 10%, 20%, 30%, 50%, 100% or more.
  • nucleic acid refers to a sequence of deoxyribonucleotides and/or ribonucleotides.
  • the nucleic acids can be DNA (cDNA or gDNA), RNA, or a mixture thereof. It can be in single stranded form or in duplex form or a mixture thereof. It can be of recombinant, artificial and/or synthetic origin and it can comprise modified nucleotides, comprising for example a modified bond, a modified purine or pyrimidine base, or a modified sugar.
  • the invention also encompasses nucleic acids which hybridize, under stringent conditions, to a nucleic acid encoding an esterase as defined above.
  • stringent conditions include incubations of hybridization filters at about 42° C for about 2.5 hours in 2 X SSC/0.1%SDS, followed by washing of the filters four times of 15 minutes in 1 X SSC/0.1% SDS at 65° C. Protocols used are described in such reference 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)).
  • nucleic acids according to the invention may be deduced from the sequence of the esterase according to the invention and codon usage may be adapted according to the host cell in which the nucleic acids shall be transcribed. These steps may be carried out according to methods well known to one skilled in the art and some of which are described in the reference manual Sambrook et al. (Sambrook et al., 2001). Nucleic acids of the invention may further comprise additional nucleotide sequences, such as regulatory regions, i.e., promoters, enhancers, silencers, terminators, signal peptides and the like that can be used to cause or regulate expression of the polypeptide in a selected host cell or system.
  • regulatory regions i.e., promoters, enhancers, silencers, terminators, signal peptides and the like that can be used to cause or regulate expression of the polypeptide in a selected host cell or system.
  • the present invention further relates to an expression cassette comprising a nucleic acid according to the invention operably linked to one or more control sequences that direct the expression of said nucleic acid in a suitable host cell.
  • expression refers to any step involved in the production of a polypeptide including, but being not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
  • expression cassette denotes a nucleic acid construct comprising a coding region, i.e. a nucleic acid of the invention, and a regulatory region, i.e. comprising one or more control sequences, operably linked.
  • the invention also relates to a vector comprising a nucleic acid or an expression cassette as defined above.
  • vector refers to a DNA or RNA molecule that comprises an expression cassette of the invention, used as a vehicle to transfer recombinant genetic material into a host cell.
  • the major types of vectors are plasmids, bacteriophages, viruses, cosmids, and artificial chromosomes.
  • the vector itself is generally a DNA sequence that consists of an insert (a heterologous nucleic acid sequence, transgene) and a larger sequence that serves as the “backbone” of the vector.
  • the purpose of a vector which transfers genetic information to the host is typically to isolate, multiply, or express the insert in the target cell.
  • Vectors called expression vectors are specifically adapted for the expression of the heterologous sequences in the target cell, and generally have a promoter sequence that drives expression of the heterologous sequences encoding a polypeptide.
  • the regulatory elements that are present in an expression vector include a transcriptional promoter, a ribosome binding site, a terminator, and optionally present operator.
  • an expression vector also contains an origin of replication for autonomous replication in a host cell, a selectable marker, a limited number of useful restriction enzyme sites, and a potential for high copy number. Examples of expression vectors are cloning vectors, modified cloning vectors, specifically designed plasmids and viruses.
  • Expression vectors providing suitable levels of polypeptide expression in different hosts are well known in the art.
  • the choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
  • the expression vector is a linear or circular double stranded DNA molecule.
  • the present invention thus relates to the use of a nucleic acid, expression cassette or vector according to the invention to transform, transfect or transduce a host cell.
  • the choice of the vector will typically depend on the compatibility of the vector with the host cell into which it must be introduced.
  • the host cell may be transformed, transfected or transduced in a transient or stable manner.
  • the expression cassette or vector of the invention is introduced into a host cell so that the cassette or vector is maintained as a chromosomal integrant or as a selfreplicating extra-chromosomal 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 useful in the production of a variant of the present invention, e.g., a prokaryote or a eukaryote.
  • the prokaryotic host cell may be any Gram-positive or Gram-negative bacterium.
  • the host cell may also be an eukaryotic cell, such as a yeast, fungal, mammalian, insect or plant cell.
  • the host cell is selected from the group of Escherichia coli, Bacillus, Streptomyces, Trichoderma, Aspergillus, Saccharomyces, Pichia, Vibrio or Yarrowia.
  • the nucleic acid, expression cassette or expression vector according to the invention may be introduced into the host cell by any method known by the skilled person, 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.
  • more than one copy of a nucleic acid, cassette or vector of the present invention may be inserted into a host cell to increase production of the variant.
  • the host cell is a recombinant microorganism.
  • the invention indeed allows the engineering of microorganisms with improved capacity to degrade polyester containing material.
  • the sequence of the invention may be used to complement a wild type strain of a fungus or bacterium already known as able to degrade polyester, in order to improve and/or increase the strain capacity.
  • the present invention relates to in vitro methods of producing an esterase of the present invention comprising (a) contacting a nucleic acid, cassette or vector of the invention with an in vitro expression system; and (b) recovering the esterase produced.
  • in vitro expression systems are well-known by the person skilled in the art and are commercially available.
  • the method of production comprises
  • the host cell is a recombinant Bacillus, recombinant E. coli, recombinant Aspergillus, recombinant Trichoderma, recombinant Streptomyces, recombinant Saccharomyces, recombinant Pichia, recombinant Vibrio or recombinant Yarrowia.
  • the host cells are cultivated in a nutrient medium suitable for production of polypeptides, using methods known in the art.
  • the cell may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed- batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the enzyme to be expressed and/or isolated.
  • the cultivation takes place in a suitable nutrient medium, from commercial suppliers or prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the esterase is excreted into the nutrient medium, the esterase can be recovered directly from the culture supernatant. Conversely, the esterase can be recovered from cell lysates or after permeabilisation.
  • the esterase may be recovered using any method known in the art.
  • the esterase may be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.
  • the esterase may be partially or totally purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction to obtain substantially pure polypeptides.
  • chromatography e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion
  • electrophoretic procedures e.g., preparative isoelectric focusing
  • differential solubility e.g., ammonium sulfate precipitation
  • the esterase may be used as such, in purified form, either alone or in combinations with additional enzymes, to catalyze enzymatic reactions involved in the degradation and/or recycling of polyester(s) and/or polyester containing material, such as plastic products containing polyester.
  • the esterase may be in soluble form, or on solid phase. In particular, it may be bound to cell membranes or lipid vesicles, or to synthetic supports such as glass, plastic, polymers, filter, membranes, e.g., in the form of beads, columns, plates and the like.
  • composition of the invention may comprise from 0.1% to 99.9%, preferably from 0.1% to 50%, more preferably from 0.1% to 30%, even more preferably from 0.1% to 5% by weight of esterase, based on the total weight of the composition.
  • the composition may comprise between 5 and 10% by weight of esterase of the invention.
  • the composition may be in liquid or dry form, for instance in the form of a powder.
  • the composition is a lyophilizate.
  • the composition may further comprise excipients and/or reagents etc.
  • excipients encompass buffers commonly used in biochemistry, agents for adjusting pH, preservatives such as sodium benzoate, sodium sorbate or sodium ascorbate, conservatives, protective or stabilizing agents such as starch, dextrin, arabic gum, salts, sugars e.g. sorbitol, trehalose or lactose, glycerol, polyethyleneglycol, polypropylene glycol, propylene glycol, sequestering agent such as EDTA, reducing agents, amino acids, a carrier such as a solvent or an aqueous solution, and the like.
  • the composition of the invention may be obtained by mixing the esterase with one or several excipients.
  • the composition comprises from 0.1% to 99.9%, preferably from 50% to 99.9%, more preferably from 70% to 99.9%, even more preferably from 95% to 99.9% by weight of excipient(s), based on the total weight of the composition.
  • the composition may comprise from 90% to 95% by weight of excipient(s).
  • composition may further comprise additional polypeptide(s) exhibiting an enzymatic activity.
  • additional polypeptide(s) exhibiting an enzymatic activity.
  • esterase of the invention will be easily adapted by those skilled in the art depending e.g., on the nature of the polyester to degrade and/or the additional enzymes/polypeptides contained in the composition.
  • the esterase of the invention may be solubilized in an aqueous medium together with one or several excipients, especially excipients which are able to stabilize or protect the polypeptide from degradation.
  • the esterase of the invention may be solubilized in water, eventually with additional components, such as glycerol, sorbitol, dextrin, starch, glycol such as propanediol, salt, etc.
  • additional components such as glycerol, sorbitol, dextrin, starch, glycol such as propanediol, salt, etc.
  • the resulting mixture may then be dried so as to obtain a powder.
  • Methods for drying such mixture are well known to the one skilled in the art and include, without limitation, lyophilisation, freeze-drying, spray-drying, supercritical drying, downdraught evaporation, thin-layer evaporation, centrifugal evaporation, conveyer drying, fluidized bed drying, drum drying or any combination thereof.
  • the composition may be under powder form and may comprise esterase and a stabilizing/solubilizing amount of glycerol, sorbitol or dextrin, such as maltodextrine and/or cyclodextrine, starch, glycol such as propanediol, and/or salt.
  • esterase a stabilizing/solubilizing amount of glycerol, sorbitol or dextrin, such as maltodextrine and/or cyclodextrine, starch, glycol such as propanediol, and/or salt.
  • the composition of the invention may comprise at least one recombinant cell expressing an esterase of the invention, or an extract thereof.
  • An “extract of a cell” designates any fraction obtained from a cell, such as cell supernatant, cell debris, cell walls, DNA extract, enzymes or enzyme preparation or any preparation derived from cells by chemical, physical and/or enzymatic treatment, which is essentially free of living cells. Preferred extracts are enzymatically-active extracts.
  • the composition of the invention may comprise one or several recombinant cells of the invention or extract thereof, and optionally one or several additional cells.
  • the composition consists or comprises a culture medium of a recombinant microorganism expressing and excreting an esterase of the invention.
  • the composition comprises such culture medium lyophilized.
  • the esterases of the invention are particularly useful for degrading PET and PET containing material, particularly at a pH between 6 and 10.
  • esterase of the invention or corresponding recombinant cell or extract thereof having an esterase activity, or composition for the enzymatic degradation of a polyester.
  • the polyester targeted by the esterase is selected from polyethylene terephthalate (PET), polytrimethylene 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), polyethylene adipate) (PEA), polyethylene naphthalate (PEN), “polyolefin-like” polyesters and blends/mixtures of these materials, preferably polyethylene terephthalate.
  • PET polyethylene terephthalate
  • PTT polytrimethylene terephthalate
  • PBT polybutylene terephthalate
  • PEIT polyethylene isosorbide terephthalate
  • PLA polylactic acid
  • PBS poly
  • 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), bi s(2-hydroxy ethyl) terephthalate (BHET), 1 -(2 -Hydroxy ethyl) 4-methyl terephthalate (HEMT) and dimethyl terephthalate (DMT)) are optionally recovered.
  • monomers e.g., monoethylene glycol or terephthalic acid
  • oligomers e.g., methyl-2-hydroxyethyl terephthalate (MHET), bi s(2-hydroxy ethyl) terephthalate (BHET), 1 -(2 -Hydroxy ethyl) 4-methyl terephthalate (HEMT) and dimethyl terephthalate (DMT)
  • esterase of the invention or corresponding recombinant cell or extract thereof, or composition for the enzymatic degradation of at least one polyester of a polyester containing material, particularly at a pH between 6 and 10.
  • polyester(s) is (are) depolymerized up to monomers and/or oligomers.
  • the invention provides a method for degrading PET of a PET containing material, wherein the PET containing material is contacted with an esterase or host cell or composition of the invention, preferably at a pH between 6 and 10, thereby degrading the PET.
  • at least one polyester is degraded into repolymerizable monomers and/or oligomers, which may be advantageously retrieved in order to be reused.
  • the retrieved monomers/oligomers may be used for recycling (e.g., repolymerizing polyesters) or methanization.
  • At least one polyester is PET, and monoethylene glycol, terephthalic acid, methyl -2-hydroxy ethyl terephthalate (MEET), bi s(2-hydroxy ethyl) terephthalate (BEET), 1 -(2 -Hydroxy ethyl) 4-methyl terephthalate (HEMT) and/or dimethyl terephthalate (DMT) are retrieved.
  • MEET methyl -2-hydroxy ethyl terephthalate
  • BEET bi s(2-hydroxy ethyl) terephthalate
  • HEMT 1 -(2 -Hydroxy ethyl) 4-methyl terephthalate
  • DMT dimethyl terephthalate
  • polyester(s) of the polyester containing material is (are) fully degraded.
  • the time required for degrading a polyester containing material may vary depending on the polyester containing material itself (i.e., nature and origin of the polyester containing material, its composition, shape etc.), the type and amount of esterase used, as well as various process parameters (i.e., temperature, pH, additional agents, etc.).
  • process parameters i.e., temperature, pH, additional agents, etc.
  • One skilled in the art may easily adapt the process parameters to the polyester containing material and the envisioned degradation time.
  • the degrading process is implemented 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.
  • the degrading process is implemented at 60°C.
  • the degrading process is implemented at 65°C.
  • the degrading process is implemented at 70°C.
  • the temperature is maintained below an inactivating temperature, which corresponds to the temperature at which the esterase is inactivated (i.e., temperature at which the esterase has lost more than 80% of activity as compared to its activity at its optimum temperature) and/or the recombinant microorganism does no more synthesize the esterase.
  • the temperature is maintained below the glass transition temperature (Tg) of the targeted polyester.
  • the process is implemented in a continuous flow process, at a temperature at which the esterase can be used several times and/or recycled.
  • the degrading process is implemented at a pH comprised between 5 to 9, preferably in a range of pH from 6 to 9, more preferably in a range of pH from 6.5 to 9, even more preferably in a range of pH from 6.5 to 8, even more preferably at a pH comprised between 7 and 9, in particular at pH 8.
  • the polyester containing material may be pretreated prior to be contacted with the esterase, in order to physically change its structure, so as to increase the surface of contact between the polyester and the esterase. It is another object of the invention to provide a method of producing monomers and/or oligomers from a polyester containing material, comprising exposing a polyester containing material to an esterase of the invention, or corresponding recombinant cell or extract thereof, or composition, particularly at a pH between 6 and 10, and optionally recovering monomers and/or oligomers.
  • Monomers and/or oligomers resulting from the depolymerization may be recovered, sequentially or continuously.
  • a single type of monomers and/or oligomers or several different types of monomers and/or oligomers may be recovered, depending on the starting polyester containing material.
  • the method of the invention is particularly useful for producing monomers selected from monoethylene glycol and terephthalic acid, and/or oligomers selected from methyl-2- hydroxy ethyl terephthalate (MHET), bis(2-hydroxy ethyl) terephthalate (BHET), l-(2- Hydroxyethyl) 4-methyl terephthalate (HEMT) and dimethyl terephthalate (DMT), from PET, and/or plastic product comprising PET.
  • MHET methyl-2- hydroxy ethyl terephthalate
  • BHET bis(2-hydroxy ethyl) terephthalate
  • HEMT l-(2- Hydroxyethyl) 4-methyl terephthalate
  • DMT dimethyl terephthalate
  • the recovered monomers and/or oligomers may be further purified, using all suitable purifying methods and conditioned in a re-polymerizable form.
  • Recovered repolymerizable monomers and/or oligomers may be reused for instance to synthesize polyesters.
  • polyesters of same nature are repolymerized.
  • the recovered monomers may be used as chemical intermediates in order to produce new chemical compounds of interest.
  • the invention also relates to a method of surface hydrolysis or surface functionalization of a polyester containing material, comprising exposing a polyester containing material to an esterase of the invention, or corresponding recombinant cell or extract thereof, or composition, particularly at a pH between 6 and 10.
  • the method of the invention is particularly useful for increasing hydrophilicity, or water absorbency, of a polyester material. Such increased hydrophilicity may have particular interest in textiles production, electronics and biomedical applications.
  • the invention also relates to a method for treating water, waste water or sewage, particularly at a pH between 6 and 10.
  • the esterase according to the invention can be used to degrade microplastic particles consisting of polyester (preferable PET) like polymer filaments, fibres or other kinds of polyester-based product debris and fragments, preferably PET -based product debris and fragments.
  • processes for preparing such polyester containing material including an esterase of the invention are disclosed in the patent applications WO2013/093355, WO 2016/198650, WO 2016/198652, WO 2019/043145 and WO 2019/043134.
  • the invention provides a polyester containing material containing an esterase of the invention and/or a recombinant cell and/or a composition or extract thereof and at least PET.
  • the invention provides a plastic product comprising PET and an esterase of the invention having a PET degrading activity.
  • the invention provides a polyester containing material containing an esterase of the invention and/or a recombinant cell and/or a composition or extract thereof and at least PBAT.
  • the invention provides a plastic product comprising PBAT and an esterase of the invention having a PBAT degrading activity.
  • the invention provides a polyester containing material containing an esterase of the invention and/or a recombinant cell and/or a composition or extract thereof and at least PBS.
  • the invention provides a plastic product comprising PBS and an esterase of the invention having a PBS degrading activity.
  • the invention provides a polyester containing material containing an esterase of the invention and/or a recombinant cell and/or a composition or extract thereof and at least PCL.
  • the invention provides a plastic product comprising PCL and an esterase of the invention having a PCL degrading activity.
  • an esterase of the invention may be used in detergent, food, animal feed, paper making, textile and pharmaceutical applications. More particularly, the esterase of the invention may be used as a component of a detergent composition.
  • Detergent compositions include, without limitation, hand or machine laundry detergent compositions, such as laundry additive composition suitable for pre-treatment of stained fabrics and rinse added fabric softener composition, detergent composition for use in general household hard surface cleaning operations, detergent compositions for hand or machine dishwashing operations.
  • an esterase of the invention may be used as a detergent additive.
  • the invention thus provides detergent compositions comprising an esterase of the invention.
  • the esterase of the invention may be used as a detergent additive in order to reduce pilling and greying effects during textile cleaning.
  • the present invention is also directed to methods for using an esterase of the invention in animal feed, as well as to feed compositions and feed additives comprising an esterase of the invention.
  • the esterase of the invention may also be used to hydrolyze proteins, and to produce hydrolysates comprising peptides. Such hydrolysates may be used as feed composition or feed additives.
  • esterase of the invention may be used to remove stickies from the paper pulp and water pipelines of paper machines.
  • Esterase according to the invention have been generated using the plasmidic construction pET26b-LCC-His.
  • This plasmid consists in cloning a gene encoding the esterase of SEQ ID NO: 1
  • the strains StellarTM (Clontech, California, USA) and E. coli One Shot® BL21 DE3 (Life technologies, Carlsbad, California, USA) have been successively employed to perform the cloning and recombinant expression in 50 mL LB-Miller medium or ZYM auto inducible medium (Studier et al., 2005- Prot. Exp. Pur. 41, 207-234).
  • the induction in LB-Miller medium has been performed at 16°C, with 0.5 mM of isopropyl P-D-l -thiogalactopyranoside (IPTG, Euromedex, Souffelweyersheim, France).
  • the cultures have been stopped by centrifugation (8000 rpm, 20 minutes at 10°C) in an Avanti J-26 XP centrifuge (Beckman Coulter, Brea, USA).
  • the cells have been suspended in 20 mL of Talon buffer (Tris-HCl 20 mM, NaCl 300 mM, pH 8). Cell suspension was then sonicated during 2 minutes with 30% of amplitude (2sec ON and Isec OFF cycles) by FB 705 sonicator (Fisherbrand, Illkirch, France). Then, a step of centrifugation has been realized: 30 minutes at 11000 rpm, 10°C in an Eppendorf centrifuge. The soluble fraction has been collected and submitted to affinity chromatography.
  • the degrading activity of the esterases has been determined and compared to the activity of esterase of SEQ ID NO: 1.
  • 100 mg of amorphous PET under powder form (prepared according to WO 2017/198786 to reach a crystallinity below 20%) were weighted and introduced in a 100 mL glass bottle.
  • 1 mL of esterase preparation comprising esterase of SEQ ID NO: 1 (as reference control) or esterase of the invention, prepared at 0.69pM or 3.45pM in Talon buffer (Tris-HCl 20 mM, NaCl 0.3M, pH 8) were introduced in the glass bottle (0.2 mg en zyme/gPET or 1.0 mgenzyme/gPEr). Finally, 49 mL of 0.1 M potassium phosphate buffer pH 8 were added.
  • the depolymerization started 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 reaction in mg of equivalent TA generated / hour, was determined by samplings performed at different time during the first 24 hours and analyzed by Ultra High Performance Liquid Chromatography (UHPLC). If necessary, samples were diluted in 0.1 M potassium phosphate buffer pH 8. Then, 150 pL of methanol and 6.5 pL of HC1 6 N were added to 150 pL of sample or dilution.
  • TA, MHET and BHET were separated using a gradient of MeOH (30 % to 90 %) in 1 mM of H2SO4 at ImL/min. Injection was 20 pL of sample.
  • TA, MHET and BHET were measured according to standard curves prepared from commercial TA and BHET and in house synthetized MHET in the same conditions than samples.
  • the specific activity of PET hydrolysis (mg of equivalent TA/hour/mg of enzyme) was determined in the linear part of the hydrolysis curve of the reaction (i.e. at the beginning of the reaction), such curve being set up by samplings performed at different time during the first 24, 48, 72, 96 hours.
  • Equivalent TA corresponds to the sum of TA measured and of TA contained in measured MHET and BHET. Said measurement of equivalent TA can also be used to calculate the yield of a PET depolymerization assay at a given time and/or after a defined period of time (e.g. 24h, 48h, 72h or 96h).
  • the diameters or the surface area of the halos formed due to the polyester degradation by parent esterase and variants were measured and compared after 2 to 24 hours at 40°C, 45 °C, 50°C, 55°C, 60°C, 65°C or 70°C.
  • the pH value was regulated to pH 8.0 by addition of a 20% NaOH (w/w) solution using the ROSITA 2.0 software (AD Biotec, France).
  • the kinetic of the PET depolymerization was followed based on NaOH consumption considering an exclusive production of TPA and MEG (2 mols of NaOH are consumed to titrate 1 mol of the diacid TPA).
  • the final yield of the PET depolymerization assay was determined either by the determination of residual PET weight or by the determination of equivalent TA generated, or through the base consumption.
  • Weight determination of residual PET was assessed by the filtration, at the end of the reaction, of the reactional volume through a 12 to 15 gm grade 11 ashless paper filter (Dutscher SAS, Brumath, France) and drying of such retentate before weighting it.
  • the determination of equivalent TA generated was realized using UHPLC methods described in 2.1, and the percentage of hydrolysis was calculated based on the ratio of molar concentration at a given time (TA + MHET + BHET) versus the total amount of TA contained in the initial sample.
  • PET depolymerization produced acid monomers that will be neutralized with the base to be able to maintain the pH in the reactor.
  • the determination of equivalent TA produced was calculating using the corresponding molar base consumption, and the percentage of hydrolysis was calculated based on the ratio of molar concentration at a given time of equivalent TA versus the total amount of TA contained in the initial sample.
  • 100 mg of amorphous PET under powder form (prepared according to WO 2017/198786 to reach a crystallinity below 20%) were weighted and introduced in a 100 mL glass bottle.
  • 1 mL of esterase preparation comprising esterase of SEQ ID NO: 1 (as reference control) or esterase of the invention, prepared at 0.69pM or 3.45pM in Talon buffer (Tris-HCl 20 mM, NaCl 0.3M, pH 8) were introduced in the glass bottle (0.2 mg en zyme/gPET or 1.0 mgenzyme/gPEr).
  • 49 mL of lOOmM potassium phosphate buffer pH 8.0 were added.
  • the depolymerization started 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).
  • reaction could be miniaturized in deepwell (ThermoSci entific, Abgene, AB-0661, Illkirch, France), 22 mg of amorphous PET under powder form (prepared according to WO 2017/198786 to reach a crystallinity below 20%) were weighted and introduced in each well of a deepwell plate.
  • esterase preparation comprising esterase of SEQ ID NO: 1 (as reference control) or esterase of the invention, prepared at 0.138pM in Talon buffer (Tris-HCl 20 mM, NaCl 0.3M, pH 8 or lOOmM potassium phosphate buffer pH 8.0) were introduced in each well of the deepwell. Finally, 0.9 mL of lOOmM potassium phosphate buffer pH 8.0 were added. The depolymerization 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 incubator shaker (Infors HT, Bottmingen, cilantro).
  • the initial rate of depolymerization reaction, in pmol of soluble degradation products generated / hour was determined by samplings performed at different time during the first 24 hours and analyzed by absorbance reading at 242 nm using an Eon Microplate Spectrophotometer (BioTek, USA).
  • the increase in absorbance of the reaction mixtures in the ultraviolet region of the light spectrum (at 242 nm) indicates the release of soluble TA or its esters (BHET and MHET) from an 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 enzyme-specific activity is determined as total equivalent TA produced. If necessary, samples were diluted in lOOmM potassium phosphate buffer pH8.0.
  • the specific activity of PET hydrolysis was determined in the linear part of the hydrolysis curve of the reaction (i.e. at the beginning of the reaction), such curve being set up by samplings performed at different time during the first 24 hours.
  • Said measurement of equivalent TA can also be used to calculate the yield of a PET depolymerization assay at a given time and/or after a defined period of time (e.g. 24h, 48h, 72h or 96h).
  • the specific degrading activity of esterases (variants) of the invention was measured at 55°C, 60°C or 65°C, pH 8 at the beginning of the reaction as exposed in Example 2.4. The results are shown in Table 1 below.
  • the specific degrading activity of the esterase of SEQ ID NO: 1 is used as a reference and considered as 100% specific degrading activity.
  • Table 1 Specific degrading activity of esterases of the invention at pH 8 compared to SEQ ID NO: 1.
  • Variants have the exact amino acid sequence of SEQ ID NO: 1 except the substitutions listed in Table 1.
  • PET depolymerization yield under basic conditions as compared to esterase of SEQ ID NO:1 The yield of PET depolymerization, of esterase (variants) of the invention was measured according to Example 2.4, after 6 hours or after 24 hours at 55°C, 60°C or 65°C, pH 8. In the context of the present invention, the PET depolymerization yield is used to evaluate the degrading activity. Results (average values) are shown in Table 2 and Table 3 , respectively.
  • the yield of PET depolymerization of the esterase of SEQ ID NO: 1 after 6 hours or after 24 hours, pH 8 is used as a reference and considered as 100% degrading activity, respectively.
  • Variants have the exact amino acid sequence of SEQ ID NO: 1 except the substitutions listed in Table 2.
  • Table 3 PET depolymerization yield of esterases of the invention after 24 hours.
  • Variants have the exact amino acid sequence of SEQ ID NO: 1 except the substitutions listed in Table 3.
  • the yield of PET depolymerization of esterase (variants) of the invention was measured in reactors according to Example 2.3, at 68°C, pH 8.
  • the PET depolymerization yield is used to evaluate the degrading activity. Results are shown in Table 4 below.
  • the time to reach 90% yield of PET depolymerization with the esterase of SEQ ID NO: 11, pH 8 is 22.2 hours. PET depolymerization was performed using 1.0 mg en zyme/gPET .
  • Table 4 Time to reach 90% of PET depolymerization yield with esterases of the invention.
  • Variants have the exact amino acid sequence of SEQ ID NO: 1 except the substitutions listed in Table 4.
  • the yield of PET depolymerization of esterase (variants) of the invention was measured in reactors according to Example 2.3, at 68°C, pH 8 with the only difference that the quantity of PET was 14% w/w except of 20%.
  • the PET depolymerization yield is used to evaluate the degrading activity. Results are shown in Table 5 below.
  • the time to reach 90% yield of PET depolymerization with the esterase of SEQ ID NO: 1, pH 8 is 20 hours. PET depolymerization was performed using 1.0 mg en zyme/gPET . Table 5: Time to reach 90% of PET depolymerization yield with esterases of the invention
  • Variants have the exact amino acid sequence of SEQ ID NO: 1 except the substitutions listed in Table 5. PET depolymerization yield under basic conditions as compared to esterase of SEQ ID NO: 2
  • the yield of PET depolymerization, of esterase (variants) of the invention was measured according to Example 2.4, after 6 hours or after 24 hours at 55°C, pH 8.
  • the PET depolymerization yield is used to evaluate the degrading activity. Results (average values) are shown in Table 6 and Table 7 , respectively after 6 hours or after 24 hours.
  • the yield of PET depolymerization of the esterase of SEQ ID NO:2 after 6 hours or after 24 hours, pH 8 is used as a reference and considered as 100% degrading activity, respectively.
  • thermostability of esterases of the invention has been determined and compared to the thermostability of the esterase of SEQ ID NO: 1 or SEQ ID NO:2.
  • thermostability Different methodologies have been used to estimate thermostability:
  • Circular dichroism has been performed with a Jasco 815 device (Easton, USA) to compare the melting temperature (Tm) of the esterase of SEQ ID NO: 1 with the Tm of the esterases of the invention.
  • Tm melting temperature
  • Technically 400pL protein sample was prepared at 0.5 mg / mL in Talon buffer and used for CD.
  • a first scan from 280 to 190 nm was realized to determine two maxima intensities of CD corresponding to the correct folding of the protein.
  • the T m obtained reflects the thermostability of the given protein. The higher the T m is, the more stable the variant is at high temperature.
  • Enzymatic reaction was performed at 30°C under agitation, during 15 minutes and absorbance at 405 nm was acquired by microplate spectrophotometer (Versamax, Molecular Devices, Sunnyvale, CA, USA).
  • Activity of pNP-B hydrolysis (initial velocity expressed in pmol of pNPB/min) was determined using a standard curve for the liberated para nitro phenol in the linear part of the hydrolysis curve.
  • 150 pL of buffer were sampled regularly. When required, samples were diluted in 0.1 M potassium phosphate buffer pH 8. Then, 150 pL of methanol and 6.5 pL of HC1 6 N were added to 150 pL of sample or dilution. After mixing and filtering on 0.45 pm syringe filter, samples were loaded on UHPLC to monitor the liberation of terephthalic acid (TA), MEET and BEET. Chromatography system used was an Ultimate 3000 UHPLC system (Thermo Fisher Scientific, Inc. Waltham, MA, USA) including a pump module, an autosampler, a column oven thermostated at 25°C, and an UV detector at 240 nm.
  • TA, MEET and BEET were separated using a gradient of MeOH (30 % to 90 %) in 1 mM of H2SO4 at ImL/min. Injection was 20 pL of sample.
  • TA, MEET and BEET were measured according to standard curves prepared from commercial TA and BEET and in house synthetized MEET in the same conditions than samples.
  • Activity of PET hydrolysis pmol of PET hydrolysed/min or mg of equivalent TA produced/hour was determined in the linear part of the hydrolysis curve, such curve being set up by samplings performed at different time during the first 24 hours.
  • Equivalent TA corresponds to the sum of TA measured and of TA contained in measured MEET and BEET.
  • the solution was mixed v/v with 0.2 M potassium phosphate buffer pH 8 containing 3% agar. Around 30 mL of the mixture was used to prepare each omni tray and stored at 4°C. The diameter or the surface area of the halos formed due to the polyester degradation by parent esterase and variants of the invention were measured and compared after
  • the half-life of the enzyme at a given temperature corresponds to the time required to decrease by a 2-fold factor the diameter of the halo.
  • the ability of the esterase to perform successive rounds of polyester’s depolymerization assays was evaluated in an enzymatic reactor.
  • a Minibio 500 bioreactor (Applikon Biotechnology B.V., Delft, The Netherlands) was started with 3 g of amorphous PET (prepared according to WO 2017/198786 to reach a crystallinity below 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. Bioreactor was thermostated at 50°C, 55°C, 60°C, 65°C or 70°C by immersion in an external water bath. pH was regulated at 8 by addition of KOH at 3 M.
  • Ethylene glycol was monitored using refractometer.
  • the percentages of hydrolysis were calculated based on the ratio of molar concentration at a given time (TA +MHET + BEET) versus the total amount of TA contained in the initial sample, or based on the ratio of molar concentration at a given time (EG +MHET + 2 x BEET) versus the total amount of EG contained in the initial sample.
  • Rate of degradation is calculated in mg of total liberated TA per hour or in mg of total EG per hour.
  • Half-life of enzyme was evaluated as the incubation time required to obtain a loss of 50 % of the degradation rate.
  • DSF was used to evaluate the thermostability of the parent protein (SEQ ID NO: 1) and variants thereof by determining their melting temperature (Tm), temperature at which half of the protein population is unfolded.
  • Protein samples were prepared at a concentration of 6.25 pM and stored in buffer A consisting of lOOmM potassium phosphate buffer, pH 8.
  • the SYPRO orange dye 5000x stock solution in DMSO was first diluted to 250x in water. Protein samples were loaded onto a white clear 96-well PCR plate (Bio-Rad cat# HSP9601) with each well containing a final volume of 25 pL. The final concentration of protein and SYPRO Orange dye in each well were 6 pM (0.17 mg/ml) and 10X respectively.
  • Loaded volumes per well were as follow: 24 pL of the 6.25 pM protein solution and 1 pL of the 250x Sypro Orange diluted solution.
  • the PCR plates were then sealed with optical quality sealing tape and spun at 1000 rpm for 1 min at room temperature. DSF experiments were then carried out using a CFX96 real-time PCR system set to use the 450/490 excitation and 560/ 580 emission filters. The samples were heated from 25 to 100°C at the rate of 0.3°C/second. A single fluorescence measurement was taken every 0.03 second. Melting temperatures were determined from the peak(s) of the first derivatives of the melting curve using the Bio-Rad CFX Manager software.
  • Variation of buffer type or buffer concentration may be used, with no impact on the delta Tm between the esterase of the invention and the parent, as far as the same buffer is used for the parent esterase.
  • Esterase of SEQ ID NO: 1 or SEQ ID NO:2 and esterases of the invention were then compared based on their Tm values. Due to high reproducibility between experiments on the same protein from different productions, a ATm of 0.8°C was considered as significant to compare variants. Tm values correspond to the average of at least 3 measurements. The Tm of the esterase of SEQ ID NO: 1 is evaluated at 84.7°C.

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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

NOVEL ESTERASES AND USES THEREOF
The present invention relates to novel esterases, more particularly to esterases having improved degrading activity and/or improved thermostability compared to a parent esterase at a pH comprised between 6 and 10, preferably at a pH comprised between 7 and 9. The present invention also relates to uses of said novel esterases for degrading polyester containing material, such as plastic products. The esterases of the invention are particularly suited to degrade polyethylene terephthalate, and polyethylene terephthalate containing material.
BACKGROUND
Esterases are able to catalyze the hydrolysis of a variety of polymers, including polyesters. In this context, esterases have shown promising effects in a number of industrial applications, including detergents for dishwashing and laundry applications, degrading enzymes for processing biomass and food, biocatalysts in detoxification of environmental pollutants or for the treatment of polyester fabrics in the textile industry. The use of esterases as degrading enzymes for hydrolyzing polyethylene terephthalate (PET) is of particular interest. Indeed, PET is used in a large number of technical fields, such as in the manufacture of clothes, carpets, or in the form of a thermoset resin for the manufacture of packaging or automobile plastics, etc., so that PET accumulation in landfills becomes an increasing ecological problem.
The enzymatic degradation of polyesters, and particularly of PET, is considered as an interesting solution to decrease plastic and textile waste accumulation. Indeed, enzymes may accelerate hydrolysis of polyester containing material, and more particularly of plastic and textile products, even up to the monomer level. Furthermore, the hydrolysate (i.e., monomers and oligomers) can be recycled as material for synthesizing new polymers.
In this context, several esterases have been identified as candidate degrading enzymes for polyesters, and some variants of such esterases have been developed. Among esterases, cutinases, also known as cutin hydrolases (EC 3.1.1.74), are of particular interest. Cutinases have been identified from various fungi (P.E. Kolattukudy in "Lipases", Ed. B. Borg- strom and H.L. Brockman, Elsevier 1984, 471-504), bacteria and plant pollen. Recently, metagenomics approaches have led to identification of additional esterases. For instance, a metagenome- derived cutinase (as described in Sulaiman et al., Appl Environ Microbiol. 2012 Mar, and referenced G9BY57 in SwissProt) and variants thereof (as described for instance in W02018011284, W02018011281, W02020021116, W02020021117, W02020021118) exhibit capacity to degrade polyester, and more particularly polyethylene terephthalate. However, there is still a need for esterases with improved activity and/or improved thermostability compared to already known esterases, to provide polyester degrading processes more efficient and thereby more competitive particularly at a pH between 6 and 10.
SUMMARY OF THE INVENTION
The present invention provides new variants of esterases exhibiting increased polyester degrading activity and/or increased thermostability at pH between 6 and 10 compared to a parent esterase, having the amino acid sequence as set forth in SEQ ID NO: 1.
The esterases of the present invention are particularly useful in processes for degrading plastic products under basic conditions, more particularly plastic products containing PET.
In this regard, it is thus an object of the 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 set forth in SEQ ID NO: 1, (ii) has at least one amino acid substitution selected from 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, H69M, P179N, 1185 V, F187L/Q, P192S, P213A, L227N, Q237L, L239M, R251S, S66T, F90C/V, P151V, A246R, A215E/D, D230E, L15K/R, N143D, V200I and N253D, or at least one combination of substitutions selected from 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 as compared to SEQ ID NO: 1, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1, (iii) has a polyester degrading activity and (iv) exhibits an increased thermostability and/or an increased polyester degrading activity as compared to the esterase of SEQ ID NO: 1 at a pH comprised between 6 and 10.
Preferably, the esterase comprises at least one amino acid substitution or at least one combination of substitutions selected from H183Q/L/A/Y/N/DZE/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, H85V, F187L/Q, P192S, P213A, L227N, Q237L, L239M, R251S, S66T, F90C/V, P151V, A246R, A215E/D, D230E, L15K/R, N253D, N204G + M208L + N211E, F90E + N204G + N21 IE, F90N + N204G + N21 IE, F90Q + N204G + N21 IE, F90R + N204G + N21 IE, F90W + N204G + N21 IE, F187I + N204G + N21 IE, S98R + E173Q and M208N + N211M. In a particular embodiment, the esterase comprises at least one combination of substitutions selected from N204G + H183Q + F187I, M208N + N211M or H183Q + F187I + N204G + N211M + S193E.
It is a further object of the invention to provide an esterase variant which (i) has at least 97%, 98% or 99% identity to the full length amino acid sequence set forth in SEQ ID NO: 1, (ii) has at least one amino acid substitution selected from 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, VI 151, S181K, H183S, Fl 871, 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 at least one combination of substitutions selected from N204G + M208L + N21 IE, F90E + N204G + N21 IE, F90N + N204G + N21 IE, F90Q + N204G + N21 IE, F90R + N204G + N21 IE, F90W + N204G + N21 IE, F187I + N204G + N21 IE, S98R + E173Q, M208Q + N21 IM and M208N + N211M as compared to the amino acid sequence SEQ ID NO: 1, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1, (iii) has at least the amino acids C240, C275, 1170, G92, P213, E182, L13 and E158, as in the parent esterase of SEQ ID NO: 1, (iv) has a polyester degrading activity, and (v) exhibits an increased thermostability and/or an increased polyester degrading activity as compared to the esterase of SEQ ID NO: 1 at a pH comprised between 6 and 10.
It is a further object of the invention 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 set forth in SEQ ID NO:2, (ii) comprises at least one amino acid substitution selected from 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 and V200I, or at least one combination of substitutions selected from N204G + M208L + N211E, L90E + N204G + N21 IE, L90N + N204G + N21 IE, L90Q + N204G + N21 IE, L90R + N204G + N21 IE, L90W + N204G + N211E, F187I + N204G + N211E, S98R + E173Q, M208A + N211M, M208G + N21 IM, M208K + N21 IM, M208N + N21 IM, M208P + N21 IM, M208Q + N21 IM, M208H + N211M and M208T + N211M, as compared to the amino acid sequence SEQ ID NO:2, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO:2, (iii) has a polyester degrading activity and (iv) exhibits an increased thermostability and/or an increased polyester degrading activity as compared to the esterase of SEQ ID NO:2 at a pH comprised between 6 and 10.
It is another object of the invention to provide an esterase variant which (i) has at least 97%, 98% or 99% identity to the full length amino acid sequence set forth 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 at least the combination of substitutions M208I + N211M, as compared to the amino acid sequence SEQ ID NO:2, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO:2, (iii) has at least the amino acids C240, C275, 1170, F92, P213, E182, L13 and E158, as in the parent esterase of SEQ ID NO:2, (iv) has a polyester degrading activity, and (v) exhibits an increased thermostability and/or an increased polyester degrading activity as compared to the esterase of SEQ ID NO:2 at a pH comprised between 6 and 10.
It is another object of the invention to provide a nucleic acid encoding an esterase of the invention. The present invention also relates to an expression cassette or an expression vector comprising said nucleic acid, and to a host cell comprising said nucleic acid, expression cassette or vector.
The present invention also provides a composition comprising an esterase of the present invention, a host cell of the present invention, or extract thereof.
It is a further object of the invention to provide a method of producing an esterase of the invention comprising:
(a) culturing the host cell according to the invention under conditions suitable to express a nucleic acid encoding an esterase; and optionally
(b) recovering said esterase from the cell culture.
It is a further object of the invention to provide a method of degrading a polyester or a polyester of a polyester containing material comprising
(a) contacting the polyester with an esterase according to the invention or a host cell according to the invention or a composition according to the invention; and, optionally
(b) recovering monomers and/or oligomers.
Advantageously, at least step a) is performed in basic conditions, particularly at a pH between 6 and 10, preferably at a pH between 7 and 9. Particularly, the invention provides a method of degrading PET, comprising contacting PET with at least one esterase of the invention, and optionally recovering monomers and/or oligomers of PET. Advantageously, at least the step of contacting PET with said esterase of the invention is performed in basic conditions, particularly at a pH between 6 and 10, preferably at a pH between 7 and 9.
The invention also relates to the use of an esterase of the invention for degrading PET or a plastic product containing PET. Advantageously, said use is performed in basic conditions, particularly at a pH between 6 and 10, preferably at a pH between 7 and 9.
The present invention also relates to a polyester containing material in which an esterase or a host cell or a composition of the invention is included.
The present invention also relates to a detergent composition comprising the esterase or host cell according to the invention or a composition comprising an esterase of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
The present disclosure will be best understood by reference to the following definitions.
Herein, the terms "peptide", "polypeptide", "protein", "enzyme" refer to a chain of amino acids linked by peptide bonds, regardless of the number of amino acids forming said chain. The amino acids are herein represented by their one-letter or three-letters code according to the following nomenclature: A: alanine (Ala); C: cysteine (Cys); D: aspartic acid (Asp); E: glutamic acid (Glu); F: phenylalanine (Phe); G: glycine (Gly); H: histidine (His); I: isoleucine (He); K: lysine (Lys); L: leucine (Leu); M: methionine (Met); N: asparagine (Asn); P: proline (Pro); Q: glutamine (Gin); R: arginine (Arg); S: serine (Ser); T: threonine (Thr); V: valine (Vai); W: tryptophan (Trp ) and Y: tyrosine (Tyr).
The term "esterase" refers to an enzyme which belongs to a class of hydrolases classified as EC 3.1.1 according to Enzyme Nomenclature that catalyzes the hydrolysis of esters into an acid and an alcohol. The term "cutinase" or "cutin hydrolase" refers to the esterases classified as EC 3.1.1.74 according to Enzyme Nomenclature that are able to catalyse the chemical reaction of production of cutin monomers from cutin and water.
The term "parent protein" refers to the esterase having the amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO:2. The terms “mutant” and “variant” refer to polypeptides derived from SEQ ID NO: 1 or SEQ ID NO:2 and comprising at least one modification or alteration, i.e., a substitution, insertion, and/or deletion, at one or more (e.g., several) positions as compared to SEQ ID NO: 1 or SEQ ID NO:2, and having a polyester degrading activity. The variants may be obtained by various techniques well known in the art. In particular, examples of techniques for altering the DNA sequence encoding the parent protein, include, but are not limited to, site-directed mutagenesis, random mutagenesis and synthetic oligonucleotide construction. Thus, the terms “modification” and “alteration” as used herein in relation to a particular position means that the amino acid in this particular position has been modified compared to the amino acid in this particular position in the parent protein.
A “substitution” means that an amino acid residue is replaced by another amino acid residue. Preferably, the term “substitution” refers to the replacement of an amino acid residue by another selected from the naturally-occurring standard 20 amino acid residues, rare naturally occurring amino acid residues (e.g. hydroxyproline, hydroxylysine, allohydroxylysine, 6-N-methylysine, N-ethylglycine, N-methylglycine, N-ethylasparagine, allo-isoleucine, N-methylisoleucine, N- methylvaline, pyroglutamine, aminobutyric acid, ornithine, norleucine, norvaline), and non- naturally occurring amino acid residue, often made synthetically, (e.g. cyclohexyl-alanine). Preferably, the term “substitution” refers to the replacement of an amino acid residue by another selected from the naturally-occurring standard 20 amino acid residues (G, P, A, V, L, I, M, C, F, Y, W, H, K, R, Q, N, E, D, S and T). The sign “+” indicates a combination of substitutions. In the present document, the following terminology is used to designate a substitution: L82A denotes that amino acid residue (Leucine, L) at position 82 of the parent sequence is substituted by an Alanine (A). A121 V/I/M denotes that amino acid residue (Alanine, A) at position 121 of the parent sequence is substituted by one of the following amino acids: Valine (V), Isoleucine (I), or Methionine (M). The substitution can be a conservative or non-conservative substitution. Examples of conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid 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 small amino acids (glycine, alanine and serine).
Unless otherwise specified, the amino acid positions disclosed in the present application are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 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. The sequence identity is determined by comparing the sequences when aligned so as to maximize overlap and identity while minimizing sequence gaps. In particular, sequence identity may be determined using any of a number of mathematical global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithm (e.g. Needleman and Wunsch algorithm; Needleman and Wunsch, 1970) which aligns the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g. Smith and Waterman algorithm (Smith and Waterman, 1981) or Altschul algorithm (Altschul et al., 1997; Altschul et al., 2005)). Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software available on internet web sites 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 needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, % amino acid sequence identity values refers to values generated using the pairwise sequence alignment program EMBOSS Needle that creates an optimal global alignment of two sequences using the Needleman-Wunsch algorithm, wherein all search parameters are set to default values, i.e. Scoring matrix = BLOSUM62, Gap open = 11, Gap extend = 1.
A "polymer" refers to a chemical compound or mixture of compounds whose structure is constituted of multiple monomers (repeat units) linked by covalent chemical bonds. Within the context of the invention, the term polymer includes natural or synthetic polymers, constituted of a single type of repeat unit (i.e., homopolymers) or of a mixture of different repeat units (i.e., copolymers or heteropolymers). According to the invention, “oligomers” refer to molecules containing from 2 to about 20 monomers.
In the context of the invention, a “polyester containing material” or “polyester containing product” refers to a product, such as plastic product, comprising at least one polyester in crystalline, semi-crystalline or totally amorphous forms. In a particular embodiment, the polyester containing material refers to any item made from at least one plastic material, such as plastic sheet, tube, rod, profile, shape, film, massive block, fiber, etc., which contains at least one polyester, and possibly other substances or additives, such as plasticizers, mineral or organic fillers. In another particular embodiment, the polyester containing material refers to a plastic compound, or plastic formulation, in a molten or solid state, suitable for making a plastic product. In another particular embodiment, the polyester containing material refers to textile, fabrics or fibers comprising at least one polyester. In another particular embodiment, the polyester containing material refers to plastic waste or fiber waste comprising at least one polyester. Particularly, the plastic article is a manufactured product, such as rigid or flexible packaging (bottle, trays, cups, etc.), agricultural films, bags and sacks, disposable items or the like, carpet scrap, fabrics, textiles, etc. The plastic article may contain additional substances or additives, such as plasticizers, minerals, organic fillers or dyes. In the context of the invention, the plastic article may comprise a mix of semi-crystalline and/or amorphous polymers and/or additives.
In the present description, the term “polyester(s)” encompasses but is not limited to polyethylene terephthalate (PET), polytrimethylene 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), polyethylene adipate) (PEA) , polyethylene naphthalate (PEN) and blends/mixtures of these polymers. Polyesters can also encompasses “polyolefin-like” polyesters, preferably “polyethylene-like” polyesters which correspond to polyolefin (preferably polyethylene) into which ester segments have been introduced (generally achieved by polycondensation of long-chain a, co-difunctional monomers), as defined in Lebarbe et al. Green Chemistry Issue 4 2014.
New esterases
The present invention provides novel esterases with improved activity and/or improved thermostability compared to a parent esterase in basic conditions, particularly at a pH comprised between 6 and 10. More particularly, the inventors have designed novel enzymes particularly suited for use in industrial processes in basic conditions. The esterases of the invention are particularly suited to degrade polyesters, more particularly PET, including PET containing material and particularly plastic product containing PET. In a particular embodiment, the esterases exhibit both an increased activity and an increased thermostability as compared to the parent esterase in basic conditions.
According to the present invention, “basic conditions” refer to conditions (e.g., medium, solution, etc.) at a pH comprised between 6 and 10. Particularly, “basic conditions” refer to the conditions to perform the degradation step of the polyester, i.e., the esterase is contacted with the polyester in a medium having a pH between 6 and 10.
The esterases of the present invention exhibit an increased activity and/or an increased thermostability as compared to the parent esterase in basic conditions. Particularly, the esterases of the present invention exhibit an increased activity and/or an increased thermostability as compared to the parent esterase when submitted at a pH between 6 and 10.
According to the invention, the increased activity and/or increased thermostability may be observed at specific pH between 6 and 10 and/or in a range of pH between 6 and 10. Particularly, the increased activity and/or increased thermostability may be observed at least at pH 6, at pH 6.5, at pH 7, at pH 7.5, at pH 8, at pH 8.5, at pH 9, at pH 9.5, and/or at pH 10. The increased activity and/or increased thermostability may also be observed in the whole range of pH 6 to 10, in the whole range of pH 6 to 9, in the whole range of pH 6.5 to 9, in the whole range of pH 7 to 9, in the whole range of pH 7.5 to 8.5, in the whole range of pH 7 to 8.5, in the whole range of pH 7.5 to 9.
It is therefore an object of the present invention to provide esterases that exhibit an increased activity at a pH comprised between 6 and 10, compared to the esterase having the amino acid sequence as set forth in the parent esterases at same pH. In the context of the present invention, the parent esterase may be either the esterase of SEQ ID NO: 1 or SEQ ID NO:2.
Particularly, the inventors have identified specific amino acid substitutions in SEQ ID NO: 1 and SEQ ID NO:2, which advantageously lead to an increased activity of the esterases on polymers in basic conditions, particularly on polyesters, more particularly on polyethylene terephthalate (PET).
Within the context of the invention, the term “increased activity” or “increased degrading activity” indicates an increased ability of the esterase to degrade a polyester and/or an increased ability to adsorb on a polyester, at given conditions (e.g., temperature, pH, concentration) as compared to the ability of the parent esterase to degrade and/or adsorb on same polyester under same conditions. Particularly, the esterase of the invention has an increased PET degrading activity. Such an increased activity may be at least 10% greater than the polyester degrading activity of the parent esterase , preferably at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130% or greater. Particularly, the degrading activity is a depolymerization activity leading to monomers and/or oligomers of the polyester, which can be further retrieved and optionally reused. Within the context of the invention, the esterase exhibits an increased degrading activity at least under basic conditions, in particular at a pH comprised between 6 and 10, as compared to the degrading activity of the parent esterase at same pH. 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 instance at pH 8.
The “degrading activity” of an esterase may be evaluated by the one skilled in the art, according to methods known per se in the art. For instance, the degrading activity can be assessed by measurement of the specific polymer’s depolymerization activity rate, the measurement of the rate to degrade a solid polymer compound dispersed in an agar plate, or the measurement of the polymer’s depolymerization activity rate in reactor. Particularly, the degrading activity may be evaluated by measuring the “specific degrading activity” of an esterase. The “specific degrading activity” of an esterase for PET corresponds to pmol of PET hydrolyzed/min or mg of equivalent TA produced/hour and per mg of esterase during the initial period of the reaction (i.e. the first 24 hours) and is determined from the linear part of the hydrolysis curve of the reaction, such curve being set up by several samplings performed at different time during the first 24 hours. As another example, the “degrading activity” may be evaluated by measuring, after a defined period of time (for example after 24h, 48h or 72h), the rate and/or yield of oligomers and/or monomers released under suitable conditions of temperature, pH and buffer, when contacting the polymer or the polymer-containing plastic product with a degrading enzyme.
The ability of an enzyme to adsorb on a substrate may be evaluated by the one skilled in the art, according to methods known per se in the art. For instance, the ability of an enzyme to adsorb on a substrate can be measured from a solution containing the enzyme and wherein the enzyme has been previously incubated with a substrate under suitable conditions.
The inventors have also identified target amino acid residues in the parent esterase, that may be advantageously modified to improve the stability of corresponding esterases in basic conditions at elevated temperatures (i.e., improved thermostability), and advantageously at temperature at or above 50°C and at or below 90°C, preferably at or above 60°C and at or below 80°C, more preferably at or above 65°C and at or below 75°C.
It is therefore an object of the present invention to provide new esterases that exhibit increased thermostability under basic conditions as compared to the thermostability of the esterase having the amino acid sequence set forth in the parent esterase at same pH.
Within the context of the invention, the term “increased thermostability” indicates an increased ability of an esterase to resist to changes in its chemical and/or physical structure at high temperatures, and particularly at temperature between 50°C and 90°C, as compared to the parent esterase. In a particular embodiment, the thermostability of the esterases is improved, in basic conditions, as compared to the thermostability of the parent esterase, at temperature(s) 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. Particularly, the thermostability of the esterases is improved, in acidic conditions, as compared to the thermostability of the parent esterase, at temperature(s) between 40°C and 80°C, between 50°C and 72°C, 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 esterases is improved, as compared to the thermostability of the parent esterase, at least at temperatures between 50°C and 65°C. Within the context of the invention, temperatures are given at +/- 1°C.
Particularly, the thermostability may be evaluated through the assessment of the melting temperature (Tm) of the esterase. In the context of the present invention, the “melting temperature” refers to the temperature at which half of the enzyme population considered is unfolded or misfolded. Typically, esterases of the invention show an increased Tm of about 0.8°C, 1°C, 2°C, 3°C, 4°C, 5°C, 10°C or more, as compared to the Tm of the parent esterase under acidic conditions, in particular at a pH comprised between 6 and 10. In particular, at a pH comprised between 6 and 10, esterases of the present invention can have an increased half- life at a temperature between 50°C and 90°C, as compared to the parent esterase. Particularly, esterases of the present invention can have an increased half-life at temperature 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, as compared to the esterase of SEQ ID NO: 1. Preferably, the esterases of the present invention have an increased half-life at least at temperature between 50°C and 65°C, as compared to the parent esterase.
Within the context of the invention, the esterases of the present invention exhibit an increased thermostability as compared to the thermostability of the esterase having the amino acid sequence set forth 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 an 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 instance at pH 8.
The melting temperature (Tm) of an esterase may be measured by the one skilled in the art, according to methods known per se in the art. For instance, the DSF may be used to quantify the change in thermal denaturation temperature of the esterase and thereby to determine its Tm. Alternatively, the Tm can be assessed by analysis of the protein folding using circular dichroism. Preferably, the Tm is measured using DSF or circular dichroism as exposed in the experimental part. In the context of the invention, comparisons of Tm are performed with Tm that are measured under same conditions (e.g. pH, nature and amount of polyesters, etc.).
Alternatively, the thermostability may be evaluated by measuring the esterase activity and/or the polyester depolymerization activity of the esterase after incubation at different temperatures and comparing with the esterase activity and/or polyester depolymerization activity of the parent esterase. The ability to perform multiple rounds of polyester’s depolymerization assays at different temperatures can also be evaluated. A rapid and valuable test may consist of the evaluation, by halo diameter measurement, of the esterase ability to degrade a solid polyester compound dispersed in an agar plate after incubation at different temperatures.
It is an object of the 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 set forth in SEQ ID NO: 1, (ii) has at least one amino acid substitution selected from 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, Il 85V, F187L/Q, P192S, P213A, L227N, Q237L, L239M, R251S, S66T, F90C/V, Pl 5 IV, A246R, A215E/D, D230E, L15K/R, N143D, V200I and N253D, or at least one combination of substitutions selected from N204G + M208L + N21 IE, F90E + N204G + N21 IE, F90N + N204G + N21 IE, F90Q + N204G + N21 IE, F90R + N204G + N21 IE, F90W + N204G + N21 IE, F187I + N204G + N21 IE, S98R + E173Q, M208Q + N21 IM and M208N + N21 IM as compared to SEQ ID NO: 1, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1, (iii) has a polyester degrading activity and (iv) exhibits an increased thermostability and/or an increased polyester degrading activity as compared to the esterase of SEQ ID NO: 1 at a pH comprised between 6 and 10.
In an embodiment, the esterase comprises at least one amino acid substitution or at least one combination of substitutions selected from 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 + N21 IE, F90N + N204G + N21 IE, F90Q + N204G + N21 IE, F90R + N204G + N21 IE, F90W + N204G + N21 IE, F187I + N204G + N21 IE, S98R + E173Q and M208N + N211M.
In an embodiment, the esterase comprises at least one amino acid substitution selected from 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, Il 85V, F187L/Q, P192S, P213A, L227N, Q237L, L239M, R251S, S66T, F90C/V, Pl 5 IV, A246R, A215E/D, D230E, L15K/R, or at least one combination of substitutions selected from N204G + M208L + N21 IE, F90E + N204G + N211E, F90N + N204G + N211E, F90Q + N204G + N211E, F90R + N204G + N21 IE, F90W + N204G + N21 IE, F187I + N204G + N21 IE, S98R + E173Q. In an embodiment, the esterase comprises at least one substitution selected from 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 selected from R30G, A64V, A68N, T109S, L124G, S145G, L152M, E158A, K159R, H183Q/L/A/Y/N/D/E/W, S193L/Q, W228R/S, V242E, more preferably selected from R30G, A64V, T109S, S145G, E158A, H183Q/N, W228S and V242E.
In an embodiment, the esterase comprises at least one substitution, preferably at least two substitutions selected from 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 and R251S.
In another embodiment, the esterase comprises at least one substitution selected from R30G, A64V, L124G, S145G, H183Q/L/A/Y/N/D/E/W, S193L/Q, W228S and V242E, preferably selected from R30G, A64V, L124G, S145G, H183Q/N, S193L/Q, W228S and V242E.
According to the invention, the esterase may further comprises a substitution at at least one amino acid position selected from SI, Y4, Q5, R6, N9, PIO, Ti l, 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, 154, M56, P58, G59, Y60, T61, A62, D63, A64, S65, S66, L67, A68, W69, L70, R72, R73, L74, L82, 184, N85, T86, N87, S88, R89, F90, D91, G92, P93, D94, S95, R96, S98, Q99, A103, L104, N105, L107, R108, SI 13, VI 15, LI 19, A121, N122, L124, A125, A127, G128, H129, M131, G132, G133, G134, G135, R138, AMO, Q142, N143, S145, K147, A149, V150, L152, T153, P154, W155, H156, T157, E158, K159, T160, N162, S164, V167, L168, 1170, 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, 1217, 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, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1, preferably at at least one position selected from SI, Y4, Q5, R6, PIO, Ti l, R12, L13, A14, L15, T16, A17, D18, S22, T25, V28, S29, L31, S32, V33, S34, G35, F36, G37, G38, Y43, S48, , G53, 154, M56, P58, G59, Y60, T61, A62, D63, S65, L67, W69, L70, R72, L82, 184, N85, T86, N87, S88, R89, F90, D91, G92, P93, D94, S95, R96, S98, Q99, A103, L104, N105, L107, SI 13, VI 15, LI 19, A121, N122, A125, A127, G128, H129, M131, G132, G133, G134, G135, R138, AMO, Q142, N143, K147, A149, V150, T153, P154, W155, H156, T157, T160, N162, S164, L168, 1170, 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, 1217, 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 and T163, more preferably selected from G35, W69, F90, N162, S181, N204, A216, N243, A246, V28, T157, M208, N211, A17, V200, 1170, N122, Q142, Q237, Q258, N85, N105, L13, E158, even more preferably selected from G35, W69, F90, VI 151, N162, S 181 , N204, A216, N243, A246, V28, and T157. Preferably, the esterase further comprises at least one substitution selected from G35A, W69L, F90L/Y, N162S, S181K, N204G, A216C, N243Y, A246C, V28I, T157P, T16K/R, Y4K/R, V219E/D, M208N, N21 IM, A17F, M208L, M208T, V200I, I170V, N122D, Q142E, Q237E, Q258E, N85D, N105D, LBS, E158D, preferably selected from selected from G35A, W69L, F90L/Y, V115I, N162S, S181K, N204G, A216C, N243Y, A246C, V28I, T157P, T16K/R, Y4K/R and V219E/D, more preferably selected from G35A, W69L, F90L/Y, VI 151, N162S, S181K, N204G, A216C, N243Y, A246C, V28I, and T157P. For instance, the esterase further comprises at least one combination of substitutions selected from S66N + M208T and A62T + M208T. In a particular embodiment, the esterase further comprises at least the combination of substitutions selected from LBS + E158D.
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. Particularly, the esterase variant comprises a combination of substitutions selected from A215E + T16K/R, A215D + T16R, D230E + Y4K/R and V219E/D + L15K/R.
In an embodiment, the esterase comprises at least one substitution selected from 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 R30G, A64V, A68N, T109S, L124G, S145G, L152M, E158A, 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 combination of substitutions selected from H183L + Fl 871, Hl 83 A + Fl 871, 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 + N21 IM and M208N + N21 IM, preferably selected from H183L + F187I, H183A + F187I, H183Y + F187I, H183D + F187I, H183W + F187I, H183E + Fl 871, A62T + S66N + M208T, F90A + H183S + F187L, F90L + H183E + F187L, F90S + H183S + F187L, F90Y + H183L + F187L and S22R + G39S + P179N + L239M.
Preferably the esterase comprises at least one substitution or at least one combination of substitutions selected from R30G, A64V, A68N, T109S, L124G, S145G, L152M, E158A, K159R, H183Q/N, S193L/Q, W228R/S, V242E, H183L + Fl 871, H183A + F187I, H183Y + Fl 871, H183D + Fl 871, H183W + Fl 871, H183E + Fl 871, A62T + S66N + M208T, F90A + H183S + F187L, F90L + H183E + F187L, F90S + H183S + F187L, F90Y + H183L + F187L and S22R + G39S + P179N + L239M, more preferably selected 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.
In an embodiment, the esterase comprises at least one substitution selected from R30G, A64V, L124G, S145G, H183Q/N, S193L/Q, W228S and V242E and further comprises at least one substitution selected from G35A, W69L, F90L/Y, VI 151, N162S, S181K, N204G, A216C, N243Y, A246C, V28I, T157P, A17F, F90Q, M208L, N21 IM, N122D, S193E, Q142E, N143D, Q237E, N253D, Q258E, V200I, Il 70V, N85D, N105D, N21 IE, Fl 871, M208N, M208T, LBS, E158D, preferably selected from G35A, W69L, F90L/Y, VI 151, N162S, S181K, N204G, A216C, N243Y, A246C, V28I and T157P.
In an embodiment, the esterase comprises the substitution R30G and further comprises at least one substitution selected from H183Q, N204G, Fl 871, T27A, S145G, W228S and V242E. Particularly, the esterase comprises at least one combination of substitutions selected from R30G + H183Q, N204G + H183Q + F187I + R30G and T27A + R30G + S145G + W228S + V242E.
In an embodiment, the esterase comprises at least the combination of substitutions selected from, or consists of the amino acid sequence as set forth in SEQ ID NO: 1 with the combination of substitutions, V200I + Il 70V.
In an embodiment, the esterase comprises the substitution A64V and further comprises at least one substitution selected from N243Y, VI 151, N204G, H183Q and Fl 871. Particularly, the esterase comprises at least one combination of substitutions selected from A64V + N243Y, A64V + VI 151 + N243 Y and N204G + Hl 83Q + F 1871 + A64V.
In an embodiment, the esterase comprises the substitution S145G and further comprises at least one substitution selected from T27A, R30G, W228S and V242E. Particularly, the esterase comprises at least the combination of substitutions T27A + R30G + S145G + W228S + V242E.
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, Fl 871, 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, L I 3S and E158D, preferably selected from F187I, F187L, A24Q, R30G, L74V, F187Q, Fl 871, 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 and F90Y. Particularly, the esterase comprises at least one combination of substitutions selected from, or consists of the amino acid sequence as set forth in SEQ ID NO: 1 with the combination of substitutions selected from H183L + F187I, H183A + F187I, H183Y + F187I, H183D + F187I, H183W + F187I,
Figure imgf000017_0001
+ V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I, A17T + T27S + S48T + L82I + F90L + G92F + G135A + AMOS + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + H183Q + F187I + N204G, A17T + T27S + S48T + L82I + F90L + G92F + G135A + AMOS + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I + L13S + E158D, preferably selected from 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 + Fl 871, N204G + H183Q + F187I + N162S + S181K, N204G + H183Q + F187I + N162S + S181K + S193Q and N204G + H183Q + F187I + W69L + A216C + A246C.
In a particular embodiment, the esterase comprises the substitution H183Q and further comprises at least one substitution, at least two substitutions, at least three substitutions selected from A24Q, R30G, L74V, F187Q, Fl 871, 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, LBS, E158D, preferably selected from A24Q, R30G, L74V, F187Q, F187I, L227N, R251S, Y26H, A64T, G35A, A62T, G46V, R73H, T50M, I169M, Y106F, Il 85V, R108L, V167L, N204G, G39S, A64V, L124G, N162S, S193Q, Q237L, M208T, S181K, W69L, A216C and A246C. Particularly, the esterase comprises at least one combination of substitutions selected from, or consists of the amino acid sequence as set forth in SEQ ID NO: 1 with a combination of substitutions selected from A24Q + H183Q, R30G + H183Q, L74V + H183Q, H183Q + F187Q, H183Q + Fl 871, L227N + H183Q, R251S + H183Q, Y26H + A64T + Hl 83Q, G35 A + A62T + Hl 83Q, G46V + R73H + Hl 83Q, T50M + H69M + Hl 83Q, 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 + N21 IM + S193E, M208T + N204G + H183Q + F187I + N21 IM + S193E, H183Q + F187I + N204G + N21 IM + S193E + V200I + H70V, H183Q + F187I + N204G + M208N + N21 IM + S193E + V200I + 1170V, M208T + N204G + H183Q + F187I + N211M + S193E + V200I + I170V, H183Q + F187I + N204G + N211M + S193E , N204G + H183Q + F187I + LBS + E158D, N204G + H183Q + F187I, M208T + N204G + H183Q + F187I + LBS + E158D, H183Q + F187I + N204G + LBS + E158D, M208T + N204G + H183Q + F187I + LBS + E158D, A17T + T27S + S48T + L82I + F90L + G92F + G135A + AMOS + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + H70V + M208T + N204G + H183Q + F187I, A17T + T27S + S48T + L82I + F90L + G92F + G135A + AMOS + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + H70V + H183Q + F187I + N204G, A17T + T27S + S48T + L82I + F90L + G92F + G135A + AMOS + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I + LI 3 S + E158D, preferably selected from A24Q + H183Q, R30G + H183Q, L74V + H183Q, H183Q + F187Q, H183Q + Fl 871, L227N + H183Q, R251S + H183Q, Y26H + A64T + Hl 83Q, G35 A + A62T + Hl 83Q, G46V + R73H + Hl 83Q, T50M + H69M + Hl 83Q, 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 + Fl 871 + N162S + S181K + S193Q and N204G + H183Q + F187I + W69L + A216C + A246C.
In another particular embodiment, the esterase comprises the substitution H183N and further comprises at least one substitution selected from Fl 871, 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. Particularly, the esterase comprises at least one combination of substitutions selected from, or consists of the amino acid sequence as set forth in SEQ ID NO: 1 with a combination of substitutions selected from H183N + Fl 871, F90L + H183N + F187L, F90Y + H183N, A17F + F90Q + H183N + N204G + M208L + N211M, A17F + F90Q + N122D + H183N + S193E + N204G + M208L + N21 IM, A17F + F90Q + Q142E + H183N + S193E + N204G + M208L + N211M, A17F + F90Q + N143D + H183N + S193E + N204G + M208L + N21 IM, A17F + F90Q + H183N + S193E + N204G + M208L + N211M + Q237E, A17F + F90Q + H183N + S193E + N204G + M208L + N21 IM + N253D, A17F + F90Q + H183N + S193E + N204G + M208L + N21 IM + Q258E, A17F + F90Q + H183N + S193E + N204G + M208L + N211M + V200I + 1170V, A17F + N85D + F90Q + H183N + S193E + N204G + M208L + N211M and A17F + F90Q + N105D + H183N + S193E + N204G + M208L + N211E, preferably selected from H183N + Fl 871, F90L + H183N + F187L and F90Y + H183N.
In an embodiment, the esterase comprises the substitution S193L and further comprises at least the substitution S181K. Particularly, the esterase comprises at least the combination of substitutions S181K + S193L.
In another embodiment, the esterase comprises the substitution S193Q and further comprises at least one substitution selected from N162S, S181K, N204G, H183Q and F187I. Particularly, 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. In an embodiment, the esterase comprises the substitution W228S and further comprises at least one substitution selected from T27A, R30G, S145G and V242E. Particularly, the esterase comprises at least the combination of substitutions selected from T27A + R30G + S145G + W228S + V242E.
In an embodiment, the esterase comprises the substitution V242E and further comprises at least one substitution selected from T27A, R30G, S145G and W228S. Particularly, the esterase comprises at least the combination of substitutions selected from T27A + R30G + S145G + W228S + V242E.
In an embodiment, the esterase comprises at least one combination of substitutions selected from, or consists of the amino acid sequence as set forth in SEQ ID NO: 1 with a combination of substitutions selected from, 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 + VI 151 + 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 + F 1871 + A62T, N204G + Hl 83Q + F 1871 + A64V, N204G + Hl 83 Q + F 1871 + 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.
In a particular embodiment, the esterase comprises at least the combination of substitutions N204G + H183Q + Fl 871 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, V200I, H70V, LBS, E158D, preferably selected from R30G, G39S, A62T, A64V, W69L, L124G, N162S, S181K, S193Q, M208T, A216C, Q237L and A246C.
Particularly, the esterase comprises at least one combination of substitutions selected from, or consists of the amino acid sequence as set forth in SEQ ID NO: 1 with a combination of substitutions selected from 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 + Fl 871, N204G + H183Q + F187I + N162S + S181K, N204G + H183Q + F187I + N162S + S181K + S193Q, N204G + H183Q + F187I + W69L + A216C + A246C, H183Q + F187I + N204G + M208N + N21 IM + S193E, H183Q + F187I + N204G + M208N + N21 IM + S193E + V200I + Il 70V, M208T + N204G + H183Q + F187I + N211M + S193E, M208T + N204G + H183Q + F187I + N21 IM + S193E + V200I + I170V, H183Q + F187I + N204G + N21 IM + S193E , H183Q + F187I + N204G + N211M + S193E + V200I + I170V, H183Q + F187I + N204G + L I3S + E158D, M208T + N204G + H183Q + F187I + LI 3 S + E158D, A17T + T27S + S48T + L82I + F90L + G92F + G135A + AMOS + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + Il 70V + M208T + N204G + H183Q + Fl 871, A17T + T27S + S48T + L82I + F90L + G92F + G135A + AMOS + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + H183Q + F187I + N204G, A17T + T27S + S48T + L82I + F90L + G92F + G135A + AMOS + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I + L13S + E158D, preferably selected from 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 + N21 IM + S193E, H183Q + F187I + N204G + M208T + N21 IM + S193E, H183Q + F187I + N204G + N21 IM + S193E + V200I + I170V, H183Q + F187I + N204G + M208N + N21 IM + S 193E + V200I + 1170V, H183Q + F187I + N204G + M208T + N21 IM + S193E + V200I + I170V, more preferably selected 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 + N21 IM + S193E, H183Q + F187I + N204G + M208T + N211M + S193E and H183Q + F187I + N204G + M208N + N211M + S193E + V200I + I170V.
Particularly, the esterase comprises at least the combination of substitutions N204G + H183Q + F187I and optionally further comprises at least one substitution selected from M208T, L13S and E158D. Particularly, the esterase comprises at least one combination of substitutions selected from, or consists of the amino acid sequence as set forth in SEQ ID NO:1 with a combination of substitutions selected from N204G + H183Q + Fl 871 + M208T, N204G + H183Q + F187I + M208T + L13S + E158D, N204G + H183Q + F187I + L13S + E158D. In a particular embodiment, the esterase comprises at least the combination of substitutions M208N + N211M.
In an embodiment, the esterase comprises at least the combination of substitutions selected from H183Q + F187I + N204G + N211M + S193E and further comprises at least one substitution selected from M208N/T, V200I and II 70V. In a preferred embodiment, the esterase comprises at least one combination of substitutions selected from N204G + H183Q + F187I + M208N/T + N21 IM + S193E, preferably the combination H183Q + F187I + N204G + M208N + N21 IM + S193E.
In an embodiment, the esterase comprises at least one substitution or combination of substitutions selected from 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 + N243 Y, L74V + H183Q, N162S + S193Q, S181K + S193L, H183Q + F187Q, H183Q + F187I, H183N + F187I, L227N + H183Q, R251 S + H183Q, Y26H + A64T + H183Q, G35A + A62T + Hl 83Q, G46V + R73H + Hl 83Q, T50M + 1169M + Hl 83Q, A64V + VI 151 + N243 Y, F90L + Hl 83N + F 187L, F90Y + H183N, Y106F + H85V + H183Q, R108L + V167L + H183Q, N162S + S181K + S193Q, H183Q + F187I + N204G, N204G + H183Q + F187I + R30G, N204G + H183Q + F187I + G39S, N204G + Hl 83Q + F 1871 + A62T, N204G + Hl 83Q + F 1871 + A64 V, N204G + Hl 83Q + 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 another embodiment, the esterase consists of amino acid sequence as set forth in SEQ ID NO: 1 with a substitution or a combination of substitutions selected from 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 + N243 Y, L74V + H183Q, N162S + S193Q, S181K + S193L, H183Q + F187Q, H183Q + F187I, H183N + F187I, L227N + H183Q, R251 S + H183Q, Y26H + A64T + H183Q, G35A + A62T + Hl 83Q, G46V + R73H + Hl 83Q, T50M + 1169M + Hl 83Q, A64V + VI 151 + N243 Y, F90L + Hl 83N + F 187L, F90Y + H183N, Y106F + H85V + 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 by reference to the amino acid sequence set forth in SEQ ID NO: 1.
In an embodiment, the esterase comprises at least one combination of substitutions selected from, or consists of the amino acid sequence as set forth in SEQ ID NO: 1 with one combination
Figure imgf000023_0001
+ S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I, A17T + T27S + S48T + L82I + F90L + G92F + G135A + AMOS + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + H183Q + F187I + N204G, A17T + T27S + S48T + L82I + F90L + G92F + G135A + AMOS + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I + L13S + E158D.
Advantageously, the esterase of the invention exhibits an increased polyester degrading activity and/or an increased thermostability as compared to the esterase of SEQ ID NO: 1 in a range of pH between 6 and 10. Particularly, the esterase of the invention exhibits an increased polyester degrading activity and/or an increased thermostability as compared to the esterase of SEQ ID NO: 1 in the range of pH from 6 to 10, from 6 to 9, from 6.5 to 9, from 7 to 9, from 7.5 to 8.5, from 7 to 8.5, from 7.5 to 9. According to the invention, the designation of a range of pH includes the lower and upper limit of said range.
In an embodiment, the esterase exhibits an increased polyester degrading activity as compared to the esterase of SEQ ID NO: 1, at least under basic conditions. Preferably, the esterase exhibits an increased polyester degrading activity at least at pH between 7.5 and 9, such as at pH 8.
In particular, the esterase may exhibit an increased specific degrading activity and/or an increased PET depolymerization yield after a defined period of time, for example after 24h, 48h or 72h, as compared to the esterase of SEQ ID NO: 1.
In an embodiment, the esterase comprises at least one substitution or combination of substitutions as described above and exhibits an increased polyester degrading activity as 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.
According to an embodiment, the esterase comprises at least one substitution or combination of substitutions as described above and has an increased PET depolymerization yield after 24 h compared to SEQ ID NO: 1.
In particular, the esterase comprises at least one substitution or combination of substitutions as described above and exhibits an increased specific degrading activity as compared to the esterase of SEQ ID NO: lat 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.
In another particular embodiment, the esterase comprises at least the substitution or combination of substitutions as described above and exhibits an increased PET depolymerization yield after 48h 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.
Alternatively or in addition, the esterase may exhibit an increased thermostability as compared to the esterase of SEQ ID NO: 1. Preferably, the esterase exhibits an increased polyester degrading activity at least at pH between 7 and 9, such as at pH 8.
In a particular embodiment, the esterase comprises at least one substitution selected from Fl 871 and S193Q or at least the combination of substitutions S181K + S193L and exhibits an 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 as compared to the esterase of SEQ ID NO: 1.
In an embodiment, the esterase of the invention further exhibits an increased thermostability and/or an increased polyester degrading activity as 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 comprised between 5 and 5.5, even more preferably at pH 5.2.
Particularly, the esterase comprises at least one substitution or at least one combination of substitutions as described above and exhibits an increased thermostability and/or an increased polyester degrading activity as compared to the esterase of SEQ ID NO: 1 at a pH comprised between 6 and 10, particularly between 7.5 and 9 and further exhibits an increased thermostability and/or an increased polyester degrading activity as compared to the esterase of SEQ ID NO: 1 at a pH between 3 and 6, preferably at a pH comprised between 5 and 6, more preferably comprised between 5 and 5.5, even more preferably at pH 5.2. According to the invention, the esterase may comprise at least one amino acid residue selected from S130, D175, H207, C240 or C275 as in the parent esterase of SEQ ID NO: 1, i.e., the esterase of the invention is not modified at one, two, three, etc., or all of these positions.
Particularly, the esterase may exhibit at least the amino acids S130, D175 and H207 forming the catalytic site of the esterase and/or the amino acids C240 and C275 forming disulphide bond as in the parent esterase. Preferably, the esterase comprises at least a combination of amino acid residues selected from S130 + D175 + H207, C240 + C275 and SI 30 + DI 75 + H207 + C240 + C275, as in the parent esterase, more preferably the combination S130 + D175 + H207 + C240 + C275 as in the parent esterase.
In addition, the esterase may further comprise at least one amino acid residue selected from C203 and C248 as in the parent esterase of SEQ ID NO: 1. For instance, the esterase may comprise the combination of amino acid residues C203 + C248 as in the parent esterase. Alternatively, the esterase comprises at least the substitution C203K/R, preferably C203K and the amino acid residue C248 as in the parent esterase.
It is another object of the invention to provide an esterase variant which (i) has at least 97%, 98% or 99% identity to the full length amino acid sequence set forth in SEQ ID NO: 1, (ii) has at least one amino acid substitution selected from 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, VI 151, S181K, H183S, Fl 871, 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 at least one combination of substitutions selected from N204G + M208L + N21 IE, F90E + N204G + N21 IE, F90N + N204G + N21 IE, F90Q + N204G + N21 IE, F90R + N204G + N21 IE, F90W + N204G + N21 IE, F187I + N204G + N21 IE, S98R + E173Q, M208Q + N21 IM and M208N + N211M as compared to the amino acid sequence SEQ ID NO: 1, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1, (iii) has at least the amino acids C240, C275, 1170, G92, P213, E182, L13 and E158, as in the parent esterase of SEQ ID NO: 1, (iv) has a polyester degrading activity, and (v) exhibits an increased thermostability and/or an increased polyester degrading activity as compared to the esterase of SEQ ID NO: 1 at a pH comprised between 6 and 10.
In an embodiment, the esterase comprises at least one amino acid substitution selected from 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, Il 85V, F187L/Q, P192S, L227N, Q237L, L239M, R251S, N9E, V28I, A62T, F90Y/R/W7L, VI 151, S181K, H183S, F187I, M208T, A246K, S66N, N243Y, S66T, F90C/V, P151V, A246R, A215E/D, D230E, L15K/R, T16K/R, Y4K/R, V219E/D or at least one combination of substitutions selected from N204G + M208L + N21 IE, F90E + N204G + N21 IE, F90N + N204G + N21 IE, F90Q + N204G + N21 IE, F90R + N204G + N21 IE, F90W + N204G + N21 IE, F187I + N204G + N21 IE and S98R + E173Q.
In an embodiment, the esterase comprises at least one substitution selected from N9E, V28I, A62T, F90Y, VI 151, S181K, H183S, F187I, M208T, S66N, N243Y, E182D, M208G, T16E, Q142E, Q237E, M208K, N105D, N85D, M208A, M208D preferably selected from N9E, V28I, A62T, F90Y, VI 151, 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.
In a preferred embodiment, the esterase comprises at least one substitution selected from 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, VI 151, S181K, H183S, F187I, M208T, S66N and N243Y, preferably selected from 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, VI 151, S181K, F187I, M208T and N243Y, as compared to the esterase of SEQ ID NO: 1, more preferably selected from 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.
In an embodiment, the esterase comprises one or two substitutions selected from A215E/D, D230E, L15K/R, T16K/R, Y4K/R and V219E/D. Particularly, the esterase comprises a combination of substitutions selected from A215E + T16K/R, A215D + T16R, D230E + Y4K/R and V219E/D + L15K/R.
In another embodiment, the esterase comprises at least one, preferably at least two substitutions selected from 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, VI 151, S181K, H183S, Fl 871, M208T, S66N and N243Y. Particularly, the esterase comprises a combination of substitutions selected from S66N + M208T, A62T + M208T. In an embodiment, the esterase comprises at least one substitution selected from R30G, A62T, A64V, F90Y, VI 151, L124G, S145G, L152M, K159R, H183Q/N/D/E, F187I, S193L/Q, M208T, W228R/S, V242E and N243Y, preferably selected from R30G, A62T, A64V, F90Y, VI 151, L124G, S145G, L152M, K159R, H183Q/N, F187I, S193L/Q, M208T, W228R/S, V242E and N243 Y.
According to the invention, the esterase may further comprise from one to seven substitutions at amino acid positions selected from SI, Y4, Q5, R6, N9, PIO, Ti l, 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, 154, M56, P58, G59, Y60, T61, A62, D63, A64, S65, S66, L67, A68, W69, L70, R72, R73, L74, L82, 184, N85, T86, N87, S88, R89, F90, D91, G92, P93, D94, S95, R96, S98, Q99, A103, L104, N105, L107, R108, SI 13, VI 15, LI 19, A121, N122, L124, A125, A127, G128, H129, M131, G132, G133, G134, G135, R138, AMO, N143, S145, K147, A149, V150, L152, T153, P154, W155, H156, T157, E158, K159, T160, N162, S164, V167, L168, 1170, 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, 1217, 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, preferably selected from SI, Y4, Q5, R6, PIO, T11, R12, A14, L15, T16, A17, D18, S22, T25, S29, L31, S32, V33, S34, G35, F36, G37, G38, Y43, S48, , G53, 154, M56, P58, G59, Y60, T61, D63, S65, L67, W69, L70, R72, L82, 184, N85, T86, N87, S88, R89, D91, P93, D94, S95, R96, S98, Q99, A103, L104, N105, L107, SI 13, L119, A121, N122, A125, A127, G128, H129, M131, G132, G133, G134, G135, R138, AMO, 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, 1217, 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, more 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 from one to seven substitutions selected from, 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.
According to an embodiment, the esterase comprises the substitution F90Y and further comprises at least one substitution selected from H183L/N. Particularly, the esterase comprises a combination of substitutions selected from F90Y + H183L + F187L and F90Y + H183N. According to another embodiment, the esterase comprises the substitution VI 151 and further comprises at least one substitution selected from A64V and N243Y. Particularly, the esterase comprises the combination of substitutions A64V + VI 151 + N243Y.
According to another embodiment, the esterase comprises the substitution Fl 871 and further comprises one, two or more substitutions selected from 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 selected from R30G, G39S, A62T, A64V, W69L, L124G, N162S, S181K, H183L/A/Y/Q/N/D/W/E/S, S193Q, N204G, M208T, A216C, Q237L, A246C. Particularly, the esterase comprises at least one combination of substitutions selected from, or consists of the amino acid sequence as set forth in SEQ ID NO: 1 with a combination of substitutions selected from H183L + F187I, H183A + F187I, H183Y + F187I, H183Q + F187I, H183N + F187I, H183D + F187I, H183W + F187I, H183E + F 1871, Hl 83 S + F 1871, Hl 83Q + F 1871 + N204G, N204G + Hl 83Q + F 1871 + 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 + N21 IM + S 193E, M208T + N204G + H183Q + F187I + N211M + S193E, H183Q + F187I + N204G + N21 IM + S193E , H183Q + F187I + N204G + N21 IM + S193E + V200I + I170V, M208T + N204G + H183Q + F187I, preferably selected from H183L + F187I, H183A + F187I, H183Y + F187I, H183Q + F187I, H183N + F187I, H183D + F187I, H183W + F187I, H183E + F187I, H183S + F187I, H183Q + F187I + N204G, N204G + H183Q + F 1871 + R30G, N204G + Hl 83Q + F 1871 + G39S, N204G + Hl 83 Q + F 1871 + 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 + F 1871 + N162S + S 18 IK + S 193Q and N204G + Hl 83Q + F 1871 + W69L + A216C + A246C.
According to another embodiment, the esterase comprises the substitution M208T and further comprises at least one substitution selected from A62T, S66N, H183Q, F187I, N204G, N21 IM, S193E, V200I, I170V, preferably selected from A62T, S66N, H183Q, F187I and N204G. Particularly, the esterase comprises at least the combination of substitutions selected from, or consists of the amino acid sequence as set forth in SEQ ID NO: 1 with a combination of substitutions selected from 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.
According to another embodiment, the esterase comprises the substitution N243 Y and further comprises at least one substitution selected from A64V and VI 151. Particularly, the esterase comprises at least the combination of substitutions selected from A64V + N243 Y and A64V + V115I + N243Y.
In an embodiment, the esterase comprises at least one combination of substitutions selected from, or consists of the amino acid sequence as set forth in SEQ ID NO: 1 with the combination of substitutions selected from 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 + VI 151 + 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 + Fl 871, T27A + R30G + S145G + W228S + V242E, N204G + H183Q + F187I + N162S + S181K, G7A + N9T + P192S + P213A + W228R + Q237L, N204G + H183Q + F187I + N162S + S181K + S193Q andN204G + H183Q + F187I + W69L + A216C + A246C.
In a particular embodiment, the esterase comprises at least the combination of substitutions N204G + H183Q + Fl 871 and optionally further comprises at least one substitution selected from R30G, G39S, A62T, A64V, W69L, L124G, N162S, S181K, S193Q, M208T, A216C, Q237L, A246C, M208N, N21 IM, S193E, preferably selected from R30G, G39S, A62T, A64V, W69L, L124G, N162S, S181K, S193Q, M208T, A216C, Q237L and A246C. Particularly, the esterase comprises at least one combination of substitutions selected from or consists of the amino acid sequence as set forth in SEQ ID NO: 1 with a combination of substitutions selected from 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 + N21 IM + S193E, M208T + N204G + H183Q + F187I + N211M + S193E, H183Q + F187I + N204G + N21 IM + S193E , preferably selected from 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 + N21 IM + S193E, H183Q + F187I + N204G + M208T + N211M + S193E, H183Q + F187I + N204G + N21 IM + S193E + V200I + Il 70V, more preferably selected 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 + S 193 Q, Hl 83 Q + F 1871 + N204G + M208N + N21 IM + S 193E and Hl 83 Q + F 1871 + N204G + M208T + N211M + S193E.
In a particular embodiment, the esterase comprises at least the combination of substitutions M208N + N211M.
In an embodiment, the esterase comprises at least one substitution or combination of substitutions selected from or consists of the amino acid sequence as set forth in SEQ ID NO: 1 with a combination of substitutions selected from 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, VI 151, S 18 IK, F 1871, M208T, N243 Y, 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 + F 1871, L227N + Hl 83Q, R251 S + Hl 83Q, Y26H + A64T + Hl 83Q, G35 A + A62T + Hl 83Q, G46V + R73H + H183Q, T50M + H69M + H183Q, A62T + S66N + M208T, A64V + V115I + N243 Y, F90L + Hl 83N + F 187L, F90Y + Hl 83L + F 187L, F90Y + Hl 83N, Y106F + 1185 V + H183Q, R108L + V167L + H183Q, N162S + S181K + S193Q, H183Q + F187I + N204G, N204G + H183Q + F187I + R30G, N204G + H183Q + F187I + G39S, N204G + H183Q + F 1871 + A62T, N204G + Hl 83Q + F 1871 + A64V, N204G + Hl 83 Q + F 1871 + 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. In an embodiment, the esterase comprises at least one substitution selected from Fl 871 and S193Q or at least one combination of substitutions selected from S181K + S193L and exhibits an 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 as compared to the esterase of SEQ ID NO: 1.
According to the invention, the esterase may comprise at least one amino acid residue selected from S130, D175, H207, C240 or C275 as in the parent esterase of SEQ ID NO: 1, i.e., the esterase of the invention is not modified at one, two, three, etc., or all of these positions.
Particularly, the esterase may exhibit at least the amino acids S130, D175 and H207 forming the catalytic site of the esterase and/or the amino acids C240 and C275 forming disulphide bond as in the parent esterase. Preferably, the esterase comprises at least a combination of amino acid residues selected from S130 + D175 + H207, C240 + C275 and SI 30 + DI 75 + H207 + C240 + C275, as in the parent esterase, more preferably the combination S130 + D175 + H207 + C240 + C275 as in the parent esterase.
In addition, the esterase may further comprise at least one amino acid residue selected from C203 and C248 as in the parent esterase of SEQ ID NO: 1. For instance, the esterase may comprise the combination of amino acid residues C203 + C248 as in the parent esterase. Alternatively, the esterase comprises at least the substitution C203K/R, preferably C203K and the amino acid residue C248 as in the parent esterase.
In a particular embodiment, the esterase consists of the amino acid sequence as set forth in SEQ ID NO: 1 with a combination of substitutions selected from A17T + T27S + S48T + L82I + F90L + G92F + G135A + AMOS + N143I + S145T + A149G + S164P + V167Q + H183Q + F187I + C203K + N204G + S206T + M208T + C248S + T252S, A17T + T27S + S48T + L82I + F90L + G92F + G135A + AMOS + N143I + S145T + A149G + S164P + V167Q + H183Q + F187I + C203K + N204G + S206T + C248S + T252S, A17T + T27S + S48T + L82I + F90L + G92Y + G135A + AMOS + N143I + S145T + A149G + S164P + V167Q + H183Q + F187I + C203K + N204G + M208T + S206T + C248S + T252S, A17T + T27S + S48T + L82I + F90L + G92F + G135A + AMOS + N143I + S145T + A149G + S164P + V167Q + H183Q + F187I + N204G + S206T + M208T + T252S or A17T + T27S + S48T + L82I + F90L + G92F + G135A + AMOS + N143I + S145T + A149G + S164P + V167Q + H183Q + F187I + N204G + S206T + T252S.
In particular embodiments, the esterases of the invention are derived from SEQ ID NO:2, which corresponds to the amino acid sequence of SEQ ID NO: 1, with the combination of substitutions A17T + T27S + S48T + L82I + F90L + G92F + G135A + AMOS + N143I + S145T + A149G + S164P + V167Q + 1170V + S206T + T252S as compared to SEQ ID NO: 1. That is to say that variants derived from SEQ ID NO:2 comprise at least one of these substitutions as compared to SEQ ID NO: 1 and one or more additional substitutions as described in the present application.
It is a further object of the invention 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 set forth in SEQ ID NO:2, (ii) comprises at least one amino acid substitution selected from 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 and V200I, or at least one combination of substitutions selected from N204G + M208L + N211E, L90E + N204G + N21 IE, L90N + N204G + N21 IE, L90Q + N204G + N21 IE, L90R + N204G + N21 IE, L90W + N204G + N211E, F187I + N204G + N21 IE, S98R + E173Q, M208A + N211M, M208G + N21 IM, M208K + N21 IM, M208N + N21 IM, M208P + N21 IM, M208Q + N21 IM, M208H + N211M and M208T + N211M, as compared to the amino acid sequence SEQ ID NO:2, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO:2, (iii) has a polyester degrading activity and (iv) exhibits an increased thermostability and/or an increased polyester degrading activity as compared to the esterase of SEQ ID NO:2 at a pH comprised between 6 and 10.
In an embodiment, the esterase comprises at least one substitution selected from 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, Il 85V, F187L/Q, P192S, P213A, L227N, Q237L, L239M and R251S, preferably selected from R30G, A64V, A68N, T109S, L124G, T145G, L152M, E158A, K159R, H183Q/L/A/Y/N/D/E/W, S193L/Q, W228R/S and V242E, preferably selected from R30G, A64V, T109S, T145G, E158A, H183Q/N, W228S and V242E.
In a preferred embodiment, the esterase comprises at least one substitution selected from A64V, S66N, H183N, I143D, N253D, preferably selected from H183N and S66N.
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.
According to the invention, the esterase may further comprises a substitution at at least one amino acid position selected from SI, Y4, Q5, R6, N9, PIO, Ti l, 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, 154, M56, P58, G59, Y60, T61, A62, D63, A64, S65, S66, L67, A68, W69, L70, R72, R73, L74, 182, 184, N85, T86, N87, S88, R89, L90, D91, F92, P93, D94, S95, R96, S98, Q99, A103, L104, N105, L107, R108, SI 13, VI 15, LI 19, A121, N122, L124, A125, A127, G128, H129, M131, G132, G133, G134, A135, R138, S140, 1143, 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, 1217, 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, as compared to the amino acid sequence SEQ ID NO:2, preferably at at least one position selected from SI, Y4, Q5, R6, PIO, Ti l, R12, L13, A14, L15, T16, T17, D18, S22, T25, V28, S29, L31, S32, V33, S34, G35, F36, G37, G38, Y43, T48, G53, 154, M56, P58, G59, Y60, T61, A62, D63, S65, L67, W69, L70, R72, 182, 184, N85, T86, N87, S88, R89, L90, D91, F92, P93, D94, S95, R96, S98, Q99, A103, L104, N105, L107, SI 13, V115, L119, A121, N122, A125, A127, G128, H129, M131, G132, G133, G134, A135, R138, S140, 1143, 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, 1217, 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 and T163, more preferably selected from G35, W69, L90, VI 151, N162, S 181 , N204, A216, N243, A246, V28, T157, H183, F187, M208, N211, S193, V200, T17, N85, N122, Q142, 1145, Q237, N253, Q258, S98, N105, L13, E158, preferably selected from G35, W69, L90, VI 151, N162, S181, N204, A216, N243, A246, V28, and T157. Preferably the esterase further comprises at least one substitution selected from G35A, W69L, L90L/Y, VI 151, N162S, S181K, N204G, A216C, N243Y, A246C, V28I, T157P, T16K/R, Y4K/R, V219E/D, Fl 871, L90Q, H183D, H183E, H183N, H183Q, I145D, L90Q, M208A, M208L, M208N, M208T, N105D, N122D, N211M, N253D, N85D, Q142E, Q237E, Q258E, S193E, S98R, T17F, V200I, L I 3S, E158D, preferably selected from G35A, W69L, L90L/Y, VI 151, N162S, SI 8 IK, N204G, A216C, N243Y, A246C, V28I, T157P, T16K/R, Y4K/R and V219E/D, more preferably selected from G35A, W69L, L90Y, VI 151, N162S, S181K, N204G, A216C, N243Y, A246C, V28I, and T157P. For instance, the esterase further comprises at least one combination of substitutions selected from S66N + M208T and A62T + M208T.
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, as compared to the amino acid sequence SEQ ID NO:2. Particularly, the esterase variant comprises a combination of substitutions selected from A215E + T16K/R, A215D + T16R, D230E + Y4K/R and V219E/D + L15K/R.
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 G35A, W69L, L90L/Y, VI 151, 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 and V200I, preferably selected from G35A, W69L, L90L/Y, VI 151, N162S, SI 8 IK, N204G, A216C, N243Y, A246C, V28I and T157P, as compared to the amino acid sequence SEQ ID NO:2.
According to 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/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, L90VY, as compared to the amino acid sequence SEQ ID NO:2. Particularly, the esterase comprises at least one combination of substitutions selected from H183L + Fl 871, Hl 83 A + Fl 871, H183Y + Fl 871, H183D + Fl 871, 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.
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 F187I, L90Q, I145D, L90Q, M208A, M208L, M208M, M208N, M208T, N105D, N122D, N204G, N211M, N253D, N85D, Q142E, Q237E, Q258E, S193E, S98R, T17F and V200I. Particularly, the esterase comprises at least one combination of substitutions selected from, or consists of the amino acid sequence as set forth in SEQ ID NO:2 with a combination of substitutions selected from H183D + Fl 871, H183E + Fl 871, H183N + Fl 871, M208T + N204G + H183Q + F187I + N211M + S193E, H183Q + F187I + N204G + N211M + S193E + V200I, T17F + L90Q +
Figure imgf000035_0001
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 selected from H183D + Fl 871, H183E + Fl 871, H183N + Fl 871, M208T + N204G + H183Q + F187I + N211M + S193E, H183Q + F187I + N204G + N211M + S193E + V200I, T17F + L90Q + H183N + N204G + M208L + N21 IM, T17F + L90Q + H183N + S193E + N204G + M208L + N21 IM, H183Q + F187I + N204G + M208N + N21 IM + S193E + V200I, M208T + N204G + H183Q + F187I + N21 IM + S193E + V200I, T17F + N85D + L90Q + H183N + S193E + N204G + M208L + N21 IM, T17F + L90Q + N122D + H183N + S193E + N204G + M208L + N211M, T17F + L90Q + Q142E + H183N + S193E + N204G + M208L + N211M, T17F + L90Q + I145D + H183N + S193E + N204G + M208L + N21 IM, T17F + L90Q + H183N + S193E + N204G + M208L + N21 IM + Q237E, T17F + L90Q + H183N + S193E + N204G + M208L + N21 IM + N253D, T17F + L90Q + H183N + S193E + N204G + M208L + N21 IM + Q258E, T17F + L90Q + H183N + S193E + N204G + M208L + N21 IM + V200I, T17F + L90Q + S98R + N105D + H183N + S193E + N204G + N21 IM, T17F + L90Q + H183N + S193E + N204G + M208N + N21 IM + V200I, T17F + L90Q + H183N + S193E + N204G + M208A + N21 IM + V200I, T17F + L90Q + H183N + S193E + N204G + M208M + N21 IM + V200I.
In a particular embodiment, the esterase comprises the substitution H183Q and further comprises at least one, two, three or more substitutions selected from 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, as compared to the amino acid sequence SEQ ID NO:2. Particularly, the esterase comprises at least one combination of substitutions selected from A24Q + H183Q, R30G + H183Q, L74V + H183Q, H183Q + F187Q, H183Q + F187I, L227N + H183Q, R251 S + H183Q, Y26H + A64T + H183Q, G35A + A62T + Hl 83Q, 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.
In a particular embodiment, the esterase comprises the substitution H183Q and further comprises at least one, two, three, four, five, six or more substitutions selected from Fl 871, M208N, M208T, N204G, N211M, S193E, V200I. Particularly, the esterase comprises at least one combination of substitutions selected from, or consists of the amino acid sequence as set forth in SEQ ID NO:2 with a combination of substitutions selected from H183Q + F187I + N204G + M208T + N211M + S193E, H183Q + F187I + N204G + M208N + N21 IM + S 193E + V200I, H183Q + F187I + N204G + M208T + N21 IM + S193E + V200I, H183Q + F187I + N204G + M208N + N21 IM + S193E, H183Q + F187I + N204G, H183Q + F187I + N204G + M208T, H183Q + F187I + N204G + M208T + LI 3 S + 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 + Hl 83Q + F 1871 + N204G + LI 3 S + El 58D, preferably selected from Hl 83 Q + F 1871 + N204G + M208T + N21 IM + S 193E, Hl 83Q + F 1871 + N204G + M208N + N21 IM + S 193E + V200I, H183Q + F187I + N204G + M208T + N21 IM + S193E + V200I.
In a particular embodiment, 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. Particularly, the esterase comprises at least one combination of substitutions selected from, or consists of the amino acid sequence as set forth in SEQ ID NO:2 with a combination of substitutions selected from H183N + F187I, T17F + L90Q + H183N + N204G + M208L + N211M, T17F + L90Q + H183N + S193E + N204G + M208L + N21 IM, T17F + N85D + L90Q + H183N + S193E + N204G + M208L + N211M, T17F + L90Q + N122D + H183N + S193E + N204G + M208L + N21 IM, T17F + L90Q + Q142E + H183N + S193E + N204G + M208L + N211M, T17F + L90Q + I145D + H183N + S193E + N204G + M208L + N21 IM, T17F + L90Q + H183N + S193E + N204G + M208L + N21 IM + Q237E, T17F + L90Q + H183N + S193E + N204G + M208L + N21 IM + N253D, T17F + L90Q + H183N + S193E + N204G + M208L + N21 IM + Q258E, T17F + L90Q + H183N + S193E + N204G + M208L + N21 IM + V200I, T17F + L90Q + S98R + N105D + H183N + S193E + N204G + N21 IM, T17F + L90Q + H183N + S193E + N204G + M208N + N21 IM + V200I, T17F + L90Q + H183N + S193E + N204G + M208A + N21 IM + V200I, T 17F + L90Q + H183N + S193E + N204G + M208M + N211 M + V200I.
In a particular embodiment, the esterase comprises at least the combination of substitutions N204G + H183Q + Fl 871 and optionally further comprises at least one substitution selected from R30G, G39S, A62T, A64V, W69L, L124G, N162S, S181K, S193Q, M208T, A216C, Q237L and A246C, as compared to the amino acid sequence SEQ ID NO:2. Particularly, the esterase comprises at least one combination of substitutions selected from 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 + F 1871 + N162S + S 18 IK + S 193Q and N204G + Hl 83Q + F 1871 + W69L + A216C + A246C. In addition, the esterase may further comprise at least the substitution C203K. For instance, the esterase may comprise the combination of substitutions M208T + C203K + N204G + H183Q + F187I.
In a preferred embodiment, the esterase comprises at least the combination of substitutions N204G + H183Q + Fl 871 and optionally further comprises at least one substitution selected from M208T, N211M, S193E, V200I, M208N, LBS, E158D. Particularly, the esterase comprises at least one combination of substitutions selected from, or consists of the amino acid sequence as set forth in SEQ ID NO:2 with a combination of substitutions selected from M208T + N204G + H183Q + F187I + N211M + S193E, H183Q + F187I + N204G + M208N + N211M + S193E + V200I, M208T + N204G + H183Q + F187I + N21 IM + S193E + V200I, H183Q + F187I + N204G + M208N + N21 IM + S193E, H183Q + F187I + N204G, H183Q + F187I + N204G + M208T, Hl 83Q + F 1871 + N204G + M208T + L13 S + El 58D, T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + H43N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + M208T + LBS + E158D, T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + H43N + 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 + L I 3 S + E158D, preferably selected from M208T + N204G + H183Q + F187I + N21 IM + S193E, H183Q + F187I + N204G + M208N + N21 IM + S193E + V200I, M208T + N204G + H183Q + F187I + N21 IM + S193E + V200I.
In a particular embodiment, the esterase comprises at least the combination of substitutions M208N + N211M.
In an embodiment, the esterase comprises at least one substitution or combination of substitutions selected from 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 + VI 151 + 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 + Hl 83Q + F 1871 + A62T, N204G + Hl 83Q + F 1871 + A64 V, N204G + Hl 83Q + F187I + L124G, N204G + H183Q + F187I + N162S, N204G + H183Q + F187I + S193Q, N204G + H183Q + F187I + Q237L, M208T + N204G + H183Q + F187I, S27A + R30G + T145G + W228S + V242E, N204G + Hl 83Q + F 1871 + N162S + S 18 IK, G7A + N9T + P 192S + P213A + W228R + Q237L, N204G + H183Q + F187I + N162S + S181K + S193Q, N204G + H183Q + F187I + W69L + A216C + A246C, as compared to the amino acid sequence SEQ ID N0:2.
In another embodiment, the esterase comprises at least one substitution or combination of substitutions selected from or consists of the amino acid sequence as set forth in SEQ ID NO:2 with a combination of substitutions selected from M208N + N21 IM, H183Q + F187I + N204G + M208N + N21 IM + S 193E, Hl 83Q + F 1871 + N204G, Hl 83Q + F 1871 + N204G + M208T, H183Q + F187I + N204G + M208T + L13S + E158D, T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + H43N + T145S + G149A + P164S + Q167V + T206S + S252T + V170I + H183Q + F187I + N204G + M208T + L13S + E158D, T17A + S27T + T48S + I82L + L90F + F92G + A135G + S140A + H43N + 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 + L I 3 S + E158D.
In an embodiment, the esterase comprises at least one substitution selected from Fl 871 and S193Q or at least the combination of substitutions S181K + S193L as compared to the amino acid sequence SEQ ID NO:2 and exhibits an 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 as compared to the esterase of SEQ ID NO:2.
Preferably, the esterase exhibits at least one amino acid residue selected from S130, D175, H207, C240 or C275 as in the parent esterase of SEQ ID NO:2, i.e. the esterase of the invention is not modified at one, two, three, etc., or all of these positions.
Particularly, the esterase may exhibit at least the amino acids S130, D175 and H207 forming the catalytic site of the esterase and/or the amino acids C240 and C275 forming disulphide bond as in the parent esterase. Preferably, the esterase comprises at least a combination of amino acid residues selected from S130 + D175 + H207, C240 + C275 and SI 30 + DI 75 + H207 + C240 + C275, as in the parent esterase, more preferably the combination S130 + D175 + H207 + C240 + C275 as in the parent esterase.
According to the invention, the esterase may further comprise at least one amino acid residue selected from C203 and C248 as in the parent esterase of SEQ ID NO:2. For instance, the esterase may comprise the combination of amino acid residues C203 + C248 as in the parent esterase. Alternatively, the esterase comprises at least the substitution C203K/R, preferably C203K and the amino acid residue C248 as in the parent esterase.
According to the invention, the esterase further exhibits an increased thermostability and/or an increased polyester degrading activity as compared to the esterase of SEQ ID NO: 1.
It is another object of the invention to provide an esterase variant which (i) has at least 97%, 98% or 99% identity to the full length amino acid sequence set forth 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 at least the combination of substitutions M208I + N211M, as compared to the amino acid sequence SEQ ID NO:2, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO:2, (iii) has at least the amino acids C240, C275, V170, F92, P213, E182, L13 and E158, as in the parent esterase of SEQ ID NO:2, (iv) has a polyester degrading activity, and (v) exhibits an increased thermostability and/or an increased polyester degrading activity as compared to the esterase of SEQ ID NO:2 at a pH comprised between 6 and 10.
In an embodiment, the esterase comprises at least one substitution selected from M208N, M208S, N122D, Q237E, Q258E, and S212T.
In an embodiment, the esterase comprises at least one substitution selected from M208A, M208N, N122D, Q237E and Q258E.
According to the invention, the esterase may further comprises at least one substitution at at least one amino acid position selected from SI, Y4, Q5, R6, N9, PIO, Ti l, 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, 154, M56, P58, G59, Y60, T61, A62, D63, A64, S65, S66, L67, A68, W69, L70, R72, R73, L74, 182, 184, N85, T86, N87, S88, R89, L90, D91, F92, P93, D94, S95, R96, S98, Q99, A103, L104, N105, L107, R108, SI 13, VI 15, LI 19, A121, N122, L124, A125, A127, G128, H129, M131, G132, G133, G134, A135, R138, S140, 1143, 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, 1217, 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, as compared to the amino acid sequence SEQ ID NO:2.
In an embodiment, the esterase comprises at least one substitution selected from M208A, M208N, N122D, Q237E and Q258E and further comprises at least one substitution selected from N21 IM, H183Q, Fl 871, N204G, S193E, V200I, T17F, L90Q, H183N and M208L.
In an embodiment, the esterase comprises at least the combination of substitutions selected from or consists of the amino acid sequence as set forth in SEQ ID NO:2 with the combination of substitutions selected from M208A + N21 IM or M208N + N21 IM.
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. Particularly, the esterase comprises at least one combination of substitutions selected from, or consists of the amino acid sequence as set forth in SEQ ID NO:2 with a combination of substitutions selected from M208N + N211M, H183Q + F187I + N204G + M208N + N211M + S193E + V200I and T 17F + L90Q + H183N + S193E + N204G + M208N + N211M + V200I. In another embodiment, the esterase consists of the amino acid sequence as set forth in SEQ ID NO:2 with a combination of substitutions selected from L90W, M208A, M208N, M208S, N122D, Q237E, Q258E, S212T, M208I + N211M.
Preferably, the esterase exhibits at least one amino acid residue selected from S130, D175, H207, C240 or C275 as in the parent esterase of SEQ ID NO:2, i.e. the esterase of the invention is not modified at one, two, three, etc., or all of these positions.
Particularly, the esterase may exhibit at least the amino acids S130, D175 and H207 forming the catalytic site of the esterase and/or the amino acids C240 and C275 forming disulphide bond as in the parent esterase. Preferably, the esterase comprises at least a combination of amino acid residues selected from S130 + D175 + H207, C240 + C275 and SI 30 + DI 75 + H207 + C240 + C275, as in the parent esterase, more preferably the combination S130 + D175 + H207 + C240 + C275 as in the parent esterase.
According to the invention, the esterase may further comprise at least one amino acid residue selected from C203 and C248 as in the parent esterase of SEQ ID NO:2. For instance, the esterase may comprise the combination of amino acid residues C203 + C248 as in the parent esterase. Alternatively, the esterase comprises at least the substitution C203K/R, preferably C203K and the amino acid residue C248 as in the parent esterase.
According to the invention, the esterase further exhibits an increased thermostability and/or an increased polyester degrading activity as compared to the esterase of SEQ ID NO: 1.
Polyester degrading activity of the variant
It is an object of the invention to provide new enzymes having an esterase activity under basic conditions. In a particular embodiment, the enzyme of the invention exhibits a cutinase activity.
In a particular embodiment, the esterase of the invention has a polyester degrading activity, preferably a polyethylene terephthalate (PET) degrading activity, and/or a polybutylene adipate terephthalate (PBAT) degrading activity and/or a polycaprolactone (PCL) degrading activity and/or a polybutylene succinate (PBS) activity, more preferably a polyethylene terephthalate (PET) degrading activity, and/or a polybutylene adipate terephthalate (PBAT) degrading activity. Even more preferably, the esterase of the invention has a polyethylene terephthalate (PET) degrading activity.
Advantageously, the esterase of the invention exhibits a polyester degrading activity in a range of temperatures from 20°C to 90°C, preferably from 30°C to 90°C, more preferably from 40°C to 90°C, more preferably from 50°C to 90°C, even more preferably from 60°C to 90°C, from 68°C to 90°C. Particularly, the esterase of the invention exhibits a polyester degrading activity in a range of temperatures from 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. Particularly, the esterase of the invention exhibits a polyester degrading 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 invention exhibits a polyester degrading 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. In a particular embodiment, the esterase exhibits a polyester degrading 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, a polyester degrading activity is still measurable at a temperature between 55°C and 70°C. Within the context of the invention, temperatures are given at +/- 1°C.
According to the invention, the esterase of the invention has an increased polyester degrading activity at a given temperature, compared to the parent esterase, and more particularly at a temperature between 40°C and 90°C, more preferably between 50°C and 90°C. Advantageously, the esterase of the invention has an increased polyester degrading activity compared to the esterase of SEQ ID NO: 1 and/or SEQ ID NO:2 in the whole range of temperatures 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. Particularly, the esterase of the invention exhibits an increased polyester degrading 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 invention exhibits an increased polyester degrading 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 invention exhibits an increased polyester degrading activity at least at 50°C, 54°C, 55 °C, 60°C, 65 °C or 68°C, preferably at 55 °C or at 60°C or at 65 °C. Advantageously, the esterase has a polyester degrading activity at least 5% higher than the polyester degrading activity of the parent esterase, preferably at least 10%, 20%, 50%, 100% or more.
Preferably, the esterase has a polyester degrading activity at 65°C at least 5% higher than the polyester degrading activity of the parent esterase, preferably at least 10%, 20%, 30%, 50%, 100% or more.
In another preferred embodiment, the esterase has a polyester degrading activity at 60°C at least 5% higher than the polyester degrading activity of the parent esterase, preferably at least 10%, 20%, 50%, 100% or more. Advantageously, the esterase of the invention exhibits a measurable esterase activity at least in a range of pH from 5 to 11, preferably from 6 to 9, more preferably in a range of pH from 6.5 to 9, even more preferably in a range of pH from 6.5 to 8, even more preferably at a pH comprised between 7 and 9, in particular at pH 8.
Nucleic acids, expression cassette, vector, host cell
It is a further object of the invention to provide a nucleic acid encoding an esterase as defined above.
As used herein, the term "nucleic acid", “ nucleic sequenced “polynucleotide", “oligonucleotide" and “nucleotide sequence" refer to a sequence of deoxyribonucleotides and/or ribonucleotides. The nucleic acids can be DNA (cDNA or gDNA), RNA, or a mixture thereof. It can be in single stranded form or in duplex form or a mixture thereof. It can be of recombinant, artificial and/or synthetic origin and it can comprise modified nucleotides, comprising for example a modified bond, a modified purine or pyrimidine base, or a modified sugar. The nucleic acids of the invention can be in isolated or purified form, and made, isolated and/or manipulated by techniques known per se in the art, e.g., cloning and expression of cDNA libraries, amplification, enzymatic synthesis or recombinant technology. The nucleic acids can also be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Belousov (1997) Nucleic Acids Res. 25:3440-3444.
The invention also encompasses nucleic acids which hybridize, under stringent conditions, to a nucleic acid encoding an esterase as defined above. Preferably, such stringent conditions include incubations of hybridization filters at about 42° C for about 2.5 hours in 2 X SSC/0.1%SDS, followed by washing of the filters four times of 15 minutes in 1 X SSC/0.1% SDS at 65° C. Protocols used are described in such reference 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)).
The invention also encompasses nucleic acids encoding an esterase of the invention, wherein the sequence of said nucleic acids, or a portion of said sequence at least, has been engineered using optimized codon usage.
Alternatively, the nucleic acids according to the invention may be deduced from the sequence of the esterase according to the invention and codon usage may be adapted according to the host cell in which the nucleic acids shall be transcribed. These steps may be carried out according to methods well known to one skilled in the art and some of which are described in the reference manual Sambrook et al. (Sambrook et al., 2001). Nucleic acids of the invention may further comprise additional nucleotide sequences, such as regulatory regions, i.e., promoters, enhancers, silencers, terminators, signal peptides and the like that can be used to cause or regulate expression of the polypeptide in a selected host cell or system.
The present invention further relates to an expression cassette comprising a nucleic acid according to the invention operably linked to one or more control sequences that direct the expression of said nucleic acid in a suitable host cell.
The term "expression", as used herein, refers to any step involved in the production of a polypeptide including, but being not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
The term “expression cassette” denotes a nucleic acid construct comprising a coding region, i.e. a nucleic acid of the invention, and a regulatory region, i.e. comprising one or more control sequences, operably linked.
Typically, the expression cassette comprises, or consists of, a nucleic acid according to the invention operably linked to a control sequence such as transcriptional promoter and/or transcription terminator. The control sequence may include a promoter that is recognized by a host cell or an in vitro expression system for expression of a nucleic acid encoding an esterase of the present invention. The promoter contains transcriptional control sequences that mediate the expression of the enzyme. The promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell. The control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription. The terminator is operably linked to the 3'-terminus of the nucleic acid encoding the esterase. Any terminator that is functional in the host cell may be used in the present invention. Typically, the expression cassette comprises, or consists of, a nucleic acid according to the invention operably linked to a transcriptional promoter and a transcription terminator.
The invention also relates to a vector comprising a nucleic acid or an expression cassette as defined above.
As used herein, the terms “vector” or "expression vector" refer to a DNA or RNA molecule that comprises an expression cassette of the invention, used as a vehicle to transfer recombinant genetic material into a host cell. The major types of vectors are plasmids, bacteriophages, viruses, cosmids, and artificial chromosomes. The vector itself is generally a DNA sequence that consists of an insert (a heterologous nucleic acid sequence, transgene) and a larger sequence that serves as the “backbone” of the vector. The purpose of a vector which transfers genetic information to the host is typically to isolate, multiply, or express the insert in the target cell. Vectors called expression vectors (expression constructs) are specifically adapted for the expression of the heterologous sequences in the target cell, and generally have a promoter sequence that drives expression of the heterologous sequences encoding a polypeptide. Generally, the regulatory elements that are present in an expression vector include a transcriptional promoter, a ribosome binding site, a terminator, and optionally present operator. Preferably, an expression vector also contains an origin of replication for autonomous replication in a host cell, a selectable marker, a limited number of useful restriction enzyme sites, and a potential for high copy number. Examples of expression vectors are cloning vectors, modified cloning vectors, specifically designed plasmids and viruses. Expression vectors providing suitable levels of polypeptide expression in different hosts are well known in the art. The choice of the vector will typically 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.
It is another object of the invention to provide a host cell comprising a nucleic acid, an expression cassette or a vector as described above. The present invention thus relates to the use of a nucleic acid, expression cassette or vector according to the invention to transform, transfect or transduce a host cell. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which it must be introduced.
According to the invention, the host cell may be transformed, transfected or transduced in a transient or stable manner. The expression cassette or vector of the invention is introduced into a host cell so that the cassette or vector is maintained as a chromosomal integrant or as a selfreplicating extra-chromosomal 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 useful in the production of a variant of the present invention, e.g., a prokaryote or a eukaryote. The prokaryotic host cell may be any Gram-positive or Gram-negative bacterium. The host cell may also be an eukaryotic cell, such as a yeast, fungal, mammalian, insect or plant cell. In a particular embodiment, the host cell is selected from the group of Escherichia coli, Bacillus, Streptomyces, Trichoderma, Aspergillus, Saccharomyces, Pichia, Vibrio or Yarrowia.
The nucleic acid, expression cassette or expression vector according to the invention may be introduced into the host cell by any method known by the skilled person, 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.
Optionally, more than one copy of a nucleic acid, cassette or vector of the present invention may be inserted into a host cell to increase production of the variant.
In a particular embodiment, the host cell is a recombinant microorganism. The invention indeed allows the engineering of microorganisms with improved capacity to degrade polyester containing material. For instance, the sequence of the invention may be used to complement a wild type strain of a fungus or bacterium already known as able to degrade polyester, in order to improve and/or increase the strain capacity.
Production of esterase
It is another object of the invention to provide a method of producing an esterase of the invention, comprising expressing a nucleic acid encoding the esterase and optionally recovering the esterase.
In particular, the present invention relates to in vitro methods of producing an esterase of the present invention comprising (a) contacting a nucleic acid, cassette or vector of the invention with an in vitro expression system; and (b) recovering the esterase produced. In vitro expression systems are well-known by the person skilled in the art and are commercially available.
Preferably, the method of production comprises
(a) culturing a host cell that comprises a nucleic acid encoding an esterase of the invention under conditions suitable to express the nucleic acid; and optionally
(b) recovering said esterase from the cell culture.
Advantageously, the host cell is a recombinant Bacillus, recombinant E. coli, recombinant Aspergillus, recombinant Trichoderma, recombinant Streptomyces, recombinant Saccharomyces, recombinant Pichia, recombinant Vibrio or recombinant Yarrowia.
The host cells are cultivated in a nutrient medium suitable for production of polypeptides, using methods known in the art. For example, the cell may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed- batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the enzyme to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium, from commercial suppliers or prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the esterase is excreted into the nutrient medium, the esterase can be recovered directly from the culture supernatant. Conversely, the esterase can be recovered from cell lysates or after permeabilisation. The esterase may be recovered using any method known in the art. For example, the esterase may be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. Optionally, the esterase may be partially or totally purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction to obtain substantially pure polypeptides.
The esterase may be used as such, in purified form, either alone or in combinations with additional enzymes, to catalyze enzymatic reactions involved in the degradation and/or recycling of polyester(s) and/or polyester containing material, such as plastic products containing polyester. The esterase may be in soluble form, or on solid phase. In particular, it may be bound to cell membranes or lipid vesicles, or to synthetic supports such as glass, plastic, polymers, filter, membranes, e.g., in the form of beads, columns, plates and the like.
Composition
It is a further object of the invention to provide a composition comprising an esterase, or a host cell of the invention, or extract thereof containing the esterase. In the context of the invention, the term “composition” encompasses any kind of compositions comprising an esterase or host cell of the invention, or an extract thereof containing the esterase.
The composition of the invention may comprise from 0.1% to 99.9%, preferably from 0.1% to 50%, more preferably from 0.1% to 30%, even more preferably from 0.1% to 5% by weight of esterase, based on the total weight of the composition. Alternatively, the composition may comprise between 5 and 10% by weight of esterase of the invention.
The composition may be in liquid or dry form, for instance in the form of a powder. In some embodiments, the composition is a lyophilizate.
The composition may further comprise excipients and/or reagents etc. Appropriate excipients encompass buffers commonly used in biochemistry, agents for adjusting pH, preservatives such as sodium benzoate, sodium sorbate or sodium ascorbate, conservatives, protective or stabilizing agents such as starch, dextrin, arabic gum, salts, sugars e.g. sorbitol, trehalose or lactose, glycerol, polyethyleneglycol, polypropylene glycol, propylene glycol, sequestering agent such as EDTA, reducing agents, amino acids, a carrier such as a solvent or an aqueous solution, and the like. The composition of the invention may be obtained by mixing the esterase with one or several excipients.
For instance, the composition comprises from 0.1% to 99.9%, preferably from 50% to 99.9%, more preferably from 70% to 99.9%, even more preferably from 95% to 99.9% by weight of excipient(s), based on the total weight of the composition. Alternatively, the composition may comprise from 90% to 95% by weight of excipient(s).
The composition may further comprise additional polypeptide(s) exhibiting an enzymatic activity. The amounts of esterase of the invention will be easily adapted by those skilled in the art depending e.g., on the nature of the polyester to degrade and/or the additional enzymes/polypeptides contained in the composition.
The esterase of the invention may be solubilized in an aqueous medium together with one or several excipients, especially excipients which are able to stabilize or protect the polypeptide from degradation. For instance, the esterase of the invention may be solubilized in water, eventually with additional components, such as glycerol, sorbitol, dextrin, starch, glycol such as propanediol, salt, etc. The resulting mixture may then be dried so as to obtain a powder. Methods for drying such mixture are well known to the one skilled in the art and include, without limitation, lyophilisation, freeze-drying, spray-drying, supercritical drying, downdraught evaporation, thin-layer evaporation, centrifugal evaporation, conveyer drying, fluidized bed drying, drum drying or any combination thereof.
The composition may be under powder form and may comprise esterase and a stabilizing/solubilizing amount of glycerol, sorbitol or dextrin, such as maltodextrine and/or cyclodextrine, starch, glycol such as propanediol, and/or salt.
The composition of the invention may comprise at least one recombinant cell expressing an esterase of the invention, or an extract thereof. An “extract of a cell” designates any fraction obtained from a cell, such as cell supernatant, cell debris, cell walls, DNA extract, enzymes or enzyme preparation or any preparation derived from cells by chemical, physical and/or enzymatic treatment, which is essentially free of living cells. Preferred extracts are enzymatically-active extracts. The composition of the invention may comprise one or several recombinant cells of the invention or extract thereof, and optionally one or several additional cells.
For instance, the composition consists or comprises a culture medium of a recombinant microorganism expressing and excreting an esterase of the invention. In a particular embodiment, the composition comprises such culture medium lyophilized. Uses of esterase
It is a further object of the invention to provide methods using an esterase of the invention for degrading and/or recycling in aerobic or anaerobic conditions polyester, or polyester containing material. The esterases of the invention are particularly useful for degrading PET and PET containing material, particularly at a pH between 6 and 10.
It is therefore an object of the invention to use an esterase of the invention, or corresponding recombinant cell or extract thereof having an esterase activity, or composition for the enzymatic degradation of a polyester.
Advantageously, the polyester targeted by the esterase is selected from polyethylene terephthalate (PET), polytrimethylene 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), polyethylene adipate) (PEA), polyethylene naphthalate (PEN), “polyolefin-like” polyesters and blends/mixtures of these materials, preferably polyethylene terephthalate.
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), bi s(2-hydroxy ethyl) terephthalate (BHET), 1 -(2 -Hydroxy ethyl) 4-methyl terephthalate (HEMT) and dimethyl terephthalate (DMT)) are optionally recovered.
It is also an object of the invention to use an esterase of the invention, or corresponding recombinant cell or extract thereof, or composition for the enzymatic degradation of at least one polyester of a polyester containing material, particularly at a pH between 6 and 10.
It is another object of the invention to provide a method for degrading at least one polyester of a polyester containing material, wherein the polyester containing material is contacted with an esterase or host cell or extract thereof or composition of the invention, particularly at a pH between 6 and 10, thereby degrading the at least one polyester of a polyester containing material.
Advantageously, polyester(s) is (are) depolymerized up to monomers and/or oligomers.
Particularly, the invention provides a method for degrading PET of a PET containing material, wherein the PET containing material is contacted with an esterase or host cell or composition of the invention, preferably at a pH between 6 and 10, thereby degrading the PET. Advantageously, at least one polyester is degraded into repolymerizable monomers and/or oligomers, which may be advantageously retrieved in order to be reused. The retrieved monomers/oligomers may be used for recycling (e.g., repolymerizing polyesters) or methanization. In a particular embodiment, at least one polyester is PET, and monoethylene glycol, terephthalic acid, methyl -2-hydroxy ethyl terephthalate (MEET), bi s(2-hydroxy ethyl) terephthalate (BEET), 1 -(2 -Hydroxy ethyl) 4-methyl terephthalate (HEMT) and/or dimethyl terephthalate (DMT) are retrieved.
Preferably, polyester(s) of the polyester containing material is (are) fully degraded.
The time required for degrading a polyester containing material may vary depending on the polyester containing material itself (i.e., nature and origin of the polyester containing material, its composition, shape etc.), the type and amount of esterase used, as well as various process parameters (i.e., temperature, pH, additional agents, etc.). One skilled in the art may easily adapt the process parameters to the polyester containing material and the envisioned degradation time.
Advantageously, the degrading process is implemented 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 degrading process is implemented at 60°C. In another particular embodiment, the degrading process is implemented at 65°C. In another particular embodiment, the degrading process is implemented at 70°C. More generally, the temperature is maintained below an inactivating temperature, which corresponds to the temperature at which the esterase is inactivated (i.e., temperature at which the esterase has lost more than 80% of activity as compared to its activity at its optimum temperature) and/or the recombinant microorganism does no more synthesize the esterase. Particularly, the temperature is maintained below the glass transition temperature (Tg) of the targeted polyester.
Advantageously, the process is implemented in a continuous flow process, at a temperature at which the esterase can be used several times and/or recycled.
Advantageously, the degrading process is implemented at a pH comprised between 5 to 9, preferably in a range of pH from 6 to 9, more preferably in a range of pH from 6.5 to 9, even more preferably in a range of pH from 6.5 to 8, even more preferably at a pH comprised between 7 and 9, in particular at pH 8.
The polyester containing material may be pretreated prior to be contacted with the esterase, in order to physically change its structure, so as to increase the surface of contact between the polyester and the esterase. It is another object of the invention to provide a method of producing monomers and/or oligomers from a polyester containing material, comprising exposing a polyester containing material to an esterase of the invention, or corresponding recombinant cell or extract thereof, or composition, particularly at a pH between 6 and 10, and optionally recovering monomers and/or oligomers.
Monomers and/or oligomers resulting from the depolymerization may be recovered, sequentially or continuously. A single type of monomers and/or oligomers or several different types of monomers and/or oligomers may be recovered, depending on the starting polyester containing material.
The method of the invention is particularly useful for producing monomers selected from monoethylene glycol and terephthalic acid, and/or oligomers selected from methyl-2- hydroxy ethyl terephthalate (MHET), bis(2-hydroxy ethyl) terephthalate (BHET), l-(2- Hydroxyethyl) 4-methyl terephthalate (HEMT) and dimethyl terephthalate (DMT), from PET, and/or plastic product comprising PET.
The recovered monomers and/or oligomers may be further purified, using all suitable purifying methods and conditioned in a re-polymerizable form.
Recovered repolymerizable monomers and/or oligomers may be reused for instance to synthesize polyesters. Advantageously, polyesters of same nature are repolymerized. However, it is possible to mix the recovered monomers and/or oligomers with other monomers and/or oligomers, in order for instance to synthesize new copolymers. Alternatively, the recovered monomers may be used as chemical intermediates in order to produce new chemical compounds of interest.
As an example, processes for degrading such polyester containing material including an esterase of the invention are disclosed in the 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 invention also relates to a method of surface hydrolysis or surface functionalization of a polyester containing material, comprising exposing a polyester containing material to an esterase of the invention, or corresponding recombinant cell or extract thereof, or composition, particularly at a pH between 6 and 10. The method of the invention is particularly useful for increasing hydrophilicity, or water absorbency, of a polyester material. Such increased hydrophilicity may have particular interest in textiles production, electronics and biomedical applications.
The invention also relates to a method for treating water, waste water or sewage, particularly at a pH between 6 and 10. In waste water or sewage treatment applications the esterase according to the invention can be used to degrade microplastic particles consisting of polyester (preferable PET) like polymer filaments, fibres or other kinds of polyester-based product debris and fragments, preferably PET -based product debris and fragments. It is a further object of the invention to provide a polyester containing material in which an esterase of the invention and/or a recombinant microorganism expressing and excreting said esterase is/are included. As an example, processes for preparing such polyester containing material including an esterase of the invention are disclosed in the patent applications WO2013/093355, WO 2016/198650, WO 2016/198652, WO 2019/043145 and WO 2019/043134.
It is thus an object of the invention to provide a polyester containing material containing an esterase of the invention and/or a recombinant cell and/or a composition or extract thereof and at least PET. According to an embodiment, the invention provides a plastic product comprising PET and an esterase of the invention having a PET degrading activity.
It is thus another object of the invention to provide a polyester containing material containing an esterase of the invention and/or a recombinant cell and/or a composition or extract thereof and at least PBAT. According to an embodiment, the invention provides a plastic product comprising PBAT and an esterase of the invention having a PBAT degrading activity.
It is thus another object of the invention to provide a polyester containing material containing an esterase of the invention and/or a recombinant cell and/or a composition or extract thereof and at least PBS. According to an embodiment, the invention provides a plastic product comprising PBS and an esterase of the invention having a PBS degrading activity.
It is thus another object of the invention to provide a polyester containing material containing an esterase of the invention and/or a recombinant cell and/or a composition or extract thereof and at least PCL. According to an embodiment, the invention provides a plastic product comprising PCL and an esterase of the invention having a PCL degrading activity.
Classically, an esterase of the invention may be used in detergent, food, animal feed, paper making, textile and pharmaceutical applications. More particularly, the esterase of the invention may be used as a component of a detergent composition. Detergent compositions include, without limitation, hand or machine laundry detergent compositions, such as laundry additive composition suitable for pre-treatment of stained fabrics and rinse added fabric softener composition, detergent composition for use in general household hard surface cleaning operations, detergent compositions for hand or machine dishwashing operations. For instance, an esterase of the invention may be used as a detergent additive. The invention thus provides detergent compositions comprising an esterase of the invention. Particularly, the esterase of the invention may be used as a detergent additive in order to reduce pilling and greying effects during textile cleaning. The present invention is also directed to methods for using an esterase of the invention in animal feed, as well as to feed compositions and feed additives comprising an esterase of the invention. The terms “feed” and “feed composition” refer to any compound, preparation, mixture, or composition suitable for, or intended for intake by an animal. The esterase of the invention may also be used to hydrolyze proteins, and to produce hydrolysates comprising peptides. Such hydrolysates may be used as feed composition or feed additives.
It is a further object of the invention to provide a method for using an esterase of the invention in papermaking industry. More particularly, the esterase of the invention may be used to remove stickies from the paper pulp and water pipelines of paper machines.
EXAMPLES
Example 1 -Construction, expression and purification of esterases
- Construction
Esterase according to the invention have been generated using the plasmidic construction pET26b-LCC-His. This plasmid consists in cloning a gene encoding the esterase of SEQ ID
NO: 1, optimized for Escherichia coli expression between Ndel and Xhol restriction sites. Two site directed mutagenesis kits have been used according to the recommendations of the supplier, in order to generate the esterase variants: QuikChange II Site-Directed Mutagenesis kit and QuikChange Lightning Multi Site-Directed from Agilent (Santa Clara, California, USA). - Expression and purification of the esterases
The strains Stellar™ (Clontech, California, USA) and E. coli One Shot® BL21 DE3 (Life technologies, Carlsbad, California, USA) have been successively employed to perform the cloning and recombinant expression in 50 mL LB-Miller medium or ZYM auto inducible medium (Studier et al., 2005- Prot. Exp. Pur. 41, 207-234). The induction in LB-Miller medium has been performed at 16°C, with 0.5 mM of isopropyl P-D-l -thiogalactopyranoside (IPTG, Euromedex, Souffelweyersheim, France). The cultures have been stopped by centrifugation (8000 rpm, 20 minutes at 10°C) in an Avanti J-26 XP centrifuge (Beckman Coulter, Brea, USA). The cells have been suspended in 20 mL of Talon buffer (Tris-HCl 20 mM, NaCl 300 mM, pH 8). Cell suspension was then sonicated during 2 minutes with 30% of amplitude (2sec ON and Isec OFF cycles) by FB 705 sonicator (Fisherbrand, Illkirch, France). Then, a step of centrifugation has been realized: 30 minutes at 11000 rpm, 10°C in an Eppendorf centrifuge. The soluble fraction has been collected and submitted to affinity chromatography. This purification step has been completed with Talon® Metal Affinity Resin (Clontech, CA, USA). Protein elution has been carried out with steps of Talon buffer supplemented with imidazole. Purified protein has been dialyzed against Talon buffer then quantified using Bio-Rad protein assay according to manufacturer instructions (Lifescience Bio-Rad, France) and stored at +4°C.
Example 2 - Evaluation of the degrading activity of the esterases
The degrading activity of the esterases has been determined and compared to the activity of esterase of SEQ ID NO: 1.
Multiple methodologies to assess the specific activity have been used:
(1) Specific activity based upon PET hydrolysis
(2) Degrading activity based upon the degradation of a polyester under solid form
(3) Degrading activity based upon PET hydrolysis in reactors above 100 mL
(4) Specific activity based upon PET hydrolysis and Ultraviolet Light Absorbance (UV Assay) analysis
Details on the protocol of such methods are given below.
2.1. Specific activity based upon PET hydrolysis
100 mg of amorphous PET under powder form (prepared according to WO 2017/198786 to reach a crystallinity below 20%) were weighted and introduced in a 100 mL glass bottle. 1 mL of esterase preparation comprising esterase of SEQ ID NO: 1 (as reference control) or esterase of the invention, prepared at 0.69pM or 3.45pM in Talon buffer (Tris-HCl 20 mM, NaCl 0.3M, pH 8) were introduced in the glass bottle (0.2 mgenzyme/gPET or 1.0 mgenzyme/gPEr). Finally, 49 mL of 0.1 M potassium phosphate buffer pH 8 were added. The depolymerization started 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 reaction, in mg of equivalent TA generated / hour, was determined by samplings performed at different time during the first 24 hours and analyzed by Ultra High Performance Liquid Chromatography (UHPLC). If necessary, samples were diluted in 0.1 M potassium phosphate buffer pH 8. Then, 150 pL of methanol and 6.5 pL of HC1 6 N were added to 150 pL of sample or dilution. After mixing and filtering on 0.45 pm syringe filter, samples were loaded on UHPLC to monitor the liberation of terephthalic acid (TA), MEET and BHET. Chromatography system used was an Ultimate 3000 UHPLC system (Thermo Fisher Scientific, Inc. Waltham, MA, USA) including a pump module, an autosampler, a column oven thermostated at 25°C, and an UV detector at 240 nm. The column used was a Discovery® HS C18 HPLC Column (150 x 4.6 mm, 5 pm, equipped with precolumn, Supelco, Bellefonte, USA). TA, MHET and BHET were separated using a gradient of MeOH (30 % to 90 %) in 1 mM of H2SO4 at ImL/min. Injection was 20 pL of sample. TA, MHET and BHET were measured according to standard curves prepared from commercial TA and BHET and in house synthetized MHET in the same conditions than samples. The specific activity of PET hydrolysis (mg of equivalent TA/hour/mg of enzyme) was determined in the linear part of the hydrolysis curve of the reaction (i.e. at the beginning of the reaction), such curve being set up by samplings performed at different time during the first 24, 48, 72, 96 hours. Equivalent TA corresponds to the sum of TA measured and of TA contained in measured MHET and BHET. Said measurement of equivalent TA can also be used to calculate the yield of a PET depolymerization assay at a given time and/or after a defined period of time (e.g. 24h, 48h, 72h or 96h).
2.2. Activity based upon degradation of a polyester under solid form
20 pL of enzyme preparation was deposited in a well created in an agar plate containing PET. Preparation of agar plates was realized by solubilizing 500mg of PET in hexafluoro-2-propanol (HFIP) and pouring this medium in a 250 mL aqueous solution. After HFIP evaporation at 52°C under 140 mbar, the solution was mixed v/v with 0.2 M potassium phosphate buffer pH 8 containing 3% agar. Around 30 mL of the mixture is used to prepare each plate and stored at 4°C. The diameters or the surface area of the halos formed due to the polyester degradation by parent esterase and variants were 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. Activity based upon PET hydrolysis in reactor
49 mg purified protein prepared in 195 ml of sodium potassium phosphate buffer (pH 8.0, 100 mM) were combined with 50 g foamed PET (98% purity) (prepared according to WO 2021/123299 to reach a crystallinity below 20%) in a 500 mL Benchtop Fl 0.5 MB Bioreactor (AD Biotec, France). Temperature regulation at 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 68°C or 70°C was performed in the water jacketed bioreactor and a double Rushton impeller was used to maintain constant agitation at 800 rpm. The pH value was regulated to pH 8.0 by addition of a 20% NaOH (w/w) solution using the ROSITA 2.0 software (AD Biotec, France). The kinetic of the PET depolymerization was followed based on NaOH consumption considering an exclusive production of TPA and MEG (2 mols of NaOH are consumed to titrate 1 mol of the diacid TPA). The final yield of the PET depolymerization assay was determined either by the determination of residual PET weight or by the determination of equivalent TA generated, or through the base consumption. Weight determination of residual PET was assessed by the filtration, at the end of the reaction, of the reactional volume through a 12 to 15 gm grade 11 ashless paper filter (Dutscher SAS, Brumath, France) and drying of such retentate before weighting it. The determination of equivalent TA generated was realized using UHPLC methods described in 2.1, and the percentage of hydrolysis was calculated based on the ratio of molar concentration at a given time (TA + MHET + BHET) versus the total amount of TA contained in the initial sample. PET depolymerization produced acid monomers that will be neutralized with the base to be able to maintain the pH in the reactor. The determination of equivalent TA produced was calculating using the corresponding molar base consumption, and the percentage of hydrolysis was calculated based on the ratio of molar concentration at a given time of equivalent TA versus the total amount of TA contained in the initial sample.
2.4. Specific activity based upon PET hydrolysis and Ultraviolet Light Absorbance (UV Assay) analysis
100 mg of amorphous PET under powder form (prepared according to WO 2017/198786 to reach a crystallinity below 20%) were weighted and introduced in a 100 mL glass bottle. 1 mL of esterase preparation comprising esterase of SEQ ID NO: 1 (as reference control) or esterase of the invention, prepared at 0.69pM or 3.45pM in Talon buffer (Tris-HCl 20 mM, NaCl 0.3M, pH 8) were introduced in the glass bottle (0.2 mgenzyme/gPET or 1.0 mgenzyme/gPEr). Finally, 49 mL of lOOmM potassium phosphate buffer pH 8.0 were added. The depolymerization started 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, reaction could be miniaturized in deepwell (ThermoSci entific, Abgene, AB-0661, Illkirch, France), 22 mg of amorphous PET under powder form (prepared according to WO 2017/198786 to reach a crystallinity below 20%) were weighted and introduced in each well of a deepwell plate. 0.1 mL of esterase preparation comprising esterase of SEQ ID NO: 1 (as reference control) or esterase of the invention, prepared at 0.138pM in Talon buffer (Tris-HCl 20 mM, NaCl 0.3M, pH 8 or lOOmM potassium phosphate buffer pH 8.0) were introduced in each well of the deepwell. Finally, 0.9 mL of lOOmM potassium phosphate buffer pH 8.0 were added. The depolymerization 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 incubator shaker (Infors HT, Bottmingen, Suisse). The initial rate of depolymerization reaction, in pmol of soluble degradation products generated / hour was determined by samplings performed at different time during the first 24 hours and analyzed by absorbance reading at 242 nm using an Eon Microplate Spectrophotometer (BioTek, USA). The increase in absorbance of the reaction mixtures in the ultraviolet region of the light spectrum (at 242 nm) indicates the release of soluble TA or its esters (BHET and MHET) from an 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 enzyme-specific activity is determined as total equivalent TA produced. If necessary, samples were diluted in lOOmM potassium phosphate buffer pH8.0. The specific activity of PET hydrolysis (pmol of soluble products/hour/mg of enzyme) was determined in the linear part of the hydrolysis curve of the reaction (i.e. at the beginning of the reaction), such curve being set up by samplings performed at different time during the first 24 hours. Said measurement of equivalent TA can also be used to calculate the yield of a PET depolymerization assay at a given time and/or after a defined period of time (e.g. 24h, 48h, 72h or 96h).
RESULTS
Specific degrading activity based upon PET hydrolysis under basic conditions as compared to esterase of SEQ ID NO : 1
The specific degrading activity of esterases (variants) of the invention was measured at 55°C, 60°C or 65°C, pH 8 at the beginning of the reaction as exposed in Example 2.4. The results are shown in Table 1 below. The specific degrading activity of the esterase of SEQ ID NO: 1 is used as a reference and considered as 100% specific degrading activity. Table 1 : Specific degrading activity of esterases of the invention at pH 8 compared to SEQ ID NO: 1.
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
Variants have the exact amino acid sequence of SEQ ID NO: 1 except the substitutions listed in Table 1.
PET depolymerization yield under basic conditions as compared to esterase of SEQ ID NO:1 The yield of PET depolymerization, of esterase (variants) of the invention was measured according to Example 2.4, after 6 hours or after 24 hours at 55°C, 60°C or 65°C, pH 8. In the context of the present invention, the PET depolymerization yield is used to evaluate the degrading activity. Results (average values) are shown in Table 2 and Table 3 , respectively. The yield of PET depolymerization of the esterase of SEQ ID NO: 1 after 6 hours or after 24 hours, pH 8 is used as a reference and considered as 100% degrading activity, respectively.
Table 2: PET depolymerization yield of esterases of the invention after 6 hours
Figure imgf000060_0002
Figure imgf000061_0001
Figure imgf000062_0001
Variants have the exact amino acid sequence of SEQ ID NO: 1 except the substitutions listed in Table 2.
Table 3: PET depolymerization yield of esterases of the invention after 24 hours.
Figure imgf000062_0002
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
Variants have the exact amino acid sequence of SEQ ID NO: 1 except the substitutions listed in Table 3.
PET depolymerization yield in reactor under basic conditions as compared to esterase of SEQ ID NO:1
The yield of PET depolymerization of esterase (variants) of the invention was measured in reactors according to Example 2.3, at 68°C, pH 8. In the context of the present invention, the PET depolymerization yield is used to evaluate the degrading activity. Results are shown in Table 4 below. The time to reach 90% yield of PET depolymerization with the esterase of SEQ ID NO: 11, pH 8 is 22.2 hours. PET depolymerization was performed using 1.0 mgenzyme/gPET .
Table 4: Time to reach 90% of PET depolymerization yield with esterases of the invention.
Figure imgf000065_0002
Figure imgf000066_0001
Variants have the exact amino acid sequence of SEQ ID NO: 1 except the substitutions listed in Table 4.
The yield of PET depolymerization of esterase (variants) of the invention was measured in reactors according to Example 2.3, at 68°C, pH 8 with the only difference that the quantity of PET was 14% w/w except of 20%. In the context of the present invention, the PET depolymerization yield is used to evaluate the degrading activity. Results are shown in Table 5 below. The time to reach 90% yield of PET depolymerization with the esterase of SEQ ID NO: 1, pH 8 is 20 hours. PET depolymerization was performed using 1.0 mgenzyme/gPET . Table 5: Time to reach 90% of PET depolymerization yield with esterases of the invention
Figure imgf000066_0002
Variants have the exact amino acid sequence of SEQ ID NO: 1 except the substitutions listed in Table 5. PET depolymerization yield under basic conditions as compared to esterase of SEQ ID NO: 2
The yield of PET depolymerization, of esterase (variants) of the invention was measured according to Example 2.4, after 6 hours or after 24 hours at 55°C, pH 8. In the context of the present invention, the PET depolymerization yield is used to evaluate the degrading activity. Results (average values) are shown in Table 6 and Table 7 , respectively after 6 hours or after 24 hours. The yield of PET depolymerization of the esterase of SEQ ID NO:2 after 6 hours or after 24 hours, pH 8 is used as a reference and considered as 100% degrading activity, respectively.
Table 6: PET depolymerization yield of esterases of the invention after 6 hours
Figure imgf000067_0002
Figure imgf000067_0001
Figure imgf000068_0001
Figure imgf000068_0002
The variants listed in Table 6 have the exact amino acid sequence of SEQ ID NO:2 except the substitutions listed in Table 6, respectively. Table 7: PET depolymerization yield of esterases of the invention after 24 hours.
Figure imgf000069_0001
Figure imgf000069_0002
Figure imgf000070_0001
Variants have the exact amino acid sequence of SEQ ID NO:2 except the substitutions listed in Table 7.
Example 3 - Evaluation of the thermostability of esterases of the invention
The thermostability of esterases of the invention has been determined and compared to the thermostability of the esterase of SEQ ID NO: 1 or SEQ ID NO:2.
Different methodologies have been used to estimate thermostability:
(1) Circular dichroism of proteins in solution;
(2) Residual esterase activity after protein incubation in given conditions of temperatures, times and buffers;
(3) Residual polyester’s depolymerization activity after protein incubation in given conditions of temperatures, times and buffers;
(4) Ability to degrade a solid polyester compound (such as PET or PBAT or analogues) dispersed in an agar plate, after protein incubation in given conditions of temperatures, times and buffers;
(5) Ability to perform multiple rounds of polyester’s depolymerization assays in given conditions of temperatures, buffers, protein concentrations and polyester concentrations;
(6) Differential Scanning Fluorimetry (DSF);
Details on the protocol of such methods are given below.
3.1. Circular dichroism
Circular dichroism (CD) has been performed with a Jasco 815 device (Easton, USA) to compare the melting temperature (Tm) of the esterase of SEQ ID NO: 1 with the Tm of the esterases of the invention. Technically 400pL protein sample was prepared at 0.5 mg / mL in Talon buffer and used for CD. A first scan from 280 to 190 nm was realized to determine two maxima intensities of CD corresponding to the correct folding of the protein. A second scan was then performed from 25 °C to 110°C, at length waves corresponding to such maximal intensities and providing specific curves (sigmoid 3 parameters y=a/(l+eA((x-xO)/b))) that were analyzed by Sigmaplot version 11.0 software, the Tm is determined when x=x0. The Tm obtained reflects the thermostability of the given protein. The higher the Tm is, the more stable the variant is at high temperature.
3.2. Residual esterase activity
1 mL of a solution of 40 mg/L (in Talon buffer) of the esterase of SEQ ID NO: 1 or of an esterase of the invention was incubated at different temperatures (40, 50, 60, 65, 70, 75, 80 and 90°C) up to 10 days. Regularly, a sample, was taken, diluted 1 to 500 times in a 0.1M potassium phosphate buffer pH 8.0 and para nitro phenol -butyrate (pNP-B) assay was realized. 20pL of sample are mixed with 175pL of 0.1M potassium phosphate buffer pH 8.0 and 5pL of pNP-B solution in 2-methyl-2 butanol (40 mM). Enzymatic reaction was performed at 30°C under agitation, during 15 minutes and absorbance at 405 nm was acquired by microplate spectrophotometer (Versamax, Molecular Devices, Sunnyvale, CA, USA). Activity of pNP-B hydrolysis (initial velocity expressed in pmol of pNPB/min) was determined using a standard curve for the liberated para nitro phenol in the linear part of the hydrolysis curve.
3.3. Residual polyester depolymerizing activity
10 mL of a solution of 40 mg/L (in Talon buffer) of the esterase of SEQ ID NO: 1 and of an esterase of the invention respectively were incubated at different temperatures (40°C, 50°C, 60°C, 65°C, 70°C, 75°C, 80°C and 90°C) up to 30 days. Regularly, a 1 mL sample was taken, and transferred into a bottle containing 100 mg of amorphous PET (prepared according to WO 2017/198786 to reach a crystallinity below 20%) micronized at 250-500 pm and 49 mL of 0.1M potassium phosphate buffer pH 8.0 and incubated at 50°C, 55°C, 60°C, 65°C or 70°C. 150 pL of buffer were sampled regularly. When required, samples were diluted in 0.1 M potassium phosphate buffer pH 8. Then, 150 pL of methanol and 6.5 pL of HC1 6 N were added to 150 pL of sample or dilution. After mixing and filtering on 0.45 pm syringe filter, samples were loaded on UHPLC to monitor the liberation of terephthalic acid (TA), MEET and BEET. Chromatography system used was an Ultimate 3000 UHPLC system (Thermo Fisher Scientific, Inc. Waltham, MA, USA) including a pump module, an autosampler, a column oven thermostated at 25°C, and an UV detector at 240 nm. The column used was a Discovery® HS C18 HPLC Column (150 x 4.6 mm, 5 pm, equipped with precolumn, Supelco, Bellefonte, USA). TA, MEET and BEET were separated using a gradient of MeOH (30 % to 90 %) in 1 mM of H2SO4 at ImL/min. Injection was 20 pL of sample. TA, MEET and BEET were measured according to standard curves prepared from commercial TA and BEET and in house synthetized MEET in the same conditions than samples. Activity of PET hydrolysis (pmol of PET hydrolysed/min or mg of equivalent TA produced/hour) was determined in the linear part of the hydrolysis curve, such curve being set up by samplings performed at different time during the first 24 hours. Equivalent TA corresponds to the sum of TA measured and of TA contained in measured MEET and BEET.
3.4. Degradation of a polyester under solid form
1 mL of a solution of 40 mg/L (in Talon buffer) of the esterase of SEQ ID NO: 1 and of an esterase of the invention respectively were incubated at different temperatures (40°C, 50°C, 60°C, 65°C, 70°C, 75°C, 80°C and 90°C) up to 30 days. Regularly, 20 pL of enzyme preparation was deposited in a well created in an agar plate containing PET. Preparation of agar plates containing PET was realized by solubilizing 500mg of PET in hexafluoro-2-propanol (HFE), and pouring this medium in a 250 mL aqueous solution. After HFE evaporation at 52°C under 140 mbar, the solution was mixed v/v with 0.2 M potassium phosphate buffer pH 8 containing 3% agar. Around 30 mL of the mixture was used to prepare each omni tray and stored at 4°C. The diameter or the surface area of the halos formed due to the polyester degradation by parent esterase and variants of the invention were 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 to decrease by a 2-fold factor the diameter of the halo.
3.5. Multiple rounds of polyester’s depolymerization
The ability of the esterase to perform successive rounds of polyester’s depolymerization assays was evaluated in an enzymatic reactor. A Minibio 500 bioreactor (Applikon Biotechnology B.V., Delft, The Netherlands) was started with 3 g of amorphous PET (prepared according to WO 2017/198786 to reach a crystallinity below 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. Bioreactor was thermostated at 50°C, 55°C, 60°C, 65°C or 70°C by immersion in an external water bath. pH was regulated at 8 by addition of KOH at 3 M. The different parameters (pH, temperature, agitation, addition of base) were monitored thanks to BioXpert software V2.95. 1.8 g of amorphous PET (prepared according to WO 2017/198786 to reach a crystallinity below 20%) were added every 20 h. 500 pL of reaction medium was sampled regularly. Amount of TA, MEET and BEET was determined by HPLC, as described in example 2.3. Amount of EG was determined using an Aminex HPX-87K column (Bio-Rad Laboratories, Inc, Hercules, California, United States) thermostated at 65 °C. Eluent was K2HPO4 5 mM at 0.6 mL.min'1. Injection was 20 pL. Ethylene glycol was monitored using refractometer. The percentages of hydrolysis were calculated based on the ratio of molar concentration at a given time (TA +MHET + BEET) versus the total amount of TA contained in the initial sample, or based on the ratio of molar concentration at a given time (EG +MHET + 2 x BEET) versus the total amount of EG contained in the initial sample. Rate of degradation is calculated in mg of total liberated TA per hour or in mg of total EG per hour. Half-life of enzyme was evaluated as the incubation time required to obtain a loss of 50 % of the degradation rate.
3.6. Differential Scanning Fluorimetry (DSF)
DSF was used to evaluate the thermostability of the parent protein (SEQ ID NO: 1) and variants thereof by determining their melting temperature (Tm), temperature at which half of the protein population is unfolded. Protein samples were prepared at a concentration of 6.25 pM and stored in buffer A consisting of lOOmM potassium phosphate buffer, pH 8. The SYPRO orange dye 5000x stock solution in DMSO was first diluted to 250x in water. Protein samples were loaded onto a white clear 96-well PCR plate (Bio-Rad cat# HSP9601) with each well containing a final volume of 25 pL. The final concentration of protein and SYPRO Orange dye in each well were 6 pM (0.17 mg/ml) and 10X respectively. Loaded volumes per well were as follow: 24 pL of the 6.25 pM protein solution and 1 pL of the 250x Sypro Orange diluted solution. The PCR plates were then sealed with optical quality sealing tape and spun at 1000 rpm for 1 min at room temperature. DSF experiments were then carried out using a CFX96 real-time PCR system set to use the 450/490 excitation and 560/ 580 emission filters. The samples were heated from 25 to 100°C at the rate of 0.3°C/second. A single fluorescence measurement was taken every 0.03 second. Melting temperatures were determined from the peak(s) of the first derivatives of the melting curve using the Bio-Rad CFX Manager software. Variation of buffer type or buffer concentration may be used, with no impact on the delta Tm between the esterase of the invention and the parent, as far as the same buffer is used for the parent esterase. Esterase of SEQ ID NO: 1 or SEQ ID NO:2 and esterases of the invention were then compared based on their Tm values. Due to high reproducibility between experiments on the same protein from different productions, a ATm of 0.8°C was considered as significant to compare variants. Tm values correspond to the average of at least 3 measurements. The Tm of the esterase of SEQ ID NO: 1 is evaluated at 84.7°C.

Claims

1. 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 set forth in SEQ ID NO: 1, (ii) has at least one amino acid substitution selected from 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 and N253D, or at least one combination of substitutions selected from N204G + M208L + N211E, F90E + N204G + N211E, F90N + N204G + N211E, F90Q + N204G + N211E, F90R + N204G + N21 IE, F90W + N204G + N21 IE, F187I + N204G + N21 IE, and S98R + E173Q, M208Q + N21 IM and M208N + N21 IM as compared to SEQ ID NO: 1, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1, (iii) has a polyester degrading activity and (iv) exhibits an increased thermostability and/or an increased polyester degrading activity as compared to the esterase of SEQ ID NO: 1 at a pH comprised between 6 and 10.
2. The esterase according to claim 1, wherein said esterase comprises at least one amino acid substitution or at least one combination of substitutions selected from 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 + N21 IE, F90Q + N204G + N21 IE, F90R + N204G + N21 IE, F90W + N204G + N21 IE, F187I + N204G + N21 IE, S98R + E173Q and M208N + N21 IM.
3. The esterase according to anyone of the previous claims, wherein said esterase further comprises at least one substitution selected from 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, N I 05D, L13S, E L58D.
4. The esterase according to anyone of the previous claims, wherein said esterase comprises at least one substitution selected from R30G, A64V, L124G, S145G, H183Q/N, S193L/Q, W228S and V242E and further comprises at least one substitution selected from G35A, W69L, F90L/Y, VI 151, 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, LBS, E158D, preferably selected from G35A, W69L, F90L/Y, VI 151, N162S, S181K, N204G, A216C, N243Y, A246C, V28I and T157P.
5. The esterase according to anyone of the previous claims, wherein said 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, Il 85V, 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, LBS and E158D.
6. The esterase according to claim 5, wherein said esterase comprises at least one combination of substitutions selected from or consists of the amino acid sequence as set forth in SEQ ID NO: 1 with one combination of substitutions selected from 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 + N21 IM, H183Q + Fl 871 + N204G + M208N + N211M + S193E, M208T + N204G + H183Q + F187I + N21 IM + S193E, H183Q + F187I + N204G + N21 IM + S193E + V200I + I170V, A17F + F90Q + N122D + H183N + S193E + N204G + M208L + N21 IM, A17F + F90Q + Q142E + H183N + S 193E + N204G + M208L + N21 IM, Al 7F + F90Q + N143D + Hl 83N + S 193E + N204G + M208L + N211M, A17F + F90Q + H183N + S193E + N204G + M208L + N211M + Q237E, A17F + F90Q + H183N + S193E + N204G + M208L + N21 IM + N253D, A17F + F90Q + Hl 83N + S 193E + N204G + M208L + N21 IM + Q258E, Hl 83Q + F 1871 + N204G + M208N + N211M + S193E + V200I + I170V, M208T + N204G + H183Q + F187I + N21 IM + S193E + V200I + 1170V, A17F + N85D + F90Q + H183N + S193E + N204G + M208L + N21 IM, A17F + F90Q + N105D + Hl 83N + S193E + N204G + M208L + N21 IE, H183Q + F187I + N204G + LI 3 S + E158D, M208T + N204G + H183Q + F187I + L13S + E158D, A17T + T27S + S48T + L82I + F90L + G92F + G135A + AMOS + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + H70V + M208T + N204G + H183Q + F187I, A17T + T27S + S48T + L82I + F90L + G92F + G135A + AMOS + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + H70V + H183Q + F187I + N204G, A17T + T27S + S48T + L82I + F90L + G92F + G135A + AMOS + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + H70V + M208T + N204G + H183Q + F187I + L13S + E158D.
7. The esterase according to claim 5, wherein said esterase comprises the substitution H183Q and further comprises at least one substitution, at least two substitutions, at least three substitutions selected from A24Q, R30G, L74V, F187Q, F187I, L227N, R251S, Y26H, A64T, G35A, A62T, G46V, R73H, T50M, H69M, Y106F, I185V, R108L, V167L, N204G, G39S, A64V, L124G, N162S, S193Q, Q237L, M208T, S181K, W69L, A216C, A246C, N204G, M208N, N211M, S193E, V200I, I170V, L13S, E158D.
8. The esterase according to claim 7, wherein said esterase comprises at least one combination of substitutions selected from or consists of the amino acid sequence as set forth in SEQ ID NO: 1 with one combination of substitutions selected from A24Q + H183Q, R30G + H183Q, L74V + H183Q, H183Q + F187Q, H183Q + F187I, L227N + H183Q, R251S + H183Q, Y26H + A64T + Hl 83Q, G35 A + A62T + Hl 83Q, G46V + R73H + Hl 83Q, T50M + H69M + 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 + F 1871 + Q237L, M208T + N204G + Hl 83Q + F 1871, N204G + Hl 83Q + F 1871 + N162S + S181K, N204G + H183Q + F187I + N162S + S181K + S193Q, N204G + H183Q + F187I + W69L + A216C + A246C, H183Q + F187I + N204G + M208N + N211M + S193E, M208T + N204G + Hl 83Q + F 1871 + N21 IM + S 193E, Hl 83Q + F 1871 + N204G + N21 IM + S 193E + V200I + 1170 V, H183Q + F187I + N204G + M208N + N21 IM + S 193E + V200I + I170V, M208T + N204G + H183Q + F187I + N21 IM + S193E + V200I + I170V, H183Q + F187I + N204G + N21 IM + S193E , N204G + H183Q + F187I + L13S + E158D, N204G + H183Q + F187I, M208T + N204G + H183Q + F187I + LI 3 S + E158D, H183Q + F187I + N204G + L13S + E158D, M208T + N204G + H183Q + F187I + L13S + E158D, A17T + T27S + S48T + L82I + F90L + G92F + G135A + AMOS + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + H70V + M208T + N204G + H183Q + F187I, A17T + T27S + S48T + L82I + F90L + G92F + G135A + AMOS + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + H70V + H183Q + F187I + N204G, A17T + T27S + S48T + L82I + F90L + G92F + G135A + AMOS + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + H70V + M208T + N204G + H183Q + F187I + L13S + E158D.
9. The esterase according to claim 7, wherein said esterase comprises at least the combination of substitutions 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, V200I, I170V, L13S, E158D.
10. The esterase according to claim 9, wherein said esterase comprises at least one combination of substitutions selected from or consists of the amino acid sequence as set forth in SEQ ID NO: 1 with one combination of substitutions selected from N204G + H183Q + Fl 871 + 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 + F 1871 + N162S + S 18 IK + S 193 Q, 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 + N21 IM + 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 + AMOS + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I, A17T + T27S + S48T + L82I + F90L + G92F + G135A + AMOS + 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 + L I 3 S + E158D, preferably selected from 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 + N21 IM + S193E, H183Q + F187I + N204G + N21 IM + S193E + V200I + I170V, H183Q + F 1871 + N204G + M208N + N21 IM + S 193E + V200I + Il 70 V, H183Q + F187I + N204G + M208T + N211M + S193E + V200I + I170V, more preferably selected 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, H183Q + F187I + N204G + M208T + N211M + S193E and H183Q + F187I + N204G + M208N + N211M + S193E + V200I + I170V.
11. The esterase according to claim 9, wherein said esterase comprises at least the combination of substitution selected from or consists of the amino acid sequence as set forth in SEQ ID NO: 1 with one combination of substitutions selected from H183Q + Fl 871 + N204G + N211M + S193E and further comprises at least one substitution selected from M208N/T, V200I and II 70V, preferably the esterase comprises at least one substitution selected from N204G + H183Q + F187I + M208N/T + N21 IM + S193E, preferably selected from H183Q + F187I + N204G + M208N + N21 IM + S193E.
12. The esterase according to claim 5, wherein said esterase comprises the substitution H183N and further comprises at least one substitution selected from Fl 871, F90L, F187L, F90Y, A17F, F90Q, N204G, M208L, N211M, N122D, S193E, Q142E, N143D, Q237E, N253D, Q258E, V200I, I170V, N85D, N105D, N211E, L13S, E158D.
13. The esterase according to claim 12, wherein said esterase comprises at least one combination of substitutions selected from or consists of the amino acid sequence as set forth in SEQ ID NO: 1 with one combination of substitutions selected from H183N + F187I, F90L + H183N + F187L, F90Y + H183N, A17F + F90Q + H183N + N204G + M208L + N21 IM, A17F + F90Q + N122D + H183N + S193E + N204G + M208L + N21 IM, A17F + F90Q + Q142E + H183N + S193E + N204G + M208L + N211M, A17F + F90Q + N143D + H183N + S193E + N204G + M208L + N211M, A17F + F90Q + H183N + S193E + N204G + M208L + N21 IM + Q237E, A17F + F90Q + H183N + S193E + N204G + M208L + N21 IM + N253D, A17F + F90Q + H183N + S193E + N204G + M208L + N21 IM + Q258E, A17F + F90Q + H183N + S193E + N204G + M208L + N21 IM + V200I + I170V, A17F + N85D + F90Q + H183N + S193E + N204G + M208L + N211M and A17F + F90Q + N105D + H183N + S193E + N204G + M208L + N21 IE.
14. The esterase according to claim 1 or 2, wherein said esterase comprises at least the combination of substitutions M208N + N21 IM.
15. The esterase according to claim 1, wherein said esterase comprises at least one combination of substitutions selected from or consists of the amino acid sequence as set forth in SEQ ID NO: 1 with one combination of substitutions selected from M208N + N211M, H183Q + F187I + N204G + M208N + N21 IM + S193E, H183Q + F187I + N204G, H183Q + F187I + N204G + L 13 S + El 58D, M208T + N204G + Hl 83Q + F 1871, M208T + N204G + Hl 83Q + F187I + LI 3 S + E158D, A17T + T27S + S48T + L82I + F90L + G92F + G135A + AMOS + N143I + S145T + A149G + S164P + V167Q + S206T + T252S + I170V + M208T + N204G + H183Q + F187I, A17T + T27S + S48T + L82I + F90L + G92F + G135A + AMOS + N143I + S145T + A149G+ S164P + V167Q + S206T + T252S + I170V + H183Q + F187I + N204G, A17T + T27S + S48T + L82I + F90L + G92F + G135A + AMOS + N143I + S145T + A149G+ S164P + V167Q + S206T + T252S + H70V + M208T + N204G + H183Q + F187I + L13S + E158D.
16. An esterase variant which (i) has at least 97%, 98% or 99% identity to the full length amino acid sequence set forth in SEQ ID NO: 1, (ii) has at least one amino acid substitution selected from 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, H69M, P179N, I185V, F187L/Q, P192S, L227N, Q237L, L239M, R251S, N9E, V28I, A62T, F90Y/R/W/L, VI 151, 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 at least one combination of substitutions selected from N204G + M208L + N21 IE, F90E + N204G + N21 IE, F90N + N204G + N21 IE, F90Q + N204G + N21 IE, F90R + N204G + N21 IE, F90W + N204G + N211E, F187I + N204G + N211E, S98R + E173Q, M208Q + N211M and M208N + N211M, as compared to the amino acid sequence SEQ ID NO: 1, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1, (iii) has at least the amino acids C240, C275, 1170, G92, P213, E182, L13 and E158, as in the parent esterase of SEQ ID NO: 1, (iv) has a polyester degrading activity, and (v) exhibits an increased thermostability and/or an increased polyester degrading activity as compared to the esterase of SEQ ID NO: 1 at a pH comprised between 6 and 10.
17. The esterase according to claim 16, wherein said esterase comprises at least one substitution selected from N9E, V28I, A62T, F90Y, VI 151, S181K, H183S, Fl 871, M208T, S66N, N243Y, E182D, M208G, T16E, Q142E, Q237E, M208K, N105D, N85D, M208A, M208D preferably selected from N9E, V28I, A62T, F90Y, VI 151, 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.
18. The esterase according to claim 16, wherein said esterase comprises the substitution F187I and further comprises one, two or more substitutions selected from 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 said esterase comprises at least one combination of substitutions selected from or consists of the amino acid sequence as set forth in SEQ ID NO: 1 with one combination of substitutions selected from H183L + Fl 871, Hl 83 A + Fl 871, H183Y + Fl 871, H183Q + Fl 871, Hl 83N + Fl 871, 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 + Fl 871, N204G + H183Q + F187I + N162S + S181K, N204G + H183Q + F187I + N162S + S181K + S193Q, N204G + H183Q + F187I + W69L + A216C + A246C, H183Q + F187I + N204G + M208N + N21 IM + S193E, M208T + N204G + H183Q + F187I + N21 IM + S193E, H183Q + F187I + N204G + N211M + S193E , H183Q + F187I + N204G + N211M + S193E + V200I + I170V, M208T + N204G + H183Q + F187I.
19. The esterase according to claim 16, wherein said 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 said esterase comprises at least the combination of substitutions selected from or consists of the amino acid sequence as set forth in SEQ ID NO: 1 with one combination of substitutions selected from A62T + S66N + M208T, M208T + N204G + Hl 83Q + F 1871, M208T + N204G + Hl 83Q + F 1871 + N21 IM + S193E and M208T + N204G + H183Q + F187I, preferably selected from A62T + S66N + M208T and M208T + N204G + H183Q + F187IM208T + N204G + H183Q + Fl 871.
20. The esterase according to claim 16, wherein said esterase comprises at least the combination of substitutions 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 said esterase comprises at least one combination of substitutions selected from or consists of the amino acid sequence as set forth in SEQ ID NO: 1 with one combination of substitutions selected from 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, more preferably selected from 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 + N21 IM + S 193E, Hl 83Q + F 1871 + N204G + M208T + N21 IM + S 193E, Hl 83Q + F 1871 + N204G + N21 IM + S193E + V200I + H70V, eve more preferably selected from N204G + H183Q + F187I + A62T, N204G + H183Q + F187I + A64V, N204G + H183Q + F187I + L124G, N204G + H183Q + F187I + N162S, N204G + H183Q + F187I + S193Q, N204G + H183Q + F 1871 + Q237L, M208T + N204G + Hl 83Q + F 1871, N204G + Hl 83Q + F 1871 + N162S + S181K + S193Q, H183Q + F187I + N204G + M208N + N21 IM + S193E and H183Q + F187I + N204G + M208T + N21 IM + S193E.
21. The esterase according to claim 16, wherein said esterase comprises at least the combination of substitutions M208N + N21 IM.
22. The esterase according to claim 1 to 21, wherein said esterase exhibits at least one amino acid residue selected from S130, D175, H207, C240 or C275 as in the parent esterase of SEQ ID NO: 1.
23. 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 set forth in SEQ ID NO:2, (ii) comprises at least one amino acid substitution selected from 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 and V200I, or at least one combination of substitutions selected from N204G + M208L + N21 IE, L90E + N204G + N21 IE, L90N + N204G + N21 IE, L90Q + N204G + N21 IE, L90R + N204G + N21 IE, L90W + N204G + N21 IE, F187I + N204G + N21 IE and, S98R + E173Q, M208A + N211M, M208G + N211M, M208K + N211M, M208N + N21 IM, M208P + N21 IM, M208Q + N21 IM, M208H + N21 IM and M208T + N21 IM, as compared to the amino acid sequence SEQ ID NO: 2, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO:2, (iii) has a polyester degrading activity and (iv) exhibits an increased thermostability and/or an increased polyester degrading activity as compared to the esterase of SEQ ID NO: 2 at a pH comprised between 6 and 10.
24. The esterase according to claim 23, wherein said esterase comprises at least one substitution selected from A64V, S66N, H183N, I143D, N253D, preferably selected from H183N and S66N.
25. The esterase according to claim 23, wherein said 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 Fl 871, L90Q, I145D, L90Q, M208A, M208L, M208M, M208N, M208T, N105D, N122D, N204G, N211M, N253D, N85D, Q142E, Q237E, Q258E, S193E, S98R, T17F and V200I, preferably wherein said esterase comprises at least one combination of substitutions from or consists of the amino acid sequence as set forth in SEQ ID NO:2 with one combination of substitutions selected from H183D + Fl 871, H183E + Fl 871, H183N + F187I, M208T + N204G + H183Q + F187I + N211M + S193E, H183Q + F187I + N204G + N211M + S193E + V200I, T17F + L90Q + H183N + N204G + M208L + N21 IM, 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 + N21 IM, T17F + L90Q + N122D + H183N + S193E + N204G + M208L + N21 IM, T17F + L90Q + Q142E + H183N + S193E + N204G + M208L + N21 IM, T17F + L90Q + I145D + H183N + S 193E + N204G + M208L + N21 IM, T 17F + L90Q + Hl 83N + S 193E + N204G + M208L + N211M + Q237E, T17F + L90Q + H183N + S193E + N204G + M208L + N211M + N253D, T17F + L90Q + H183N + S193E + N204G + M208L + N21 IM + Q258E, T17F + L90Q + H183N + S193E + N204G + M208L + N211M + V200I, T17F + L90Q + S98R + N105D + H183N + S193E + N204G + N21 IM, T17F + L90Q + H183N + S193E + N204G + M208N + N211M + V200I, T17F + L90Q + H183N + S193E + N204G + M208A + N21 IM + V200I, T17F + L90Q + H183N + S193E + N204G + M208M + N21 IM + V200I, H183Q + F187I + N204G + M208N + N21 IM + S193E, H183Q + F187I + N204G, H183Q + F187I + N204G + M208T, H183Q + F187I + N204G + M208T + LI 3 S + 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 + QI 67 V + T206S + S252T + V170I + H183Q + F187I + N204G + L13S + E158D, preferably selected from H183D + Fl 871, H183E + Fl 871, H183N + Fl 871, M208T + N204G + H183Q + F187I + N21 IM + S193E, H183Q + F187I + N204G + N21 IM + S193E + V200I, T17F + L90Q + H183N + N204G + M208L + N21 IM, T17F + L90Q + H183N + S193E + N204G + M208L + N211M, H183Q + F187I + N204G + M208N + N211M + S193E + V200I, M208T + N204G + H183Q + F187I + N21 IM + S193E + V200I, T17F + N85D + L90Q + H183N + S193E + N204G + M208L + N21 IM, T17F + L90Q + N122D + H183N + S193E + N204G + M208L + N21 IM, T17F + L90Q + Q142E + H183N + S193E + N204G + M208L + N21 IM, T 17F + L90Q + I145D + Hl 83N + S 193E + N204G + M208L + N211M, T17F + L90Q + H183N + S193E + N204G + M208L + N21 IM + Q237E, T17F + L90Q + Hl 83N + S 193E + N204G + M208L + N21 IM + N253D, T17F + L90Q + Hl 83N + S 193E + N204G + M208L + N21 IM + Q258E, T 17F + L90Q + H183N + S193E + N204G + M208L + N21 IM + V200I, T17F + L90Q + S98R + N105D + H183N + S193E + N204G + N211M, T17F + L90Q + H183N + S193E + N204G + M208N + N21 IM + V200I, T17F + L90Q + H183N + S193E + N204G + M208A + N21 IM + V200I, T17F + L90Q + H183N + S193E + N204G + M208M + N21 IM + V200I.
26. The esterase according to 25, wherein said 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 said esterase comprises at least one combination of substitutions selected from or consists of the amino acid sequence as set forth in SEQ ID NO:2 with one combination of substitutions selected from H183Q + F187I + N204G + M208T + N21 IM + S193E, H183Q + F187I + N204G +
Figure imgf000085_0001
N204G + L13S + E158D, preferably selected from H183Q + F187I + N204G + M208T + N21 IM + S193E, H183Q + F187I + N204G + M208N + N21 IM + S193E + V200I, H183Q + F187I + N204G + M208T + N21 IM + S193E + V200I.
27. The esterase according to 25 or 26, wherein said esterase comprises at least the combination of substitutions N204G + H183Q + F187I and optionally further comprises at least one substitution selected from M208T, N211M, S193E, V200I, M208N, L13S, E158D, preferably wherein said esterase comprises at least one combination of substitutions selected from or consists of the amino acid sequence as set forth in SEQ ID NO:2 with one combination of substitutions selected from M208T + N204G + H183Q + F187I + N21 IM + S 193E, Hl 83 Q + F 1871 + N204G + M208N + N21 IM + S 193E + V200I, M208T + N204G + H183Q + F187I + N211M + S193E + V200I, H183Q + F187I + N204G + M208N + N211M + S193E, H183Q + F187I + N204G, H183Q + F187I + N204G + M208T, H183Q
Figure imgf000086_0001
+ V170I + H183Q + F187I + N204G + L13S + E158D, more preferably selected from M208T + N204G + Hl 83Q + F 1871 + N21 IM + S 193E, Hl 83Q + F 1871 + N204G + M208N + N211M + S193E + V200I, M208T + N204G + H183Q + F187I + N21 IM + S193E + V200I.
28. The esterase according to 25, wherein said esterase comprises the substitution H183N and further comprises at least one, two, three, four, five, six, seven or more substitutions selected from F187I, L90Q, H45D, 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 or consists of the amino acid sequence as set forth in SEQ ID NO:2 with one combination of substitutions selected from H183N + F187I, T17F + L90Q + H183N + N204G + M208L + N211M, T17F + L90Q + H183N + S193E + N204G + M208L + N211M, T17F + N85D + L90Q + H183N + S193E + N204G + M208L + N21 IM, T17F + L90Q + N122D + H183N + S 193E + N204G + M208L + N21 IM, T 17F + L90Q + Q 142E + Hl 83N + S 193E + N204G + M208L + N211M, T17F + L90Q + H45D + H183N + S193E + N204G + M208L + N211M, T17F + L90Q + H183N + S193E + N204G + M208L + N21 IM + Q237E, T17F + L90Q + H183N + S193E + N204G + M208L + N21 IM + N253D, T17F + L90Q + H183N + S 193E + N204G + M208L + N21 IM + Q258E, T 17F + L90Q + H183N + S193E + N204G + M208L + N21 IM + V200I, T17F + L90Q + S98R + N105D + H183N + S193E + N204G + N211M, T17F + L90Q + H183N + S193E + N204G + M208N + N21 IM + V200I, T17F + L90Q + H183N + S193E + N204G + M208A + N21 IM + V200I, T17F + L90Q + H183N + S193E + N204G + M208M + N21 IM + V200I.
29. The esterase according to 25, wherein said esterase comprises at least the combination of M208N + N211M.
30. The esterase according to 25, wherein said esterase comprises at least one substitution or combination of substitutions selected from M208N + N21 IM, H183Q + F187I + N204G + M208N + N21 IM + S 193E, Hl 83Q + F 1871 + N204G, Hl 83Q + F 1871 + N204G + M208T,
Figure imgf000087_0001
31. An esterase variant which (i) has at least 97%, 98% or 99% identity to the full length amino acid sequence set forth 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 at least the combination of substitutions M208I + N21 IM, as compared to the amino acid sequence SEQ ID NO:2, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO:2, (iii) has at least the amino acids C240, C275, V170, F92, P213, E182, L13 and E158, as in the parent esterase of SEQ ID NO:2, (iv) has a polyester degrading activity, and (v) exhibits an increased thermostability and/or an increased polyester degrading activity as compared to the esterase of SEQ ID NO:2 at a pH comprised between 6 and 10.
32. The esterase according to claim 31, wherein said esterase comprises at least the substitution M208N and further comprises at least one substitution selected from N21 IM, H183Q, F187I, N204G, S193E, V200I, T17F, L90Q and H183N, preferably wherein said esterase comprises at least one combination of substitutions selected from or consists of the amino acid sequence as set forth in SEQ ID NO:2 with one combination of substitutions selected from M208N + N2 1 IM, Hl 83 Q + F 1871 + N204G + M208N + N21 IM + S 193E + V200I and T 17F + L90Q + H183N + S193E + N204G + M208N + N21 IM + V200I.
33. The esterase according to claim 23 to 32, wherein said esterase exhibits at least one amino acid residue selected from S130, D175, H207, C240 or C275 as in the parent esterase of SEQ ID NO:2.
34. A method of degrading a polyester comprising a. contacting the polyester with an esterase according to any one of claims 1 to 33 or a host cell comprising and expressing a nucleic acid encoding the esterase according to any one of claims 1 to 33 or extract thereof comprising said esterase or a composition comprising the esterase according to any one of claims 1 to 33; and, optionally b. recovering monomers and/or oligomers.
35. A method of degrading at least one polyester of a polyester containing material comprising a. contacting the polyester containing material with an esterase according to any one of claims
1 to 33, or a host cell comprising and expressing a nucleic acid encoding the esterase according to any one of claims 1 to 33, or extract thereof comprising said esterase or a composition comprising the esterase according to any one of claims 1 to 33; and, optionally b. recovering monomers and/or oligomers.
36. The method of claim 34 or 35, wherein the polyester is selected from polyethylene terephthalate (PET), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polylactic acid (PLA), polyhydroxy alkanoate (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.
37. A polyester containing material containing an esterase according to any one of claims 1 to 33 or a host cell comprising and expressing a nucleic acid encoding the esterase according to any one of claims 1 to 33 or extract thereof comprising said esterase or a composition comprising the esterase according to any one of claims 1 to 33.
38. A detergent composition comprising the esterase according to any one of claims 1 to 33 or a host cell comprising and expressing a nucleic acid encoding the esterase according to any one of claims 1 to 33, or extract thereof comprising said esterase or a composition comprising the esterase according to any one of claims 1 to 33.
PCT/EP2024/063319 2023-05-15 2024-05-15 Novel esterases and uses thereof Pending WO2024236008A2 (en)

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