RU2819730C2 - New esterases and use thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed is an esterase, which has at least 90%, 95%, 96%, 97%, 98% or 99% identity to the full-length amino acid sequence presented in SEQ ID NO: 1 comprises at least combination of substitutions N207C and S256C and has high activity of decomposition of polyesters and/or high thermal stability compared to esterase SEQ ID NO: 1. Also disclosed are a nucleic acid coding said esterase, an expression cassette containing said nucleic acid, a host cell containing said nucleic acid or a cassette, composition for breaking down polyester-containing materials containing said esterase or a host cell. Also disclosed is a method of breaking down at least one polyester of a polyester-containing material using said esterase or host cell or composition.

EFFECT: invention enables to obtain esterases exhibiting higher activity and/or higher thermal stability compared to esterase having the amino acid sequence specified in SEQ ID NO: 1.

14 cl, 5 tbl, 3 ex

Description

The present invention relates to new esterases, more particularly to esterases having improved activity and/or improved thermostability compared to the parent esterase. The present invention also relates to the use of these new esterases for the destruction of materials containing polyesters, such as plastic products. The esterases of the invention are particularly suitable for the degradation of polyethylene terephthalate and materials containing polyethylene terephthalate.

BACKGROUND OF THE ART

Esterases are capable of catalyzing the hydrolysis of various polymers, including polyesters. In this context, esterases have shown promising effects in a number of industrial applications, including as dishwashing and laundry detergents, as degrading enzymes for biomass and food processing, as biocatalysts in the detoxification of environmental pollutants, or for the treatment of polyesters. fabrics in the textile industry. Of particular interest is the use of esterases as degrading enzymes for the hydrolysis of polyethylene terephthalate (PET). Indeed, PET is used in a large number of technical fields such as clothing, carpets, or as a thermoset resin for packaging or automotive plastics, etc., so the accumulation of PET in landfills is becoming a growing environmental problem.

Enzymatic degradation of polyesters, and especially PET, is considered an interesting solution to reduce the accumulation of plastic waste. Indeed, enzymes can accelerate the hydrolysis of polyester-containing material, and in particular plastic products, even to the monomeric level. In addition, the hydrolyzate (ie monomers and oligomers) can be recycled as a material for the synthesis of new polymers.

In this context, several esterases have been identified as candidate polyester degrading enzymes, and several variants of such esterases have been developed. Of particular interest among esterases are cutinases, also known as cutine hydrolases (EC 3.1.1.74). Cutinases have been identified in various fungi (P.E. Kolattukudy in "Lipases", Ed. B. Borg-strόm and H.L. Brockman, Elsevier 1984, 471-504), bacteria and pollen. Recently, metagenomics approaches have led to the identification of additional esterases.

However, there is still a need for esterases with improved activity and/or improved thermal stability compared to already known esterases in order to provide more efficient and therefore more competitive polyester degradation processes.

SUMMARY OF THE INVENTION

The present invention relates to novel esterases exhibiting increased activity and/or increased thermostability compared to the parental or wild-type esterase having the amino acid sequence set forth in SEQ ID No. 1. This wild-type esterase corresponds to amino acids 28 to 290 of the esterase amino acid sequence, described in Yoshida S. et al., 2016 (A bacterium that degrades and assimilates poly(ethylene terephthalate) Science 351 (6278), 1196-1199 (2016)), where methionine was added at position 1. The esterases of the present invention are particularly useful in the processes of destruction of plastic products, in particular plastic products containing PET.

In this regard, it is an object of the invention to provide an esterase that (i) has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the full-length amino acid sequence specified in SEQ ID No. 1, and (ii) has at least one amino acid substitution at a position corresponding to residues selected from S256, T13, A15, S16, L17, E18, A19, S20, A21, S66, I68, W70, P73, R74, D86, D92, G136, A222, L223, G225, K226, K227, F245, A246, C247, N249, P250, N251, S252, V255 and C263, where positions are numbered with reference to the amino acid sequence , presented in SEQ ID No. 1, and (iii) demonstrates increased polyester degrading activity and/or increased thermal stability compared to the esterase of SEQ ID No. 1.

Another object of the invention is to provide an esterase that (i) has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the full-length amino acid sequence set forth in SEQ. ID No. 1, and (ii) has at least one amino acid substitution at a position corresponding to residues selected from S256, T13, A15, S16, L17, E18, A19, S20, A21, S66, I68, W70, P73 R74, D86, D92, G136, F175, S187, I192, A222, L223, I224, G225, K226, K227, T244, F245, A246, C247, E248, N249, P250, N251, S252, T253, V255, C263, C263, C263, C263, C263, C263, C263, C263, C263, C263, C263, C263, C263, C263, C263, C263, C265, C263 , G60, S161, S162, P184, G208, or G209, wherein the positions are numbered according to the amino acid sequence set forth in SEQ ID No. 1, and (iii) exhibits increased polyester degrading activity and/or increased thermal stability compared to the esterase of SEQ ID N 1.

It is also an object of the invention to provide an esterase that (i) has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the full-length amino acid sequence represented by in SEQ ID No. 1, (ii) contains at least one replacement selected from the group consisting of S256C, S212I/W, N207C, S181N, S66T, T62M, N215D/M, Q93G/P, N220P/D, S187E, A14H, T172Q, or K226E, wherein the positions are numbered according to the amino acid sequence set forth in SEQ ID No. 1, and (iii) exhibits increased polyester degrading activity and/or increased thermal stability compared to the esterase SEQ ID No. 1, preferably selected from S256C, S212I/W, N207C, S181N, S66T, T62M, N215D/M or Q93G/P.

Preferably, the esterase of the invention contains at least one substitution at position S256, preferably S256C, and optionally a substitution at position N207, preferably N207C. More preferably, the esterase further contains at least one substitution at position S212, preferably S212I/W.

Preferably, the esterase further contains at least one amino acid residue selected from S134, D180, H211, preferably from the combination S134+D180+H211.

Another object of the invention is to provide a nucleic acid encoding an esterase according to the invention. The present invention also relates to an expression cassette or expression vector containing the specified nucleic acid, and to a host cell containing the specified nucleic acid, expression cassette or vector.

The present invention also provides a composition containing an esterase of the present invention, a host cell of the present invention, or an extract thereof.

Another object of the invention is to provide a process for producing the esterase of the invention, comprising:

(a) culturing the host cell of the invention under conditions suitable for expression of the nucleic acid encoding the esterase; and optional

(b) isolating said esterase from the cell culture.

Another object of the invention is to provide a method for degrading polyester, comprising

(a) contacting the polyester with an esterase of the invention, or a host cell of the invention, or a composition of the invention; and, optionally

(b) reduction of monomers and/or oligomers.

In particular, the invention provides a method for degrading PET, comprising contacting PET with at least one esterase of the invention and optionally reducing PET monomers and/or oligomers.

The present invention also relates to a method for degrading at least one polyester of a polyester-containing material, comprising the following steps:

(a) contacting the polyester-containing material with an esterase or host cell of the invention, thereby destroying at least one polyester or polyester-containing material; and optional

(b) reducing monomers and/or oligomers of said at least one polyester.

The invention also relates to the use of the esterase of the invention to degrade PET or a plastic product containing PET.

The present invention also relates to a polyester-containing material in which an esterase or host cell or composition of the invention is included.

The present invention also relates to a detergent composition containing an esterase or a host cell of the invention, or a composition containing an esterase of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

This disclosure will be best understood by reference to the following definitions.

As used 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. Amino acids herein are represented by their one-letter or three-letter 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 (Ile); K: lysine (Lys); L: leucine (Leu); M: methionine (Met); N: asparagine (Asn); P: proline (Pro); Q: glutamine (Gln); R: arginine (Arg); S: serine (Ser); T: threonine (Thr); V: valine (Val); W: tryptophan (Trp) and Y: tyrosine (Tyr).

The term esterase refers to an enzyme belonging to the class of hydrolases, classified as EC 3.1.1 according to Enzyme Nomenclature, which catalyzes the hydrolysis of esters into acid and alcohol. The term cutinase or cutin hydrolase refers to esterases classified as EC 3.1.1.74 by Enzyme Nomenclature that are capable of catalyzing the chemical reaction to form cutin monomers from cutin and water.

The terms wild-type protein or parent protein refer to the non-mutated version of the polypeptide as it occurs in nature. Here, the parent esterase refers to an esterase having the amino acid sequence specified in SEQ ID No. 1.

The terms "mutant" and "variant" refer to polypeptides derived from SEQ ID No. 1 and containing at least one modification or change, i.e., substitution, insertion and/or deletion, in one or more (for example, several) positions, and having polyester degrading activity. The variants can be prepared by various methods well known in the art. In particular, examples of methods for altering the DNA sequence encoding a wild-type protein include, but are not limited to, site-directed mutagenesis, random mutagenesis, and the design of synthetic oligonucleotides. Thus, the terms modification and change , as used herein in relation to a particular position, mean that the amino acid at that particular position has been modified compared to the amino acid at that particular position in the wild-type protein.

"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 with another selected from the naturally occurring standard 20 amino acid residues, rare naturally occurring amino acid residues (e.g., hydroxyproline, hydroxylysine, allohydroxylysine, 6-N-methyllysine, N-ethylglycine, N -methylglycine, N-ethylasparagine, allo-isoleucine, N-methylisoleucine, N-methylvaline, pyroglutamine, aminobutyric acid, ornithine, norleucine, norvaline) and non-naturally occurring amino acid residues, often obtained synthetically (for example, cyclohexylalanine). Preferably, the term "substitution" refers to the replacement of an amino acid residue with 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. As used herein, the following terminology is used to denote substitution: L82A means that the amino acid residue (leucine, L) at position 82 of the original sequence is replaced by alanine (A). A121V/I/M means that the amino acid residue (alanine, A) at position 121 of the original sequence is replaced by one of the following amino acids: valine (V), isoleucine (I) or methionine (M). The replacement may be a conservative or non-conservative replacement. Examples of conservative substitutions are 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 indicated, the provisions described in this application are numbered in accordance with the amino acid sequence presented in SEQ ID No. 1.

As used herein, the term sequence identity or identity refers to the number (or percentage, expressed) of matches (identical amino acid residues) between two polypeptide sequences. Sequence identity is determined by comparing sequences aligned to maximize overlap and identity while minimizing sequence gaps. In particular, sequence identity can be determined using any of a number of mathematical algorithms for global or local alignment depending on the length of the two sequences. Sequences of equal length are preferably aligned using a global alignment algorithm (e.g., Needleman and Wunsch's algorithm; Needleman and Wunsch, 1970), which optimally aligns sequences along their entire length, while sequences of significantly different lengths are preferably aligned using a local alignment algorithm (see . for example, the Smith and Waterman algorithm (Smith and Waterman, 1981) or the Altschul algorithm (Altschul et al., 1997; Altschul et al., 2005)). Alignment to determine the proportion of amino acid sequence identity can be achieved in a variety of ways known to those skilled in the art, for example, using publicly available computer software available on Internet websites such as http://blast.ncbi.nlm. nih.gov/ or http://www.ebi.ac.uk/Tools/emboss/). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. For the purposes of this document, % amino acid sequence identity values refer to those obtained using the pairwise sequence alignment program EMBOSS Needle, which generates an optimal global alignment of two sequences using the Needleman-Wunsch algorithm with all search parameters set to default values, i.e. .e. Scoring Matrix=BLOSUM62, Gap Penalty=10, Gap Continue Penalty=0.5, End Gap Penalty=false, End Gap Penalty=10 and End Gap Penalty=0.5.

"Polymer" refers to a chemical compound or mixture of compounds whose structure consists of many monomers (repeating units) linked by covalent chemical bonds. In the context of the invention, the term polymer includes natural or synthetic polymers consisting of one type of repeating units (ie, homopolymers) or a mixture of different repeating units (ie, copolymers or heteropolymers). According to the invention, "oligomers" refer to molecules containing from 2 to about 20 monomers.

In the context of the invention, “ polyester containing material” or “ polyester containing product” refers to a product, such as a plastic product, containing at least one polyester in crystalline, semi-crystalline or completely amorphous form. In a specific embodiment, polyester containing material refers to any product made from at least one plastic material, such as a plastic sheet, pipe, rod, profile, mold, film, solid block, etc. which contains at least one polyester, and possibly other substances or additives such as plasticizers, mineral or organic fillers. In another specific embodiment, the polyester-containing material refers to a plastic compound or plastic composition in a molten or solid state suitable for making a plastic product. In another specific embodiment, polyester-containing material refers to textiles, fabrics or fibers containing at least one polyester. In another specific embodiment, the polyester containing material refers to plastic waste or fiber waste containing at least one polyester.

As used herein, the term "polyester(s)" includes, 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 adipaterephthalate (PBAT), polyethylene furanoate (PEF), polycaprolactone (PCL), poly(ethylene adipate) (PEA), polyethylene naphthalate (PEN) and mixtures/blends of these polymers.

New esterases

The present invention provides new esterases with improved activity and/or improved thermostability compared to the original esterase. More specifically, the inventors have developed new enzymes particularly suitable for use in industrial processes. The esterases of the invention are particularly suitable for degrading polyesters, more particularly PET, including PET-containing materials and in particular plastic products containing PET. In a particular embodiment, the esterases exhibit both increased activity and increased thermal stability.

Thus, it is an object of the present invention to provide esterases that exhibit increased activity compared to esterases having the amino acid sequence specified in SEQ ID No. 1.

In particular, the inventors have identified certain amino acid residues in SEQ ID No. 1, which are designed for contact with the polymer substrate in the X-ray crystal structure (ie, folded 3D structure) of esterases, which can be successfully modified to promote contact of the substrate with esterases and lead to increased adsorption of the polymer and/or thereby to increased esterase activity on this polymer.

In the context of the invention, the term "increased activity" or "increased degradative activity" indicates increased ability of the esterase to degrade polyester and/or increased ability to adsorb to polyester at a given temperature compared to the ability of the esterase of SEQ ID NO 1 to degrade and/or adsorb on the same polyester at the same temperature. In particular, the esterase of the invention has increased PET degradation activity. Such an increase may be at least 10% greater than the PET degrading esterase activity of SEQ ID NO: 1, preferably at least 20%, 30%, 40%, 50%, 60%, 70% , 80%, 90%, 100%, 110%, 120%, 130% or more. In particular, the degradation activity is a depolymerization activity resulting in polyester monomers and/or oligomers, which can be further recovered and optionally reused.

One skilled in the art can evaluate the "destructive activity" of the esterase according to methods known per se in the art. For example, degradation activity can be assessed by measuring the rate of depolymerization activity of a particular polymer, measuring the rate of degradation of a solid polymer compound dispersed in an agar plate, or measuring the rate of depolymerization activity of a polymer in a reactor. In particular, degradation activity can be assessed by measuring the "specific degradation activity" of the esterase. The “specific degradation activity” of an esterase for PET corresponds to µmol of hydrolyzed PET/min or mg of equivalent TA produced/hour, and per mg of esterase in the initial period of the reaction (i.e. the first 24 hours), and is determined from the linear part of the reaction hydrolysis curve, where such a curve is constructed from several samples taken at different times during the first 24 hours. As another example, "destructive activity" can be assessed by measuring, over a period of time, the rate and/or yield of oligomers and/or monomers released under suitable temperature, pH and buffer conditions upon contact with a polymer or polymer-containing plastic product with a destructive enzyme.

The ability of an enzyme to adsorb to a substrate can be assessed by one skilled in the art according to methods known per se in the art. For example, the ability of an enzyme to adsorb to a substrate can be measured in a solution containing the enzyme and where the enzyme has been pre-incubated with the substrate under suitable conditions.

The inventors have also identified target amino acid residues in SEQ ID No. 1 that can be advantageously modified to improve the stability of the corresponding esterases at high temperatures (ie, improved thermal stability) and preferably at temperatures above 40°C, preferably above 50°C.

It is therefore an object of the present invention to provide novel esterases that exhibit increased thermal stability compared to that of an esterase having the amino acid sequence set forth in SEQ ID No. 1.

In the context of the invention, the term "increased thermal stability" indicates the increased ability of the esterase to resist changes in its chemical and/or physical structure at high temperatures and, in particular, at temperatures from 40°C to 80°C, compared to the esterase SEQ ID No. 1 In particular, thermal stability can be assessed by assessing the melting temperature (Tm) of the esterase. In the context of the present invention, "melting temperature" refers to the temperature at which half of the enzyme population in question is unfolded or misfolded. Typically, the esterases of the invention exhibit an increased Tm of about 1°C, 2°C, 3°C, 4°C, 5°C, 10°C or more compared to the Tm of the esterases of SEQ ID NO: 1. In particular, The esterases of the present invention may have an increased half-life at temperatures from 40°C to 80°C compared to the esterase of SEQ ID No. 1.

The melting point (Tm) of the esterase can be measured by one skilled in the art in accordance with methods known per se in the art. For example, DSF can be used to quantify the thermal denaturation temperature change of an esterase and thus determine its Tm. Alternatively, Tm can be estimated by analyzing protein folding using circular dichroism. Preferably, Tm is measured using DSF or circular dichroism as shown in the experimental section. In the context of the invention, Tm is compared with Tm measured under the same conditions (eg pH, nature and amount of polyesters, etc.).

Alternatively, thermal stability can be assessed by measuring the esterase activity and/or polyester depolymerization activity of the esterase after incubation at various temperatures and compared to the esterase activity and/or polyester depolymerization activity of the parent esterase. The feasibility of performing multiple rounds of polyester depolymerization analysis at different temperatures can also be evaluated. A quick and valuable test may consist of assessing, by measuring the diameter of the halo, the ability of the esterase to degrade the solid polyester compound dispersed in an agar plate after incubation at various temperatures.

The object of the present invention is to provide an esterase that (i) has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the full length of the amino acid sequence indicated in SEQ ID No. 1, (ii) contains at least one amino acid substitution at a position corresponding to residues selected from the group consisting of S256, T13, A15, S16, L17, E18, A19, S20, A21, S66, I68, W70, P73, R74, D86, D92, G136, A222, L223, G225, K226, K227, F245, A246, C247, N249, P250, N251, S252, V255, C263, G60, S162, P184 or G209 compared to the amino acid sequence of SEQ ID No. 1 and (iii) exhibits increased polyester degrading activity and/or increased thermal stability compared to the esterase of SEQ ID No. 1.

In a specific embodiment, the esterase has the amino acid sequence set forth in SEQ ID No. 1, with one amino acid substitution at a position corresponding to residues selected from S256, T13, A15, S16, L17, E18, A19, S20, A21, S66, I68 , W70, P73, R74, D86, D92, G136, A222, L223, G225, K226, K227, F245, A246, C247, N249, P250, N251, S252, V255 C263, G60, S162, P184 or G209.

Unless otherwise indicated, the provisions described in this application are numbered in accordance with the amino acid sequence presented in SEQ ID No. 1.

In particular, it is also an object of the present invention to provide an esterase that (i) has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with full-length amino acid sequence specified in SEQ ID No. 1, (ii) contains at least one amino acid substitution at a position corresponding to residues selected from the group consisting of S256, T13, A15, S16, L17, E18, A19, S20, A21, S66, I68, W70, P73, R74, D86, D92, G136, A222, L223, G225, K226, K227, F245, A246, C247, N249, P250, N251, S252, V255 and C263, compared to amino acid sequence of SEQ ID No. 1, and (iii) exhibits increased polyester degrading activity and/or increased thermal stability compared to the esterase of SEQ ID No. 1.

Unless otherwise indicated, the provisions described in this application are numbered in accordance with the amino acid sequence presented in SEQ ID No. 1.

According to the invention, the target amino acid can be replaced by any of 19 other amino acids.

In a specific embodiment, the esterase has the amino acid sequence set forth in SEQ ID No. 1, with one amino acid substitution at a position corresponding to residues selected from S256, T13, A15, S16, L17, E18, A19, S20, A21, S66, I68 , W70, P73, R74, D86, D92, G136, A222, L223, G225, K226, K227, F245, A246, C247, N249, P250, N251, S252, V255 and C263.

In one embodiment, the esterase has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the full-length amino acid sequence set forth in SEQ ID No. 1 , and contain at least one substitution at a position corresponding to residues selected from S256, C247 and C263. In a preferred embodiment, the esterase contains at least one substitution at position S256. The preferred replacement is S256C.

In one embodiment, the esterase contains at least one substitution at position S66. The preferred replacement is the S66T. In another embodiment, the esterase contains at least one substitution at position K226, preferably selected from K226E.

In one embodiment, the esterase may further contain at least one substitution or combination of substitutions at a position corresponding to residues selected from A14, Y61, T62, A63, R64, S67, K69, L91, Q93, S95, W133, M135, W159, S161, F175, C177, S181, I182, A183, S187, I192, G208, S212, C213, A214, N215, I224, T244, E248, T253, R254, T87, T90, P94, I142, I206, N207, S210, S216, G217, N218, S219, N220, Q221, G60, S162, P184 or G209. For example, the esterase further contains at least one substitution at a position corresponding to residues selected from A14, Y61, T62, A63, R64, S67, K69, L91, Q93, S95, W133, M135, W159, S161, F175, C177, S181, I182, A183, S187, I192, G208, S212, C213, A214, N215, I224, T244, E248, T253 or R254. In particular, the esterase further contains at least one substitution selected from A14T/S/R/H, Y61A/F, T62A/M, A63R, R64A/E, S67M, K69A/E/N, L91F, Q93A/ G/Y/P, S95D/E, W133A/H/F, M135A, W159H/A, S161A/W/Q, F175I, C177A/S, S181T/N, I182A/V/F, A183I, S187Q/E, I192F, G208N, S212F/A/I/V/W, C213A/S, A214P, N215A/F/D/M, I224L, T244S, E248P/S, T253P/Y or R254A/N.

In one embodiment, the esterase may further comprise at least one substitution at a position corresponding to residues selected from T87, T90, P94, I142, I206, N207, S210, S216, G217, N218, S219, N220, or Q221. In particular, the esterase further contains at least one substitution selected from N220P/D, preferably N220P.

In one embodiment, the esterase further contains at least one substitution at a position corresponding to residues selected from T62, Q93, F175, S181, S187, N207, S212, N215 or N220, preferably selected from T62, Q93, F175, S181, N207, S212 or N215. Preferably, the replacement is selected from T62M, Q93G/P, F175I, S181N, S187E, N207C, S212I/W, N215D/M or N220P/D, more preferably from T62M, Q93G/P, F175I, S181N, N207C, S212I/W or N215D/M. Preferably, the esterase further contains at least an N207C substitution.

In a specific embodiment, the esterase has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the full-length amino acid sequence represented by SEQ ID. N 1 and at least a combination of substitutions at positions N207+S256. Preferably, the combination is N207C+S256C. In a specific embodiment, the esterase has the amino acid sequence set forth in SEQ ID NO: 1 with the substitution combination N207C+S256C. In one embodiment, the esterase contains an amino acid sequence that consists of the amino acid sequence set forth in SEQ ID No. 1 with the substitution combination N207C+S256C. Advantageously, the esterase contains the combination N207C+S256C and exhibits both increased thermal stability and increased destructive activity compared to the esterase of SEQ ID No. 1.

In particular, the esterase contains at least a combination of substitutions at positions N207+S256 and at least one amino acid substitution at a position selected from T62, S66, Q93, F175, S181, S187, S212, N215 or N220, preferably , selected from Q93, S187, S212 or N220. In particular, the esterase contains at least a combination of substitutions at position, preferably N207C+S256C, and at least one amino acid substitution selected from T62M, S66T, Q93G/P, F175I, S181N, S187E, S212I/W, N215D /M or N220P/D, preferably selected from Q93G/P, S187E, S212I/W or N220P/D.

More preferably, the esterase contains at least a combination of substitutions at positions S212+N207+S256, preferably selected from S212I/W+N207C+S256C.

In one embodiment, the esterase contains at least four substitutions at positions selected from S212, N207, S256, F175, S181, S66, T62, N215, Q93, S187 or N220. Preferably, at least four substitutions are selected from S212I/W, N207C, S256C, F175I, S181N, S66T, T62M, N215D/M, Q93G/P, S187E and N220P/D. In particular, the esterase contains at least four substitutions at positions selected from S212, N207, S256, F175, S181, S66, T62, N215 or Q93. Preferably, at least four substitutions are selected from S212I/W, N207C, S256C, F175I, S181N, S66T, T62M, N215D/M or Q93G/P.

In a specific embodiment, the esterase contains at least a combination of substitutions at positions S212+N207+S256 and one or two substitutions at positions selected from F175, S181, S66, T62, N215, Q93, S187 or N220, preferably selected from F175, S181, S66, T62, N215 or Q93. In particular, the esterase contains at least a combination of substitutions at positions S212I/W+N207C+S256C and one or two substitutions selected from F175I, S181N, S66T, T62M, N215D/M, Q93G/P, S187E or N220P/D , preferably selected from F175I, S181N, S66T, T62M, N215D/M or Q93G/P.

In one embodiment, the esterase contains at least a combination of substitutions at positions S212+N207+S256 and at least one substitution at a position selected from F175, S181, S66, T62, N215, Q93, S187 or N220, preferably in a position selected from F175, S181, S66, T62, N215 or Q93. Preferably, the esterase contains at least a combination of substitutions at positions S212I/W+N207C+S256C and at least one substitution selected from F175I, S181N, S66T, T62M, N215D/M, Q93G/P, S187E or N220P/ D, preferably selected from F175I, S181N, S66T, T62M, N215D/M or Q93G/P. In particular, the esterase contains at least a combination of substitutions at positions S212+N207+S256+Q93. Preferably, the esterase contains at least a combination of substitutions selected from S212I/W+N207C+S256C+Q93G/P. Advantageously, the esterase contains the combination S212I/W+N207C+S256C+Q93G and exhibits both increased thermal stability and increased destructive activity compared to the esterase of SEQ ID No. 1.

In a specific embodiment, the esterase contains a combination of substitutions selected from S212+N207+S256+Q93+N220+S187, preferably selected from S212I+N207C+S256C+Q93G+N220P+S187E.

In particular, the esterase contains at least a combination of substitutions selected from N207C+S256C, S212I/W+N207C+S256C+Q93G or S212I+N207C+S256C+Q93G+N220P+S187E. In one embodiment, the esterase contains an amino acid sequence consisting of the amino acid sequence set forth in SEQ ID No. 1 with a combination of substitutions selected from N207C+S256C or S212I/W+N207C+S256C+Q93G, and exhibits both increased degradative activity and and increased thermal stability compared to esterase SEQ ID No. 1.

In a specific embodiment, the esterase may further contain at least one substitution at a position corresponding to residues selected from A7, R8, N11, T25, V26, R27, G38, P45, T51, W71, R97, S98, S99, R106 , S110, N112, G113, T114, S115, G121, A126, M128, M131, G132, A145, N146, L150, A154, P155, Q156, A157, D160, T163, F165, V168, L173, S188 L190, 91, A200, K201, Q202, T240, S243, T260, N262, V108, G113, T114, S115, G121, K122, T125, A126, G129, G139, S140, A154, P155, D160, T172, N186, S188, 0, E205, S264. Preferably, the replacement is selected from A7Q/E, R8E, N11A, T25P/S, V26Y, R27E/Q, G38R, T51E/P, W71L, R97A, S98C, S99R, R106G/S, S110T, N112S, G113N/R, G121N/ T, A126S, M128L, A145R, L150I, P155A/G/S, Q156L, D160H/F/I/L/V/S, T163K, V168L, S188H, L190D, P191T, A200P, K201R, Q202E, T240R, S243T, T260V, N262A, V108L, G113N, T114D, S115P, G121T, K122W, T125P, A126S, G129A, G139A, S140T, A154I, P155A, D160H, T172Q/V, N186R, S188H, A200P, 205M or S264P.

In one embodiment, the esterase of the invention contains at least one amino acid residue selected from S134, D180, H211, as in the original esterase, i.e. The esterase of the invention is not modified at one, two or all of these positions. Preferably, the esterase contains the combination S134+D180+H211, as in the original esterase.

Alternatively or additionally, the esterase contains at least one amino acid residue selected from C177, C213, C247 or C263, as in the original esterase. Preferably, the esterase contains the combination C247+C263, as in the original esterase. Preferably, the esterase contains at least one combination of C247+C263 or C177+C213, as in the original esterase, even more preferably, the esterase contains C247+C263+C177+C213. In particular, the esterase contains S134+D180+H211+C247+C263+C177+C213, as in the original protease.

Another object of the present invention is to provide an esterase that (i) has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the full-length amino acid sequence set forth in SEQ ID No. 1 , (ii) contains at least one amino acid substitution at a position corresponding to residues selected from the group consisting of S256, T13, A15, S16, L17, E18, A19, S20, A21, S66, I68, W70, P73 R74, D86, D92, G136, F175, S187, I192, A222, L223, I224, G225, K226, K227, T244, F245, A246, C247, E248, N249, P250, N251, S252, T253, V255, C263, C263, C263, C263, C263, C263, C263, C263, C263, C263, C263, C263, C263, C263, C263, C263, C265, C263 , G60, S161, S162, P184, G208 and G209 compared to the amino acid sequence of SEQ ID No. 1, and (iii) exhibits increased polyester degrading activity and/or increased thermal stability compared to the esterase of SEQ ID No. 1. In particular, contains at least one amino acid substitution at a position corresponding to residues selected from the group consisting of S256, T13, A15, S16, L17, E18, A19, S20, A21, S66, I68, W70, P73, R74, D86 , D92, G136, F175, S187, I192, A222, L223, I224, G225, K226, K227, T244, F245, A246, C247, E248, N249, P250, N251, S252, T253, V255 and C263.

In a specific embodiment, the esterase has the amino acid sequence set forth in SEQ ID No. 1, with one amino acid substitution at a position corresponding to residues selected from S256, T13, A15, S16, L17, E18, A19, S20, A21, S66, I68 , W70, P73, R74, D86, D92, G136, F175, S187, I192, A222, L223, I224, G225, K226, K227, T244, F245, A246, C247, E248, N249, P250, N251, S252, T253 , V255, C263, G60, S161, S162, P184, G208 or G209. In particular, the esterase has a single amino acid substitution at a position corresponding to residues selected from the group consisting of S256, T13, A15, S16, L17, E18, A19, S20, A21, S66, I68, W70, P73, R74, D86, D92, G136, F175, S187, I192, A222, L223, I224, G225, K226, K227, T244, F245, A246, C247, E248, N249, P250, N251, S252, T253, V255 and C263. In one embodiment, the esterase contains an amino acid sequence consisting of the amino acid sequence set forth in SEQ ID No. 1, with one amino acid substitution at a position corresponding to residues selected from S256, T13, A15, S16, L17, E18, A19, S20, A21, S66, I68, W70, P73, R74, D86, D92, G136, F175, S187, I192, A222, L223, I224, G225, K226, K227, T244, F245, A246, C247, E248, N249, P250, N251, S252, T253, V255 and C263.

In a specific embodiment, the esterase contains at least one amino acid substitution at a position corresponding to residues selected from the group consisting of S256, T13, A15, S16, L17, E18, A19, S20, A21, S66, I68, W70 , P73, R74, D86, D92, G136, A222, L223, G225, K226, K227, F245, A246, C247, N249, P250, N251, S252, V255, C263, G60, S162, P184 or G209 compared to amino acid sequence of SEQ ID No. 1, and (iii) exhibits increased polyester degrading activity and/or increased thermal stability compared to the esterase of SEQ ID No. 1. In particular, the esterase contains at least one amino acid substitution at a position corresponding to the residues selected from the group consisting of S256, T13, A15, S16, L17, E18, A19, S20, A21, S66, I68, W70, P73, R74, D86, D92, G136, A222, L223, G225, K226, K227, F245, A246, C247, N249, P250, N251, S252, V255 or C263.

In a specific embodiment, the esterase has the amino acid sequence set forth in SEQ ID No. 1, with one amino acid substitution at a position corresponding to residues selected from S256, T13, A15, S16, L17, E18, A19, S20, A21, S66, I68 , W70, P73, R74, D86, D92, G136, A222, L223, G225, K226, K227, F245, A246, C247, N249, P250, N251, S252, V255, C263, G60, S162, P184 or G209. In particular, the esterase has a single amino acid substitution at a position corresponding to residues selected from the group consisting of S256, T13, A15, S16, L17, E18, A19, S20, A21, S66, I68, W70, P73, R74, D86, D92, G136, A222, L223, G225, K226, K227, F245, A246, C247, N249, P250, N251, S252, V255 or C263.

In an embodiment of the invention, the esterase has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the full-length amino acid sequence set forth in SEQ ID No. 1 and contains at least at least one substitution in a position corresponding to residues selected from S256, T13, A15, S16, L17, E18, A19, S20, A21, S66, I68, W70, P73, R74, D86, D92, G136, F175, S187, I192, A222, L223, I224, G225, K226, K227, T244, F245, A246, C247, E248, N249, P250, N251, S252, T253, V255 and C263. In a specific embodiment, the esterase contains at least one substitution at a position corresponding to residues selected from C247 and C263.

In one embodiment, the esterase has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the full-length amino acid sequence set forth in SEQ ID NO: 1 and contains at least at least one substitution at a position corresponding to residues selected from G60, S161, S162, P184, G208 or G209.

In one embodiment, the esterase has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the full-length amino acid sequence set forth in SEQ ID NO: 1 and contains at least at least one change in position S256. Preferably, the replacement is S256C.

In another preferred embodiment, the esterase has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the full-length amino acid sequence set forth in SEQ ID No. 1, and contains, at least one replacement in a position selected from S66 and F175. Preferably, the replacement is selected from S66T and F175I. In another specific embodiment, the esterase contains at least one substitution at position S187, preferably selected from S187E. In another embodiment, the esterase contains at least one substitution at position K226, preferably selected from K226E.

In a particular embodiment, the esterase may further contain at least one substitution at a position corresponding to residues selected from A14, Y61, T62, A63, R64, S67, K69, L91, Q93, S95, W133, M135, W159, C177, S181, I182, A183, S212, C213, A214, N215, R254, T87, T90, P94, I142, I206, N207, S210, S216, G217, N218, S219, N220 or Q221.

In one embodiment, the esterase contains at least one substitution at a position corresponding to residues selected from A14, Y61, T62, A63, R64, S67, K69, L91, Q93, S95, W133, M135, W159, C177, S181, I182, A183, S212, C213, A214, N215 or R254. In particular, the esterase contains at least one substitution selected from A14H/T/S/R, Y61A/F, T62A/M, A63R, R64A/E, S67M, K69A/E/N, L91F, Q93A/G /Y/P, S95D/E, W133A/H/F, M135A, W159H/A, C177A/S, S181T/N, I182A/V/F, A183I, S212F/A/V/I/W, C213A/S , A214P, N215A/F/D/M or R254A.

In one embodiment, the esterase contains at least one substitution at a position corresponding to residues selected from T87, T90, P94, I142, I206, N207, S210, S216, G217, N218, S219, N220, or Q221. In particular, the esterase further contains at least one substitution selected from N220P/D, preferably N220P.

In one embodiment, the esterase contains at least one substitution at a position corresponding to residues selected from T62, Q93, S181, N207, S212, N215 or N220, preferably T62, Q93, S181, N207, S212 or N215 . Preferably, the replacement is selected from T62M, Q93G/P, S181N, N207C, S212I/W, N215D/M or N220P/D, more preferably from T62M, Q93G/P, S181N, N207C, S212I/W or N215D/M. Preferably, the esterase contains the N207C substitution.

In a specific embodiment, the esterase contains at least a combination of substitutions at positions N207+S256. Preferably, the combination is N207C+S256C. In a preferred embodiment, the esterase contains the combination N207C+S256C and exhibits increased thermal stability and increased degradation activity compared to the esterase of SEQ ID No. 1. In a particular embodiment, the esterase contains at least a combination of substitutions at positions N207+S256 and, at at least one amino acid substitution at a position selected from S212, F175, S181, S66, T62, N215, Q93, S187 or N220, preferably selected from Q93, S187, S212 or N220. In particular, the esterase contains at least a combination of substitutions at position preferably N207C+S256C and at least one amino acid substitution selected from S212I/W, F175I, S181N, S66T, T62M, N215D/M, Q93G/P, S187E or N220P/D, preferably selected from Q93G/P, S187E, S212I/W or N220P/D.

More preferably, the esterase contains at least a combination of substitutions at positions S212+N207+S256, preferably selected from S212I/W+N207C+S256C.

In one embodiment, the esterase contains at least four substitutions at positions selected from S212, N207, S256, F175, S181, S66, T62, N215, Q93, S187 or N220, preferably selected from S212, N207, S256 , F175, S181, S66, T62, N215 or Q93. Preferably, the esterase contains at least four substitutions selected from S212I/W, N207C, S256C, F175I, S181N, S66T, T62M, N215D/M, Q93G/P, S187E or N220P, preferably selected from S212I/W, N207C, S256C, F175I, S181N, S66T, T62M, N215D/M or Q93G/P.

In one embodiment, the esterase contains at least a combination of substitutions at positions S212+N207+S256 and at least one substitution at a position selected from F175, S181, S66, T62, N215, Q93, S187 or N220, preferably in a position selected from F175, S181, S66, T62, N215 or Q93. Preferably, the esterase contains at least a combination of substitutions at positions S212I/W+N207C+S256C and at least one substitution selected from F175I, S181N, S66T, T62M, N215D/M, Q93G/P, S187E or N220P /D, preferably selected from F175I, S181N, S66T, T62M, N215D/M or Q93G/P. In a specific embodiment, the esterase contains at least a combination of substitutions at positions S212+N207+S256 and one or two substitutions at positions selected from F175, S181, S66, T62, N215, Q93, S187 or N220, preferably in positions selected from F175, S181, S66, T62, N215 or Q93. Preferably, the esterase contains at least a combination of substitutions at positions S212I/W+N207C+S256C and one or two substitutions selected from F175I, S181N, S66T, T62M, N215D/M, Q93G/P, S187E or N220P/D, preferably selected from F175I, S181N, S66T, T62M, N215D/M or Q93G/P. In particular, the esterase contains at least a combination of substitutions at positions S212+N207+S256+Q93. Preferably, the esterase contains at least a combination of substitutions selected from S212I/W+N207C+S256C+Q93G/P. Advantageously, the esterase contains the combination S212I/W+N207C+S256C+Q93G and exhibits both increased thermal stability and increased degradation activity compared to the esterase of SEQ ID No. 1.

In a specific embodiment, the esterase contains a combination of substitutions at positions S212+N207+S256+Q93+N220+S187, preferably selected from S212I+N207C+S256C+Q93G+N220P+S187E. In a specific embodiment, the esterase may further contain at least one substitution at positions corresponding to residues selected from R8, P45, W71, R97, S98, T114, S115, M131, G132, N146, A154, P155, Q156, A157, F165. or L173. Preferably, the replacement is selected from R8E, W71L, R97A, S98C, P155A/G/S, Q156L or T260V. In another specific embodiment, the esterase further contains at least one substitution selected from A7Q, R27E, R106G or D160H/F/I/L/V.

Alternatively or additionally, the esterase further contains at least one substitution at a position corresponding to residues selected from V108, G113, T114, S115, G121, K122, T125, A126, G129, G139, S140, A154, P155, D160, T172, N186, S188, A200, E205, S264, preferably selected from V108L, G113N, T114D, S115P, G121T, K122W, T125P, A126S, G129A, G139A, S140T, A154I, P155A, D160H, 2Q, T172V, N186R, S188H, A200P, E205M or S264P.

In one embodiment, the esterase of the invention contains at least one amino acid residue selected from S134, D180, H211, as in the original esterase, i.e. The esterase of the invention is not modified at one, two or all of these positions. Preferably, the esterase contains the combination S134+D180+H211, as in the original esterase.

In one embodiment, the esterase contains at least one amino acid residue selected from C177, C213, C247 or C263, as in the original esterase. Preferably, the esterase contains the combination C247+C263, as in the original esterase. Preferably, the esterase contains at least one combination of C247+C263 or C177+C213, as in the original esterase, even more preferably, the esterase contains C247+C263+C177+C213. In particular, the esterase contains S134+D180+H211+C247+C263+C177+C213, as in the original protease.

It is also an object of the invention to provide an esterase that (i) has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the full-length amino acid sequence represented by in SEQ ID No. 1, (ii) contains at least one replacement selected from the group consisting of S256C, S212I/W, N207C, S181N, S66T, T62M, N215D/M, Q93G/P, N220P/D, S187E, A14H, T172Q or K226E, and (iii) has increased degradation activity and/or increased thermal stability compared to the esterase of SEQ ID No. 1.

Preferably, the esterase contains at least one substitution selected from the group consisting of S256C, S212I/W, N207C, S181N, S66T, T62M, N215D/M or Q93G/P.

In particular, the esterase contains at least one substitution or combination of substitutions selected from the group consisting of N207C+S256C, S212I/W, S181N, S66T, T62M, N215D/M, N220P, S187E, Q93G/P, A14H, T172Q or K226E. In particular, the esterase contains at least one substitution or combination of substitutions selected from the group consisting of N207C+S256C, S212I/W, S181N, S66T, T62M, N215D/M or Q93G/P.

In a specific embodiment, said esterase further comprises an F175I substitution.

In a specific embodiment, the esterase has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the full-length amino acid sequence represented by SEQ ID. N 1 and at least a combination of substitutions N207C+S256C. In particular, the esterase contains at least a combination of substitutions N207C+S256C and at least one amino acid substitution selected from T62M, S66T, Q93G/P, F175I, S181N, S187E, S212I/W, N215D/M N220P/ D, A14H, T172Q or K226E, preferably selected from T62M, S66T, Q93G/P, F175I, S181N, S212I/W or N215D/M, more preferably selected from Q93G/P, S187E, S212I/W or N220P/D , even more preferably S212I/W. More preferably, the esterase contains at least a combination of substitutions selected from S212I/W+N207C+S256C.

In one embodiment, the esterase contains at least four substitutions at positions selected from S212I/W, N207C, S256C, F175I, S181N, S66T, T62M, N215D/M, Q93G/P, S187E and N220P/D, preferably , selected from S212I/W, N207C, S256C, F175I, S181N, S66T, T62M, N215D/M or Q93G/P.

In a specific embodiment, the esterase contains at least the combination of substitutions S212I/W+N207C+S256C and at least one substitution at positions selected from F175I, S181N, S66T, T62M, N215D/M, Q93G/P, S187E, N220P/D, A14H, T172Q or K226E, preferably selected from F175I, S181N, S66T, T62M, N215D/M or Q93G/P. In a specific embodiment, the esterase contains at least the combination of substitutions S212I/W+N207C+S256C and one or two substitutions selected from F175I, S181N, S66T, T62M, N215D/M, Q93G/P, S187E or N220P/D , preferably selected from F175I, S181N, S66T, T62M, N215D/M or Q93G/P. Advantageously, the esterase contains the combination S212I/W+N207C+S256C+Q93G and exhibits both increased thermal stability and increased degradation activity compared to the esterase of SEQ ID NO 1. In a particular embodiment, the esterase contains a combination of substitutions selected from S212+N207+ S256+Q93+N220+S187, preferably selected from S212I+N207C+S256C+Q93G+N220P+S187E.

Preferably, the esterase contains at least one substitution or combination of substitutions selected from the group consisting of S212I/W, N207C, S256C, S181N, S66T, T62M, N215D/M, N220P, S187E, Q93G/P, N207C+S256C , S212I/W+N207C+S256C, S212I/W+N207C+S256C+Q93G/P or S212I+N207C+S256C+Q93G+N220P+S187E.

In particular, the esterase contains at least a combination of substitutions selected from N207C+S256C, S212I/W+N207C+S256C+Q93G or S212I+N207C+S256C+Q93G+N220P+S187E. In one embodiment, the esterase contains an amino acid sequence consisting of the amino acid sequence set forth in SEQ ID No. 1 with a substitution combination selected from N207C+S256C or S212I/W+N207C+S256C+Q93G, and exhibits both increased degradation activity and and increased thermal stability compared to esterase SEQ ID No. 1.

In one embodiment, the esterase further contains at least one amino acid residue selected from S134, D180, H211, as in the original esterase, i.e. The esterase of the invention is not modified at one, two or all of these positions. Preferably, the esterase contains the combination S134+D180+H211, as in the original esterase.

In another embodiment, the esterase contains at least one amino acid residue selected from C177, C213, C247 or C263, as in the original esterase. Preferably, the esterase contains the combination C247+C263, as in the original esterase. Preferably, the esterase contains at least one combination of C247+C263 or C177+C213, as in the original esterase, even more preferably, the esterase contains C247+C263+C177+C213. In particular, the esterase contains S134+D180+H211+C247+C263+C177+C213, as in the original protease.

In one embodiment, the esterase further contains at least one substitution or combination of substitutions at a position corresponding to residues selected from T13, A14, A15, S16, L17, E18, A19, S20, A21, G60, Y61, A63, R64, S67, I68, K69, W70, P73, R74, D86, T87, T90, L91, D92, P94, S95, W133, M135, G136, I142, W159, S161, S162, C177, I182, A183, P184, S187, I192, I206, G208, G209, S210, C213, A214, S216, G217, N218, S219, N220, Q221, A222, L223, I224, G225, K226, K227, T244, F245, A246, C247, 8, N249, P250, N251, S252, T253, R254, V255 or C263, preferably selected from T13, A15, S16, L17, E18, A19, S20, A21, G60, Y61, A63, R64, S67, I68, K69, W70, P73, R74, D86, T87, T90, L91, D92, P94, S95, W133, M135, G136, I142, W159, S161, S162, C177, I182, A183, P184, S187, I192, I206, G208, G209, S210, C213, A214, S216, G217, N218, S219, N220, Q221, A222, L223, I224, G225, K227, T244, F245, A246, C247, E248, N249, P250, N251, S252, 3, R254, V255 or C263.

In a specific embodiment, the esterase may further contain at least one substitution at a position corresponding to residues selected from A7, R8, N11, T25, V26, R27, G38, P45, T51, W71, R97, S98, S99, R106, S110, N112, G113, T114, S115, G121, A126, M128, M131, G132, A145, N146, L150, A154, P155, Q156, A157, D160, T163, F165, V168, L173, S188 L190 , P191 , A200, K201, Q202, T240, S243, T260, N262, V108, G113, T114, S115, G121, K122, T125, A126, G129, G139, S140, A154, P155, D160, T172, N186, S188, 200 , E205, S264. Preferably, the replacement is selected from A7Q/E, R8E, N11A, T25P/S, V26Y, R27E/Q, G38R, T51E/P, W71L, R97A, S98C, S99R, R106G/S, S110T, N112S, G113R, G121N, A126S M128L, A145R, L150I, P1555A/G/S, Q156L, D160H/F/I/L/V/S, T163K, V168L, S188H, L190D, P191T, A200P, K201R, Q202E, T240R, S243T, T260V, N262AA, N262AA, N262AA, N262AA, N262AA , V108L, G113N, T114D, S115P, G121T, K122W, T125P, A126S, G129A, G139A, S140T, A154I, P155A, D160H, T172Q/V, N186R, S188H, A200P, E205M or S264P .

Another object of the present invention is to provide an esterase that (i) has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the full-length amino acid sequence, specified in SEQ ID No. 1, (ii) contains at least one amino acid substitution at a position corresponding to residues selected from the group consisting of A14, Y61, T62, A63, R64, S67, K69, L91, Q93, S95 , W133, M135, W159, S161, F175, C177, S181, I182, A183, S187, I192, G208, S212, C213, A214, N215, I224, T244, E248, T253 and R254, and where replacements differ from A14T/ S/R, Y61A/F, T62A, A63R, R64A/E, S67M, K69E/N, L91F, Q93A, S95D/E, W133A/H/F, M135A, W159H/A, S161A, F175I, C177A/S, S181T, I182A/V/F, A183I, S187Q, I192F, G208N, S212F/A/V, C213A/S, A214P, N215A/F, I224L, T244S, E248S, T253Y or R254A/N.

In a specific embodiment, the esterase further comprises a substitution at a position selected from S256, T13, A15, S16, L17, E18, A19, S20, A21, S66, I68, W70, P73, R74, D86, D92, G136, A222, L223, G225, K226, K227, F245, A246, C247, N249, P250, N251, S252, V255, C263, G60, S162, P184 or G209.

In a particular embodiment, the esterase may further comprise at least one substitution at a position selected from T87, T90, P94, I142, I206, N207, S210, S216, G217, N218, S219, N220, or Q221.

In a specific embodiment, the esterase contains at least one amino acid residue selected from S134, D180, H211, as in the original esterase, i.e. The esterase of the invention is not modified at one, two or all of these positions. Preferably, the esterase contains the combination S134+D180+H211, as in the original esterase.

In another embodiment, the esterase contains at least one amino acid residue selected from C177, C213, C247 or C263, as in the original esterase, i.e. The esterase of the invention is not modified at one, two or all of these positions. Preferably, the esterase contains the combination C247+C263, as in the original esterase.

Activity of the variant to destroy polyester

The purpose of the invention is to provide new enzymes having esterase activity. In a specific embodiment, the enzyme of the invention exhibits cutinase activity.

In a specific embodiment, the esterase of the invention has a polyester degrading activity, preferably a polyethylene terephthalate (PET) degrading activity and/or a polybutylene adipaterephthalate (PBAT) degrading activity and/or a polycaprolactone (PCL) degrading activity and/or a polycaprolactone (PCL) degrading activity ) or polybutylene succinate (PBS) degradation activity, more preferably polyethylene terephthalate (PET) degradation activity and/or polybutylene adipaterephthalate (PBAT) degradation activity. Even more preferably, the esterase of the invention has polyethylene terephthalate (PET) degrading activity.

Advantageously, the esterase of the invention exhibits polyester degrading activity at least in the temperature range of 20°C to 90°C, preferably 40°C to 80°C, more preferably 35°C to 55°C. In a specific embodiment, the esterase exhibits polyester degradation activity at 40°C. In a specific embodiment, the esterase exhibits polyester degradation activity at 50°C. In a particular embodiment, the polyester degrading activity is still measurable at temperatures between 55°C and 65°C. In another embodiment, the polyester degrading activity is still measurable at a temperature of 40°C to 50°C. In another embodiment, the polyester degrading activity is still measurable at temperatures between 60°C and 80°C. In another embodiment, the polyester degrading activity is still measurable at a temperature of 65°C to 75°C.

In a specific embodiment, the esterase of the invention has increased polyester degrading activity at a given temperature compared to the esterase of SEQ ID No. 1, and more specifically at a temperature of from 40°C to 80°C, more preferably from 40°C to 60°C, from 40°C to 50°C, from 60°C to 80°C, from 65°C to 75°C.

In a specific embodiment, the esterase has a polyester degrading activity at 50°C that is at least 5% higher than the polyester degrading activity of the esterase SEQ ID No. 1, preferably at least 10%, 20%, 50 %, 100% or more.

In another specific embodiment, the esterase has a polyester degrading activity at 40°C that is at least 5% higher than the polyester degrading activity of the esterase SEQ ID No. 1, preferably at least 10%, 20%, 50%, 100% or more.

In a specific embodiment, the esterase of the invention exhibits measurable esterase activity in at least the pH range of 5 to 11, preferably in the pH range of 6 to 9, more preferably in the pH range of 6.5 to 9, even more preferably in the pH range from 6.5 to 8.

Nucleic acids, expression cassette, vector, host cell

It is yet another object of the invention to provide a nucleic acid encoding an esterase as defined above.

As used herein, the terms nucleic acid , nucleic sequence , polynucleotide , oligonucleotide and nucleotide sequence refer to a sequence of deoxyribonucleotides and/or ribonucleotides. Nucleic acids can be DNA (cDNA or gDNA), RNA, or a mixture of both. It may be in single-stranded or duplex form or a mixture thereof. It may be of recombinant, artificial and/or synthetic origin and may contain modified nucleotides containing, for example, a modified linkage, a modified purine or pyrimidine base or a modified sugar. The nucleic acids of the invention may be in isolated or purified form and may be prepared, isolated and/or processed by methods known per se in the art, for example, cloning and expression of cDNA libraries, amplification, enzymatic synthesis or recombinant technology. Nucleic acids can also be synthesized in vitro using well known chemical synthesis methods, as described, for example, in Belousov (1997) Nucleic Acids Res. 25:3440-3444.

The invention also covers nucleic acids that hybridize, under stringent conditions, with a nucleic acid encoding an esterase as defined above. Preferably, such stringent conditions include incubating the hybridization filters at about 42°C for about 2.5 hours in 2X SSC/0.1% SDS, followed by washing the filters four times for 15 minutes in 1X SSC/0.1% SDS at 65°C. The protocols used are described in references such as Sambrook et al. (Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor N.Y. (1988)) and Ausubel (Current Protocols in Molecular Biology (1989)).

The invention also covers nucleic acids encoding an esterase of the invention, wherein the sequence of said nucleic acids, or at least a portion of said sequence, has been engineered using an optimized codon.

Alternatively, the nucleic acids of the invention can be transcribed from the esterase sequence of the invention, and the codon usage can be adapted according to the host cell in which the nucleic acids are to be transcribed. These steps can be carried out in accordance with methods well known to those skilled in the art, some of which are described in the reference manual Sambrook et al. (Sambrook et al., 2001).

The 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, which can be used to induce or regulate polypeptide expression in a selected host cell or system.

The present invention further provides an expression cassette comprising a nucleic acid of the invention operably linked to one or more control sequences that direct expression of said nucleic acid in a suitable host cell.

The term expression as used herein refers to any step associated with the production of a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification and secretion.

The term expression cassette refers to a nucleic acid construct containing a coding region, i.e. a nucleic acid according to the invention, and a regulatory region, i.e. containing one or more control sequences that are operably linked.

Typically, an expression cassette contains or consists of a nucleic acid of the invention operably linked to a control sequence, such as a transcription promoter and/or a transcription terminator. The control sequence may include a promoter recognized by a host cell or an in vitro expression system for expressing a nucleic acid encoding an esterase of the present invention. The promoter contains transcriptional control sequences that mediate the expression of the enzyme. A promoter can be any polynucleotide that exhibits transcriptional activity in a host cell, including mutant, truncated, and hybrid promoters, and can be derived from genes encoding extracellular or intracellular polypeptides homologous or heterologous to the host cell. The control sequence may also be a transcription terminator that is recognized by the host cell to terminate transcription. The terminator is operably linked to the 3' end of the nucleic acid encoding the esterase. Any terminator that functions in a host cell can be used in the present invention. Typically, an expression cassette contains or consists of a nucleic acid of the invention operably linked to a transcription promoter and a transcription terminator.

The invention also relates to a vector containing a nucleic acid or expression cassette as defined above.

As used herein, the terms “vector” or “ expression vector” refer to a DNA or RNA molecule containing an expression cassette of the invention used as a carrier for transfer of recombinant genetic material into a host cell. The main types of vectors are plasmids, bacteriophages, viruses, cosmids and artificial chromosomes. The vector itself, as a rule, is a DNA sequence consisting of an insert (a heterologous nucleic acid sequence, a transgene) and a larger sequence that serves as the “skeleton” of the vector. The purpose of a vector that conveys genetic information to a host is typically to isolate, propagate, or express an insert in a target cell. Vectors, called expression vectors (expression constructs), are specifically adapted to express heterologous sequences in a target cell and typically have a promoter sequence that drives the expression of the heterologous polypeptide-encoding sequences. Typically, regulatory elements present in the expression vector include a transcriptional promoter, a ribosome binding site, a terminator, and an optional operator. Preferably, the expression vector also contains an origin of replication for autonomous replication in the host cell, a selectable marker, a limited number of useful restriction enzyme sites, and high copy number capability. Examples of expression vectors are cloning vectors, modified cloning vectors, specially constructed plasmids and viruses. Expression vectors that provide suitable levels of polypeptide expression in different hosts are well known in the art. The choice of vector will generally depend on the compatibility of the vector with the host cell into which the vector is to be introduced. Preferably, the expression vector is a linear or circular double-stranded DNA molecule.

Another object of the invention is to provide a host cell containing a nucleic acid, expression cassette or vector as described above. Thus, the present invention relates to the use of a nucleic acid, expression cassette or vector according to the invention for transformation, transfection or transduction of a host cell. The choice of vector usually depends on the compatibility of the vector with the host cell into which it is to be introduced.

In accordance with the invention, the host cell can be transformed, transfected or transduced in a temporary or stable manner. The expression cassette or vector of the invention is introduced into a host cell such that the cassette or vector is maintained as a chromosomally integrated element or as a self-replicating extrachromosomal vector. The term "host cell" also covers any progeny of the parent host cell that is not identical to the parent host cell due to mutations occurring during replication. The host cell can be any cell suitable for producing a variant of the present invention, for example, prokaryotes or eukaryotes. The prokaryotic host cell can be any gram-positive or gram-negative bacterium. The host cell may also be a eukaryotic cell, such as a yeast, fungal, mammalian, insect or plant cell. In a specific 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 of the invention can be introduced into a host cell by any method known to one skilled in the art, such as electroporation, conjugation, transduction, competent cell transformation, protoplast transformation, protoplast fusion, biolistic gene gun transformation ", PEG-mediated transformation, lipid-mediated transformation or transfection, chemically mediated transfection, transformation, lithium acetate-mediated transformation, liposome-mediated transformation.

Optionally, more than one copy of the nucleic acid, cassette or vector of the present invention can be inserted into a host cell to increase the effectiveness of the variant.

In a specific embodiment, the host cell is a recombinant microorganism. The invention does make it possible to design microorganisms with improved ability to degrade polyester-containing material. For example, the sequence of the invention may be used to complement a wild-type fungal or bacterial strain already known to degrade polyester to improve and/or increase the efficiency of the strain.

Esterase production

Another object of the invention is to provide a method for producing the esterase of the invention, comprising expressing a nucleic acid encoding the esterase and, optionally, isolating the esterase.

In particular, the present invention relates to in vitro methods for producing esterase of the present invention,comprising (a) contacting a nucleic acid, cassette or vector of the invention with an expression systemin vitro; and (b) isolating the resulting esterase. Expression systemsin vitrowell known to those skilled in the art and commercially available.

Preferably, the production method includes

(a) culturing a host cell that contains a nucleic acid encoding an esterase of the invention under conditions suitable for expression of the nucleic acid; and optional

(b) isolating said esterase from the cell culture.

Preferably, the host cell is recombinant Bacillus, recombinant E. coli, recombinant Aspergillus, recombinant Trichoderma, recombinant Streptomyces, recombinant Saccharomyces, recombinant Pichia, recombinant Vibrio or recombinant Yarrowia .

Host cells are cultured in a culture medium suitable for the production of polypeptides using methods known in the art. For example, cells can be cultured by shake flask culture or small-scale or large-scale fermentation (including continuous, batch, fed or solid state fermentation) in laboratory or industrial fermenters, carried out in a suitable environment and under conditions allowing expression and/or release of the enzyme. Cultivation takes place in a suitable culture medium obtained from commercial suppliers or prepared in accordance with published compositions (eg, American Type Culture Collection catalogs).

If esterase is released into the culture medium, it can be isolated directly from the culture supernatant. Conversely, esterase can be isolated from cell lysates or after permeabilization. The esterase can be isolated by any method known in the art. For example, the esterase can be isolated from the culture medium by conventional methods, including, but not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation or precipitation. Optionally, the esterase may be partially or completely purified by various methods 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 (eg, ammonium sulfate precipitation), SDS-PAGE, or extraction to obtain substantially pure polypeptides.

The esterase can be used alone, in purified form, alone or in combination with additional enzymes, to catalyze enzymatic reactions involved in the degradation and/or recycling of polyesters and/or polyester-containing material, such as polyester-containing plastic products. Esterase can be in soluble form or in solid phase. In particular, it can be associated with cell membranes or fat vesicles or with synthetic supports such as glass, plastics, polymers, filters, membranes, for example in the form of beads, columns, plates and the like.

Composition

Another object of the invention is to provide a composition containing an esterase or a host cell according to the invention, or an extract thereof. In the context of the invention, the term "composition" covers any kind of compositions containing an esterase or a host cell according to the invention.

The composition of the invention may contain 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 contain from 5 to 10% by weight of the esterase of the invention.

The composition may be liquid or dry, for example in powder form. In some embodiments, the composition is a lyophilisate.

The composition may further contain excipients and/or reagents, etc. Suitable excipients include buffers commonly used in biochemistry, pH adjusting agents, preservatives such as sodium benzoate, sodium sorbate or sodium ascorbate, preservatives, protecting or stabilizing agents such as starch, dextrin, gum acacia, salts, sugars such as sorbitol , trehalose or lactose, glycerin, polyethylene glycol, polypropylene glycol, propylene glycol, a complexing agent such as EDTA, reducing agents, amino acids, a carrier such as a solvent or aqueous solution, and the like. The composition of the invention can be prepared by mixing the esterase with one or more excipients.

In a specific embodiment, the composition contains 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 excipients based on the total weight of the composition. Alternatively, the composition may contain from 90% to 95% by weight excipients.

In a particular embodiment, the composition may further comprise additional polypeptides exhibiting enzymatic activity. The amounts of esterase of the invention are readily adaptable by those skilled in the art depending, for example, on the nature of the polyester being degraded and/or additional enzymes/polypeptides contained in the composition.

In a specific embodiment, the esterase of the invention is solubilized in an aqueous medium along with one or more excipients, especially excipients that are capable of stabilizing or protecting the polypeptide from degradation. For example, the esterase of the invention can be dissolved in water, ultimately with additional components such as glycerol, sorbitol, dextrin, starch, glycol such as propanediol, salt, etc. The resulting mixture can then be dried to obtain a powder. Methods for drying such a mixture are well known to those skilled in the art and include, but are not limited to, lyophilization, freeze drying, spray drying, supercritical drying, downdraft evaporation, thin film evaporation, centrifugal evaporation, belt drying, fluid bed drying, drum drying or any combination thereof.

In a specific embodiment, the composition is in powder form and contains an esterase and a stabilizing/solubilizing amount of glycerol, sorbitol or dextrin, such as maltodextrin and/or cyclodextrin, starch, glycol, such as propanediol, and/or salt.

In a specific embodiment, the composition of the invention contains at least one recombinant cell expressing an esterase of the invention, or an extract thereof. “ Cell extract” means any fraction obtained from a cell, such as cell supernatant, cell debris, cell walls, DNA extract, enzymes or enzyme preparation, or any preparation obtained from cells by chemical, physical and/or enzymatic treatment that contains practically no living cells. Preferred extracts are enzymatically active extracts. The composition of the invention may contain one or more recombinant cells of the invention or an extract thereof and, optionally, one or more additional cells.

In one embodiment, the composition consists of or contains a culture medium of a recombinant microorganism expressing and secreting an esterase according to the invention. In a specific embodiment, the composition contains such a lyophilized culture medium.

Usage esterase

Another object of the invention is to provide methods using the esterase of the invention to degrade and/or process, under aerobic or anaerobic conditions, polyester or polyester-containing material. The esterases of the invention are particularly useful for degrading PET and materials containing PET.

It is therefore an object of the invention to use the esterase of the invention or a corresponding recombinant cell or extract or composition thereof for the enzymatic degradation of a polyester.

In a specific embodiment, the esterase-targeted polyester 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 adipaterephthalate (PBAT), polyethylene furanoate (PEF), polycaprolactone (PCL), poly(ethylene adipate) (PEA), polyethylene naphthalate (PEN) and mixtures/blends of these materials, preferably polyethylene terephthalate.

In a preferred embodiment, the polyester is PET and at least the isolated monomers (eg, monoethylene glycol or terephthalic acid) and/or oligomers (eg, methyl 2-hydroxyethyl terephthalate (MHET), bis(2-hydroxyethyl) terephthalate (BHET) ), 1-(2-hydroxyethyl) 4-methyl terephthalate (HEMT) and dimethyl terephthalate (DMT).

It is also an object of the invention to use an esterase according to the invention, or a corresponding recombinant cell, or an extract or composition thereof, to enzymatically degrade at least one polyester of a polyester-containing material.

Another object of the invention is to provide a method for degrading at least one polyester of a polyester-containing material, wherein the polyester-containing material contacts an esterase or a host cell or an extract thereof or a composition of the invention, thereby degrading at least one polyester of a polyester-containing material.

Advantageously, the polyesters are depolymerized to monomers and/or oligomers.

In particular, the invention provides a method for degrading PET material containing PET, wherein the material containing PET is contacted with an esterase or host cell or composition of the invention, thereby degrading PET.

In one embodiment, the at least one polyester is cleaved to repolymerizable monomers and/or oligomers, which can be advantageously recovered for reuse. The recovered monomers/oligomers can be used for recycling (eg repolymerizable polyesters) or methanization. In a specific embodiment, at least one polyester is PET, and monoethylene glycol, terephthalic acid, methyl 2-hydroxyethyl terephthalate (MHET), bis(2-hydroxyethyl) terephthalate (BHET), 1-(2-hydroxyethyl) 4 -methyl terephthalate (HEMT) and/or dimethyl terephthalate (DMT).

In an embodiment, the polyesters of the polyester-containing material are completely destroyed.

The time required for the polyester containing material to degrade may vary depending on the polyester containing material itself (i.e., the nature and origin of the polyester containing material, its composition, shape, etc.), type and quantity the esterase used, as well as various process parameters (i.e. temperature, pH, additional agents, etc.). One skilled in the art can easily adapt the process parameters to the polyester containing material and the expected degradation time.

Preferably, the destruction process is carried out at a temperature of from 20°C to 90°C, preferably from 40°C to 80°C, more preferably from 40°C to 50°C. In a specific embodiment, the destruction process is carried out at 40°C. In another specific embodiment, the destruction process is carried out at 50°C. More generally, the temperature is maintained below the inactivation temperature, which corresponds to the temperature at which the esterase is inactivated (ie, loses more than 80% of its activity compared to its activity at the optimal temperature) and/or the recombinant microorganism no longer synthesizes the esterase. In particular, the temperature is maintained below the glass transition temperature (Tg) of the targeted polyester.

Advantageously, the process is carried out in a continuous flow at a temperature at which the esterase can be used several times and/or recycled.

Advantageously, the destruction process is carried out at a pH of 5 to 11, preferably at a pH of 6 to 9, more preferably at a pH of 6.5 to 9, even more preferably at a pH of 6.5 to 8.

In a particular embodiment, the polyester-containing material may be pre-treated prior to contact with the esterase to physically change its structure so as to increase the contact surface between the polyester and the esterase.

Another object of the invention is to provide a method for producing monomers and/or oligomers from a polyester containing material, comprising exposing the polyester containing material to the esterases of the invention or a corresponding recombinant cell, or an extract or composition thereof, and optionally isolating the monomers and /or oligomers.

Monomers and/or oligomers resulting from depolymerization can be isolated, sequentially or continuously. One type of monomers and/or oligomers, or several different types of monomers and/or oligomers, can be isolated, depending on the starting material containing the polyester.

The process of the present invention is particularly useful for preparing monomers selected from monoethylene glycol and terephthalic acid, and/or oligomers selected from methyl 2-hydroxyethyl terephthalate (MHET), bis(2-hydroxyethyl) terephthalate (BHET), 1-(2 hydroxyethyl) 4-methyl terephthalate (HEMT) and dimethyl terephthalate (DMT), from PET and/or plastic products containing PET.

The reduced monomers and/or oligomers can be further purified using any suitable purification methods, and conditioned into a repolymerizable form. Examples of purification methods include stripping process, aqueous separation, selective steam condensation, filtration and concentration of bioprocess media, separation, distillation, vacuum evaporation, extraction, electrodialysis, adsorption, ion exchange, precipitation, crystallization, concentration and acid addition dehydration and precipitation , nanofiltration, acid catalyst treatment, semi-continuous or continuous distillation, solvent extraction, evaporative concentration, evaporative crystallization, liquid/liquid extraction, hydrogenation, azeotropic distillation process, adsorption, column chromatography, single vacuum distillation and microfiltration, in combination or not.

The isolated repolymerizable monomers and/or oligomers can be reused, for example, for the synthesis of polyesters. Advantageously, polyesters of the same nature are repolymerized. However, it is possible to mix the isolated monomers and/or oligomers with other monomers and/or oligomers, for example, to synthesize new copolymers. Alternatively, the isolated monomers can be used as chemical intermediates to obtain new chemical compounds of interest.

The invention also relates to a method for surface hydrolysis or surface functionalization of a polyester containing material, comprising exposing the polyester containing material to an esterase according to the invention, or a corresponding recombinant cell, or an extract or composition thereof. The method of the invention is particularly useful for increasing the hydrophilicity or hygroscopicity of a polyester material. This increased hydrophilicity may be of particular interest in textiles, electronics, and biomedical applications.

Another object of the invention is to provide a polyester containing material containing an esterase according to the invention and/or a recombinant microorganism expressing and secreting said esterase. By way of example, methods for producing such polyester containing material, including the esterases of the present invention, are described in patent applications WO2013/093355, WO 2016/198650, WO 2016/198652, WO 2019/043145 and WO 2019/043134.

It is therefore 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 relates to a plastic article containing PET and an esterase according to the invention having PET degrading activity.

Thus, another object of the invention is 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 relates to a plastic article containing PBAT and an esterase according to the invention having PBAT degrading activity.

Thus, another object of the invention is 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 relates to a plastic article containing PBS and an esterase according to the invention having PBS degrading activity.

Thus, another object of the invention is 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 relates to a plastic article containing PCL and an esterase according to the invention having PCL degrading activity.

Classically, the esterase of the invention can be used in detergents, food, animal feed, paper, textiles and pharmaceuticals. More specifically, the esterase of the invention can be used as a component of a detergent composition. Detergent compositions include, but are not limited to, detergent compositions for hand or machine washing, such as a laundry additive composition suitable for pre-treating dyed fabrics, and a fabric softener composition with the addition of rinse aid, a detergent composition for use in general household hard surface cleaning operations , washing compositions for manual or machine washing of dishes. In a specific embodiment, the esterase of the invention can be used as a detergent additive. Thus, the invention provides detergent compositions containing the esterase of the invention. In particular, the esterase of the invention can be used as a detergent additive to reduce the effect of pilling and graying during textile cleaning.

The present invention also relates to methods of using the esterase of the invention in animal feed, as well as feed compositions and feed additives containing the esterase of the invention. The terms “feed” and “feed composition” refer to any compound, preparation, mixture or composition suitable or intended for consumption by an animal. In another specific embodiment, the esterase of the invention is used to hydrolyze proteins and produce hydrolysates containing peptides. Such hydrolysates can be used as feed compositions or feed additives.

Another object of the invention is to provide a method for using the esterase of the invention in the paper industry. More specifically, the esterase of the invention can be used to remove stickiness from pulp and water lines of paper machines.

EXAMPLES

Example 1 - Construction, Expression and Purification of Esterases

- Construction

The esterases of the invention are generated using the plasmid construct pET21b-IsPETase-His or pET26b-IsPETase-His. These plasmids consist of cloning the gene encoding the esterase of SEQ ID NO 1, optimized for Escherichia coli expression between the NdeI and XhoI restriction sites. A PelB leader sequence, to target the expressed protein to the bacterial periplasm, is added above SEQ ID No. 1 (after the methionine at position 1) for some variants (V2, V3 and V4). According to the supplier's recommendations, two site-directed mutagenesis kits are used to generate esterase variants: QuikChange II Site-Directed Mutagenesis kit and QuikChange Lightning Multi Site-Directed from Agilent (Santa Clara, California, USA).

- Expression and purification of esterases

Strains Stellar™ (Clontech, California, USA) and E. coli Tuner™ (DE3) (Merck Millipore, Guyancourt, France) or E. coli BL21(DE3) (New England Biolabs, Evry, France) have been successfully used for cloning and recombinant expression in 50 ml of LB-Miller medium or autoinducible ZYM medium (Studier et al ., 2005- Prot. Exp. Pur. 41, 207-234). Induction in LB-Miller medium was carried out at 16°C using 0.5 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG, Euromedex, Souffelweyersheim, France). Cultures were stopped by centrifugation (8000 rpm, 20 minutes at 10°C) in an Avanti J-26 XP centrifuge (Beckman Coulter, Brea, USA). Cells are suspended in 20 ml Talon buffer (Tris-HCl 20 mM, NaCl 300 mM, pH 8). The cell suspension is then sonicated for 2 minutes at 30% amplitude (cycles 2 with ON and 1 with OFF) with an FB 705 sonicator (Fisherbrand, Illkirch, France). Then a centrifugation step is carried out: 30 minutes at 11000 rpm, 10°C in an Eppendorf centrifuge. The soluble fraction is collected and subjected to affinity chromatography. This cleaning step is completed with Talon® Metal Affinity Resin (Clontech, CA, USA). Protein elution is carried out using Talon buffer with the addition of imidazole. The purified protein was dialyzed against Talon buffer, then quantified using the Bio-Rad protein assay according to the manufacturer's instructions (Lifescience Bio-Rad, France) and stored at +4°C.

Example 2 - Evaluation of the activity of destruction of esterases according to the invention

The esterase degradation activity is determined and compared to the esterase degradation activity of SEQ ID NO: 1.

Several methodologies are used to evaluate a specific activity:

(1) Specific activity and final yield based on PET hydrolysis.

(2) Activity based on the destruction of polyester in solid form.

(3) Activity based on PET hydrolysis in reactors above 100 ml

2.1. Specific activity and final yield based on PET hydrolysis

100 mg of amorphous PET powder (prepared in accordance with WO 2017/198786 to achieve crystallinity below 20%) is weighed and placed in a 100 ml glass bottle. 1 ml of an esterase preparation containing the esterase of SEQ ID No. 1 (as a reference control) or the esterase of the invention, prepared at a concentration of 0.02 mg/ml in Talon buffer (Tris-HCl 20 mM, NaCl 0.3 M, pH 8 ), added to a glass bottle. Finally, 49 ml of 0.1 M potassium phosphate buffer pH 8 is added.

Depolymerization begins by incubating each glass bottle at 30°C, 40°C, 50°C, 55°C or 60°C and 150 rpm in a Max Q 4450 incubator (Thermo Fisher Scientific, Inc. Waltham, MA, USA) .

The initial rate of the depolymerization reaction, expressed in mg TA equivalent formed/hour, is determined by sampling at various times during the first 72 hours and analyzing by ultra-high performance liquid chromatography (UHPLC). If necessary, samples are diluted in 0.1 M potassium phosphate buffer pH 8. Then 150 µl of methanol and 6.5 µl of HCl 6 N are added to 150 µl of sample or dilution. After mixing and filtering on a 0.45 µm syringe filter, samples loaded onto UHPLC to monitor the release of terephthalic acid (TA), MHET and BHET. The chromatography system used was an Ultimate 3000 UHPLC system (Thermo Fisher Scientific, Inc. Waltham, MA, USA), comprising a pump module, an autosampler, a column oven thermostatically controlled at 25°C, and a UV sensor at 240 nm. The column used is Discovery® HS C18 HPLC Column (150 x 4.6 mm, 5 µm, equipped with a pre-column, Supelco, Bellefonte, USA). TA, MHET and BHET are separated using a MeOH gradient (30% to 90%) in 1 mM H ₂ SO ₄ at 1 ml/min. The injection is 20 µl of sample. TA, MHET and BHET are measured according to standard curves obtained from commercial TA and BHET, as well as from in-house synthesized MHET under the same conditions as the samples. The specific activity of PET hydrolysis (mg TA equivalent/hour/mg enzyme) is determined from the linear portion of the reaction hydrolysis curve constructed from samples taken at different times during the first 72 hours. The equivalent TA corresponds to the sum of the measured TA and the TA contained in the measured MHET and BHET.

2.2. Activity based on breaking down polyester in solid form

20 μl of the enzyme preparation is placed in a well created on an agar plate containing PET. Agar plates are prepared by dissolving 500 mg of PET in hexafluoro-2-propanol (HFIP) and pouring this medium into 250 ml of an aqueous solution. After evaporation of HFIP at 52°C and 140 mbar, the solution is mixed v/v. with 0.2 M potassium phosphate buffer, pH 8, containing 3% agar. About 30 ml of the mixture is used to prepare each plate, and stored at 4°C.

The diameters or surface area of halos formed by polyester degradation by wild-type and variant esterases are measured and compared after 2-24 hours at 30°C, 40°C, 50°C, 55°C, or 60°C.

2.3. Activity based on PET hydrolysis in reactor

0.69 µmol to 2.07 µmol of purified esterase, prepared in 80 ml of 100 mM potassium phosphate buffer pH 8, is mixed with 20 g of amorphous PET (prepared in accordance with WO 2017/198786 to achieve crystallinity below 20%) in 500 ml Minibio bioreactor (Applikon Biotechnology, Delft, The Netherlands). Temperature control at 30°C, 40°C, 50°C, 55°C or 60°C is accomplished by immersion in a water bath, and one vessel impeller is used to maintain constant mixing at 250 rpm. The pH of the PET depolymerization assay was adjusted to pH 8 with 6N NaOH and provided by the my-Control biocontrol system (Applikon Biotechnology, Delft, The Netherlands). Base consumption is recorded during the assay and can be used to characterize the PET depolymerization assay.

The final yield of the PET depolymerization assay is determined either by determining the residual mass of PET, by determining the TA equivalent formed, or by determining base consumption. The mass of residual PET is determined by filtering, at the end of the reaction, the reaction volume through a 12-15 μm ashless filter paper grade 11 (Dutscher SAS, Brumath, France) and drying such retentate before weighing it. Determination of the TA equivalent formed is carried out using the UHPLC methods described in 2.1, and the fraction of hydrolysis is calculated based on the ratio of the molar concentration at a given time (TA+MHET+BHET) to the total amount of TA contained in the original sample. Depolymerization of PET produces acidic monomers that will be neutralized by a base capable of maintaining the pH of the reactor. The determination of the TA equivalent produced is calculated using the corresponding molar uptake of the base, and the fraction of hydrolysis is calculated based on the ratio of the molar concentration of the TA equivalent at a given time to the total amount of TA contained in the original sample.

The yield of the esterase according to the invention upon depolymerization of PET after 70 hours is shown in Table 1 (at 40°C) and Table 2 (at 50°C) below. The yield of the esterase according to the invention upon depolymerization of PET after 89 hours is shown in Table 3 (at 40°C) and Table 4 (at 50°C) below. All tables indicate an improvement in the yield of the PET depolymerization variant compared to the PET depolymerization yield of the esterase of SEQ ID NO: 1 used as a reference (set to 1).

The PET depolymerization yield is measured as shown in Example 2.1.

Table 1: Increase in yield of esterase according to the invention upon depolymerization of PET after 70 hours at 40°C

Options Improvement in PET depolymerization yield compared to SEQ ID No. 1 V1 : N207C+S256C 9.8 times

Table 2: Improvement in the yield of esterase according to the invention when depolymerizing PET after 70 hours at 50°C

Options Improvement in PET depolymerization yield compared to SEQ ID No. 1 V1 : N207C+S256C 3.8 times

Table 3: Improvement in the yield of esterase according to the invention when depolymerizing PET after 89 hours at 40°C

Options Improvement in PET depolymerization yield compared to SEQ ID No. 1 V2 : S212I+N207C+S256C+Q93G 3.3 times V3 : S212W+N207C+S256C+Q93G 57.2 times

Table 4: Improvement in the yield of esterase according to the invention when depolymerizing PET after 89 hours at 50°C

Options Improvement in PET depolymerization yield compared to SEQ ID No. 1 V2 : S212I+N207C+S256C+Q93G 23.0 times V3 : S212W+N207C+S256C+Q93G 92.5 times

Example 3 - Evaluation of the thermal stability of esterases according to the invention

The thermal stability of the esterases according to the invention is determined and compared with the thermal stability of the esterase SEQ ID No. 1.

Various methodologies are used to assess thermal stability:

(1) Circular dichroism of proteins in solution;

(2) Residual esterase activity after incubation of the protein under given conditions of temperature, time, and buffers;

(3) Residual polyester depolymerization activity after protein incubation under specified conditions of temperature, time, and buffers;

(4) The ability to degrade solid polyester compound (such as PET or PBAT or analogues) dispersed in an agar plate after incubation of the protein under specified conditions of temperature, time and buffers;

(5) Ability to perform multiple runs of polyester depolymerization analysis under specified conditions of temperatures, buffers, protein concentrations, and polyester concentrations;

(6) Differential scanning fluorimetry (DSF);

The protocol details for such methods are given below.

3.1 Circular dichroism

Circular dichroism (CD) was performed on a Jasco 815 (Easton, USA) to compare the melting point (T _m ) esterase SEQ ID N 1 (Tm=46.4°C) with Tm of esterases according to the invention. Technically, a 400 μL protein sample is prepared at a concentration of 0.5 mg/mL in Talon buffer and used for CD. A first scan from 280 to 190 nm is performed to determine the two maximum CD intensities corresponding to proper protein folding. Then a second scan is carried out at temperatures from 25°C to 110°C at wavelengths corresponding to such maximum intensities and giving specific curves (sigmoid 3 parameters y=a/(1+e^((x-x0)/b))) , which are analyzed using Sigmaplot software version 11.0, Tmdetermined when x=x0. Received T _m reflects the thermostability of a given protein. The higher T _m , the more stable the option is at high temperatures.

3.2 Residual esterase activity

1 ml of a 40 mg/l solution (in Talon buffer) of esterase SEQ ID No. 1 or esterase according to the invention is incubated at various temperatures (35, 40, 45, 50, 55, 60, 65, 70, 75, 80 and 90°C) within up to 10 days. A sample is collected regularly, diluted 1 to 500 times in 0.1 M potassium phosphate buffer pH 8.0, and para-nitrophenol-butyrate (pNP-B) analysis is performed. 20 μl of sample is mixed with 175 μl of 0.1 M potassium phosphate buffer, pH 8.0, and 5 μl of pNP-B solution in 2-methyl-2-butanol (40 mM). The enzymatic reaction is carried out at 30°C with stirring for 15 minutes and the absorbance is measured at 405 nm using a microplate spectrophotometer (Versamax, Molecular Devices, Sunnyvale, CA, USA). The hydrolysis activity of pNP-B (initial rate expressed in μmol pNPB/min) is determined using a standard curve for the released para-nitrophenol in the linear part of the hydrolysis curve.

3.3 Residual polyester depolymerization activity

10 ml of a 40 mg/l solution (in Talon buffer) of the esterase SEQ ID No. 1 and the esterase according to the invention, respectively, are incubated at various temperatures (35, 40, 45, 50, 55, 60, 65, 70, 75, 80 and 90 °C) for 1 to 30 days. A 1 ml sample is taken regularly and transferred to a bottle containing 100 mg of amorphous PET (prepared in accordance with WO 2017/198786 to achieve a degree of crystallinity below 20%), micronized at 250-500 µm, and 49 ml of 0.1 M potassium phosphate buffer with pH 8.0, and incubated at different temperatures (35, 40, 45, 50, 55, 60, 65 or 70°C). 150 µl of buffer is withdrawn regularly. If necessary, samples are diluted in 0.1 M potassium phosphate buffer pH 8. Then 150 µl of methanol and 6.5 µl of HCl 6 N are added to 150 µl of sample or dilution. After mixing and filtering on a 0.45 µm syringe filter, samples are loaded onto UHPLC to monitor the release of terephthalic acid (TA), MHET and BHET. The chromatography system used was an Ultimate 3000 UHPLC system (Thermo Fisher Scientific, Inc. Waltham, MA, USA), comprising a pump module, an autosampler, a column oven thermostatically controlled at 25°C, and a UV sensor at 240 nm. The column used is Discovery® HS C18 HPLC Column (150 x 4.6 mm, 5 µm, pre-column equipped, Supelco, Bellefonte, USA). TA, MHET and BHET are separated using a MeOH gradient (30% to 90%) in 1 mM H ₂ SO ₄ at 1 ml/min. The injection is 20 µl of sample. TA, MHET and BHET are measured according to standard curves obtained from commercial TA and BHET, as well as from in-house synthesized MHET under the same conditions as the samples. PET hydrolysis activity (μmol hydrolyzed PET/min or mg TA equivalent produced/hour) is determined from the linear portion of the hydrolysis curve constructed from samples taken at different times during the first 24 hours. The equivalent TA corresponds to the sum of the measured TA and the TA contained in the measured MHET and BHET.

3.4 Destruction of polyester in the solid state

1 ml of a 40 mg/l solution (in Talon buffer) of the esterase SEQ ID No. 1 and the esterase of the invention, respectively, are incubated at various temperatures (35, 40, 45, 50, 55, 60, 65, 70, 75, 80 and 90° C) within 1 to 30 days. At regular intervals, 20 µl of the enzyme preparation is placed into a well created in an agar plate containing PET. The preparation of agar plates containing PET is carried out by dissolving 500 mg of PET in hexafluoro-2-propanol (HFIP) and pouring this medium into 250 ml of an aqueous solution. After evaporation of HFIP at 52°C and 140 mbar, the solution is mixed v/v. with 0.2 M potassium phosphate buffer, pH 8, containing 3% agar. Approximately 30 ml of the mixture is used to prepare each OmniTray rectangular immunoassay plate and stored at 4°C.

The diameter or surface area of halos resulting from polyester degradation by wild-type esterase and embodiments of the invention is measured and compared after 2-24 hours at 30°C, 35°C, 40°C, 45°C, 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 reduce the diameter of the halo by 2 times.

3.5 Several cycles of depolymerization of complex polyester

The ability of the esterase to perform successive cycles of polyester depolymerization assays was assessed in an enzymatic reactor. A Minibio 500 bioreactor (Applikon Biotechnology B.V., Delft, The Netherlands) is started with 3 g of amorphous PET (prepared in accordance with WO 2017/198786 to achieve a degree of crystallinity below 20%) and 100 ml of 10 mM potassium phosphate buffer, pH 8, containing 3 mg LC-esterase. The stirring speed is set to 250 rpm using a ship's impeller. The bioreactor is thermostated at 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C or 70°C by immersion in an external water bath. The pH was adjusted to 8 by adding KOH at a concentration of 3 M. Various parameters (pH, temperature, stirring, base addition) were controlled using BioXpert V2.95 software. Every 20 hours, 1.8 g of amorphous PET (obtained in accordance with WO 2017/198786 to achieve a degree of crystallinity below 20%) is added. 500 µl of reaction medium is removed regularly.

The amount of TA, MHET and BHET was determined using HPLC as described in Example 2.3. The amount of EG was determined on an Aminex HPX-87K column (Bio-Rad Laboratories, Inc, Hercules, California, United States) thermostated at 65°C. The eluent is K ₂ HPO ₄ 5 mm at 0.6 ml.min ^-1 . The injection is 20 µl. The ethylene glycol content is monitored using a refractometer.

Hydrolysis fractions are calculated based on the ratio of the molar concentration at a given time (TA +MHET+BHET) to the total amount of TA contained in the original sample, or based on the ratio of the molar concentration at a given time (EG +MHET+2 x BHET) compared with the total amount of EG contained in the original sample. The rate of destruction is calculated in mg of total TA released per hour or mg of total EG per hour.

The half-life of an enzyme is estimated as the incubation time required to obtain a 50% loss in degradation rate.

3.6 Differential scanning fluorimetry (DSF)

DSF is used to assess the thermal stability of the wild type protein (SEQ ID No. 1) and its variants by determining their melting temperature (Tm), the temperature at which half of the protein population unfolds. Protein samples are prepared at a concentration of 14 μM and stored in buffer A consisting of 20 mM Tris HCl pH 8.0, 300 mM NaCl. The stock solution of SYPRO Orange 5000x in DMSO is first diluted to 250x with water. Protein samples are loaded onto a white clear 96-well PCR plate (Bio-Rad, cat. no. HSP9601), where each well contains a final volume of 25 μl. The final concentration of protein and SYPRO Orange dye in each well is 5 µM (0.14 mg/ml) and 10X, respectively. Load volumes per well are as follows: 15 μl Buffer A, 9 μl 14 μM protein solution, and 1 μl 250X diluted Sypro Orange solution. The PCR plates are then sealed with optical grade sealing tape and spun at 2000 rpm for 1 min at room temperature. DSF experiments are then performed using a CFX96 real-time PCR system configured to use 450/490 excitation and 560/580 emission filters. Samples are heated from 25 to 100°C at a rate of 0.3°C/sec. One fluorescence measurement is taken every 0.03 seconds. Melting points were determined from the peak(s) of the first derivatives of the melting curve using Bio-Rad CFX Manager software.

The esterases of SEQ ID No. 1 and the esterases of the invention are then compared based on their Tm values. Due to the high reproducibility between experiments with the same protein from different products, a ΔTm of 0.8°C is considered relevant for comparison of variants. Tm values correspond to the average of at least 3 measurements. Tm esterase SEQ ID No. 1 is assessed at 46.4°C +/-0.2°C, as shown in example 3.6.

The thermal stability of the esterase variant of the invention is shown in Table 5 below, expressed in Tm values and evaluated according to Example 3.6. The increase in Tm compared to the esterase SEQ ID N 1 is indicated in parentheses.

Table 5: Tm of esterase according to the invention

Options Tm V1: N207C+S256C 60.0°С (+13.6°С) V2 : S212I+N207C+S256C+Q93G 57.0°С (+10.6°С) V3 : S212W+N207C+S256C+Q93G 59.6°С (+13.2°С) V4 : S212I+N207C+S256C+Q93G+N220P+S187E 52.1°С (+5.7°С)

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LIST OF SEQUENCES

<110> CARBIOS

<120> NEW ESTERASES AND THEIR APPLICATION

<130> B3033PC00

<160> 2

<170> Patent In version 3.5

<210> 1

<211> 264

<212> PROTEIN

<213> Ideonella sakaiensis

<400> 1

Met Gln Thr Asn Pro Tyr Ala Arg Gly Pro Asn Pro Thr Ala Ala Ser

1 5 10 15

Leu Glu Ala Ser Ala Gly Pro Phe Thr Val Arg Ser Phe Thr Val Ser

20 25 30

Arg Pro Ser Gly Tyr Gly Ala Gly Thr Val Tyr Tyr Pro Thr Asn Ala

35 40 45

Gly Gly Thr Val Gly Ala Ile Ala Ile Val Pro Gly Tyr Thr Ala Arg

50 55 60

Gln Ser Ser Ile Lys Trp Trp Gly Pro Arg Leu Ala Ser His Gly Phe

65 70 75 80

Val Val Ile Thr Ile Asp Thr Asn Ser Thr Leu Asp Gln Pro Ser Ser

85 90 95

Arg Ser Ser Gln Gln Met Ala Ala Leu Arg Gln Val Ala Ser Leu Asn

100 105 110

Gly Thr Ser Ser Ser Pro Ile Tyr Gly Lys Val Asp Thr Ala Arg Met

115 120 125

Gly Val Met Gly Trp Ser Met Gly Gly Gly Gly Ser Leu Ile Ser Ala

130 135 140

Ala Asn Asn Pro Ser Leu Lys Ala Ala Ala Pro Gln Ala Pro Trp Asp

145 150 155 160

Ser Ser Thr Asn Phe Ser Ser Val Thr Val Pro Thr Leu Ile Phe Ala

165 170 175

Cys Glu Asn Asp Ser Ile Ala Pro Val Asn Ser Ser Ala Leu Pro Ile

180 185 190

Tyr Asp Ser Met Ser Arg Asn Ala Lys Gln Phe Leu Glu Ile Asn Gly

195 200 205

Gly Ser His Ser Cys Ala Asn Ser Gly Asn Ser Asn Gln Ala Leu Ile

210 215 220

Gly Lys Lys Gly Val Ala Trp Met Lys Arg Phe Met Asp Asn Asp Thr

225 230 235 240

Arg Tyr Ser Thr Phe Ala Cys Glu Asn Pro Asn Ser Thr Arg Val Ser

245 250 255

Asp Phe Arg Thr Ala Asn Cys Ser

260

<210> 2

<211> 21

<212> PROTEIN

<213> artificial sequence

<220>

<223> PelB leader sequence

<400> 2

Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala Ala

1 5 10 15

Gln Pro Ala Met Ala

20

<---

Claims (16)

1. An esterase that (i) has at least 90%, 95%, 96%, 97%, 98% or 99% identity to the full-length amino acid sequence set forth in SEQ ID NO: 1, (ii) contains, at least a combination of substitutions N207C + S256C, and (iii) has increased polyester degrading activity and/or increased thermal stability compared to the esterase SEQ ID NO: 1. 2. An esterase according to any of the previous paragraphs, characterized in that said esterase additionally contains at least one substitution selected from S212I/W, F175I, S181N, S66T, T62M, N215D/M, Q93G/P, N220P/D , S187E, A14H, T172Q and K226E, preferably selected from Q93G/P, S187E, S212I/W or N220P/D. 3. An esterase according to any of the previous paragraphs, characterized in that said esterase additionally contains at least one amino acid substitution at a position corresponding to residues selected from T13, A14, A15, S16, L17, E18, A19, S20, A21 , G60, Y61, A63, R64, S67, I68, K69, W70, P73, R74, D86, T87, T90, L91, D92, P94, S95, W133, M135, G136, I142, W159, S161, S162, C177 , I182, A183, P184, S187, I192, I206, G208, G209, S210, C213, A214, S216, G217, N218, S219, N220, Q221, A222, L223, I224, G225, K226, K227, T244, 245 , A246, C247, E248, N249, P250, N251, S252, T253, R254, V255 or C263. 4. An esterase according to any of the previous paragraphs, characterized in that said esterase contains at least a combination of substitutions at positions S212I/W+N207C+S256C. 5. Esterase according to claim 4, characterized in that said esterase contains at least one substitution at positions selected from F175I, S181N, S66T, T62M, N215D/M, Q93G/P, S187E, N220P/D, A14H , T172Q and K226E, preferably selected from F175I, S181N, S66T, T62M, N215D/M and Q93G/P. 6. Esterase according to any of the previous paragraphs, characterized in that said esterase contains at least one combination of substitutions selected from the group consisting of S212I/W+N207C+S256C, S212I/W+N207C+S256C+Q93G/P or S212I+N207C+S256C+Q93G+N220P+S187E. 7. An esterase according to any of the previous claims, characterized in that said esterase additionally contains at least one amino acid residue selected from S134, D180 and H211, preferably the combination S134+D180+H211, and/or at least one amino acid residue selected from C177, C213, C247 or C263, preferably the combination C247+C263+C177+C213, as in the parent esterase. 8. Nucleic acid encoding an esterase according to any one of paragraphs. 1-7. 9. Expression cassette for producing esterase according to any one of paragraphs. 1-7, containing the nucleic acid according to claim 8. 10. Host cell for producing esterase according to any one of paragraphs. 1-7, containing the nucleic acid according to claim 8 or the expression cassette according to claim 9. 11. Composition for destruction of materials containing polyesters, containing esterase according to any one of paragraphs. 1-7 or a host cell according to claim 10, and additionally containing excipients and/or reagents. 12. A method for destroying at least one polyester of a polyester-containing material comprising (a) contacting the polyester-containing material with the esterase according to any one of claims. 1-7, or a host cell according to claim 10, or a composition according to claim 11; and, optionally, (b) isolating monomers and/or oligomers. 13. The method according to claim 12, characterized in that the polyester 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), poly(ethylene adipate) (PEA), polyethylene naphthalate (PEN) and mixtures/blends of these materials, preferably polyethylene terephthalate. 14. The composition according to claim 11, characterized in that said composition is a detergent composition.