CN118339284A

CN118339284A - Novel esterases and their use

Info

Publication number: CN118339284A
Application number: CN202280080181.6A
Authority: CN
Inventors: G·里皮恩斯; C·查理尔; M·本·卡勒德; I·安德烈; M·盖鲁特
Original assignee: Carbios SA
Current assignee: Carbios SA
Priority date: 2021-11-16
Filing date: 2022-11-15
Publication date: 2024-07-12

Abstract

The present invention relates to novel esterases and more particularly to esterase variants having improved activity and/or improved thermostability compared to the parent esterases of SEQ ID No. 1 or SEQ ID No. 2, and their use for degrading polyester-containing materials such as plastics products. The esterases of the invention are particularly suitable for degrading polyethylene terephthalate and materials containing polyethylene terephthalate.

Description

Novel esterases and their use

Technical Field

The present invention relates to novel esterases and more particularly to esterases which have improved activity and/or improved thermostability compared to the parent esterase. The invention also relates to the use of the novel esterases for degrading polyester-containing materials such as plastics products. The esterases of the invention are particularly suitable for degrading polyethylene terephthalate and materials containing polyethylene terephthalate.

Background

Esterases are capable of catalyzing the hydrolysis of a wide variety of polymers, including polyesters. In this context, esterases have shown promising results in a variety of industrial applications, including as detergents for dishwashing and laundry applications, as degrading enzymes for the treatment of biomass and food, as biocatalysts for the detoxification of environmental pollutants or for the treatment of polyester fabrics in the textile industry. The use of esterases as degrading enzymes for the hydrolysis of polyethylene terephthalate (PET) is of particular interest. In fact, PET is used in many technical fields, such as the manufacture of clothing, carpets, or in the form of thermosetting resins for the manufacture of packaging or automotive plastics, etc., making PET accumulation in landfills an increasingly serious ecological problem.

Enzymatic degradation of polyesters, especially PET, is considered an interesting solution to reduce the accumulation of plastic and textile waste. In fact, enzymes can accelerate hydrolysis of polyester-containing materials, especially plastics and textile products, even up to monomer levels. In addition, the hydrolysates (i.e., monomers and oligomers) can be recycled as materials for the synthesis of new polymers.

In this context, several esterases have been identified as candidate degrading enzymes for polyesters, and some variants of such esterases have been developed. Among esterases, cutinases, also known as keratolytic enzymes (EC 3.1.1.74), are of particular interest. Keratinase has been identified from various fungi (P.E. Kolattukudy, "Lipases", ed.B. Borg-strom and H.L. Brockman, elsevier 1984, 471-504), bacteria and plant pollen. Recently, metagenomic approaches have led to the identification of additional esterases.

However, there remains a need for esterases with improved activity and/or improved thermostability compared to previously known esterases to provide a more efficient polyester degradation process and thus more competitive.

Disclosure of Invention

The present invention provides novel esterases which exhibit increased activity and/or increased thermostability compared to a parent or wild-type esterase having the amino acid sequence shown in SEQ ID NO. 1. This wild-type esterase corresponds to amino acids 36-293 of the amino acid sequence of the metagenome-derived cutinase described in Sulaiman et al, appl Environ microbiol 2012mar, and is called G9BY57 in SwissProt and is described as having polyester degrading activity. The esterases of the invention are particularly useful in processes for degrading plastic products, especially PET-containing plastic products.

It is therefore an object of the present invention to provide an esterase having (I) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the full-length amino acid sequence shown in SEQ ID NO:1, (ii) at least one amino acid substitution selected from the group consisting of V219E, N204S, N243Y, L V/Q, D158C, T160C, R K/D/E/L, N211F, S13L, A Y and S206N compared to SEQ ID NO:1, and/or having one amino acid substitution at least one amino acid position corresponding to a residue selected from the group consisting of G7, S57, T136, E141, I169, G171, V180, A184, I185, P186, Y188, E201, R234, D249, F250, R251, H77 and L191, wherein the amino acid sequence numbers shown in position SEQ ID NO:1, (iii) having polyester degradation activity, and (iv) exhibiting improved thermal stability and/or improved degradation activity compared to SEQ ID NO: 1.

Preferably, the esterase comprises at least one substitution or combination of substitutions selected from V219E、N204S、N243Y、L15Q、N211F、S13L、A14Y、S206N、V180L/I/C/N/A/T、F250N/L/V/Y/A、L15V+R89L、N204S+N105D、E141C+R138K、R138K and v219e+q182 e+r12e.

It is a further object of the present invention to provide an esterase having (I) at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the full-length amino acid sequence shown in SEQ ID NO:2, (ii) at least one amino acid substitution selected from the group consisting of V219E, N204S, N243Y, L V/Q, D158C, T160C, R K/D/E/L, N211F, S13L, A Y and S206N compared to SEQ ID NO:2, and/or having a polyester degrading activity at least one amino acid substitution at an amino acid position corresponding to a residue selected from the group consisting of G7, S57, T136, E141, I169, G171, V180, A184, I185, P186, Y188, E201, R234, D249, F250, R251, H77 and L191, wherein the positions are numbered with reference to the amino acid sequence shown in SEQ ID NO:2, (iii) exhibiting improved stability and/or improved thermal degradation activity compared to SEQ ID NO: 2. Preferably, the esterase has at least one amino acid substitution selected from the group consisting of V219E, V I/C/N/A/T/L/and F250N/L/V/Y/A, preferably selected from the group consisting of V219E, V180I/C/N/A/T and F250N/L/V/Y/A. In particular, the esterases also exhibit increased thermostability and/or increased polyester degradation activity compared to the esterases of SEQ ID No. 1.

It is another object of the present invention to provide nucleic acids encoding the esterases of the invention. The invention also relates to an expression cassette or expression vector comprising said nucleic acid, and a host cell comprising said nucleic acid, expression cassette or vector.

The invention also provides compositions comprising the esterases of the invention, host cells of the invention, or extracts thereof.

It is a further object of the present invention to provide a method for producing the esterase of the invention, comprising:

(a) Culturing a host cell according to the invention under conditions suitable for expression of a nucleic acid encoding an esterase; and optionally

(B) Recovering the esterase from the cell culture.

A further object of the present invention is to provide a process for degrading polyesters or polyesters containing polyester materials comprising

(A) Contacting the polyester with an esterase according to the invention or a host cell according to the invention or a composition according to the invention; and optionally

(B) Recovering the monomers and/or oligomers.

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

The invention also relates to the use of the esterases of the invention for degrading PET or PET-containing plastic products.

The invention also relates to polyester-containing materials comprising the esterases or host cells or compositions of the invention.

The invention also relates to a detergent composition comprising an esterase or host cell according to the invention or a composition comprising an esterase according to the invention.

Detailed Description

Definition of the definition

The disclosure will be better understood by reference to the following definitions.

Herein, the terms "peptide", "polypeptide", "protein", "enzyme" refer to a chain of amino acids linked by peptide bonds, regardless of the number of amino acids forming the chain. Amino acids are herein denoted by their single-letter or three-letter codes 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 enzymes belonging to the class of hydrolases classified under enzyme nomenclature as EC 3.1.1, which catalyze the hydrolysis of esters into acids and alcohols. The term "cutinase" or "cutinase" refers to an esterase that is classified under enzyme nomenclature as EC 3.1.1.74, capable of catalyzing a chemical reaction of monomers of cutin and water.

The term "wild-type protein" refers to a non-mutated form of a naturally occurring polypeptide. In this case, the wild-type esterase means an esterase having the amino acid sequence shown in SEQ ID NO. 1.

The term "parent protein" refers to a reference polypeptide. In this case, the parent esterase refers to an esterase having the amino acid sequence shown in SEQ ID NO. 1or SEQ ID NO. 2.

The terms "mutant" and "variant" refer to polypeptides derived from SEQ ID NO. 1 or SEQ ID NO. 2 and comprising at least one modification or change (i.e. substitution, insertion and/or deletion) at one or more (e.g. several) positions compared to SEQ ID NO. 1 or SEQ ID NO. 2, respectively, and having polyester degrading activity. Variants may be obtained by various techniques well known in the art. In particular, examples of techniques for altering the DNA sequence encoding a wild-type protein include, but are not limited to, site-directed mutagenesis, random mutagenesis, and synthetic oligonucleotide construction. Thus, the terms "modified" and "altered" as used herein with respect to a particular position refer to an amino acid at that particular position having been modified as compared to the amino acid at that particular position in the wild-type protein.

"Substitution" refers to the replacement of an amino acid residue with another amino acid residue. Preferably, the term "substitution" refers to the replacement of an amino acid residue with another amino acid residue selected from the group consisting of a naturally occurring 20 standard amino acid residue, a rare naturally occurring amino acid residue (e.g., hydroxyproline, hydroxylysine, allophanlysine, 6-N-methyllysine, N-ethylglycine, N-methylglycine, N-ethylasparagine, iso-isoleucine, N-methylisoleucine, N-methylvaline, pyroglutamine, aminobutyric acid, ornithine, norleucine, norvaline), and a non-naturally occurring amino acid residue typically synthetically prepared (e.g., cyclohexyl-alanine). Preferably, the term "substitution" refers to the replacement of an amino acid residue with another residue selected from the group consisting of the 20 standard naturally occurring 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 symbol "+" indicates a permutation combination. In this document, the following terms are used to designate the substitutions: L82A represents the substitution of the amino acid residue (leucine, L) at position 82 of the parent sequence with alanine (A). A121V/I/M represents the substitution of the amino acid residue at position 121 of the parent sequence (alanine, A) with one of the following amino acids: valine (V), isoleucine (I) or methionine (M). Substitutions may be conservative or non-conservative substitutions. Examples of conservative substitutions are within the group 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 amino acid positions disclosed in the present application refer to the amino acid sequence numbers shown in SEQ ID NO. 1.

As used herein, the term "sequence identity" or "identity" refers to the number of matches (identical amino acid residues) between two polypeptide sequences (or expressed as a fraction of percent). Sequence identity is determined by comparing sequences when aligned so as to maximize overlap and identity while minimizing sequence gaps. In particular, any of a variety of mathematical global or local alignment algorithms may be used to determine sequence identity, depending on the length of the two sequences. Sequences of similar length are preferably aligned using global alignment algorithms (e.g., needleman and Wunsch algorithm; needleman and Wunsch, 1970) that optimally align sequences over their entire length, while sequences of significantly different lengths are preferably aligned using local alignment algorithms (e.g., smith and Waterman algorithms (Smith and Waterman, 1981) or Altschul algorithm (Altschul et al, 1997; altschul et al, 2005)). Alignment for the purpose of determining percent amino acid sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using computer software as provided on an Internet website (e.g., http:// blast. Ncbi. Nlm. Nih. Gov/or http:// www.ebi.ac.uk/Tools/empower /). One skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared. For purposes herein,% amino acid sequence identity values refer to values generated using the pairwise sequence alignment program EMBOSS Needle, which uses the Needleman-Wunsch algorithm to establish the optimal global alignment of two sequences, wherein all search parameters are set to default values, i.e., scoring matrix = BLOSUM62, gap open = 11, gap extension = 1.

"Polymer" refers to a compound or mixture of compounds whose structure is made up of multiple monomers (repeating units) linked by covalent chemical bonds. In the context of the present invention, the term "polymer" includes natural or synthetic polymers composed of a single type of repeating unit (i.e. a homopolymer) or a mixture of different repeating units (i.e. a copolymer or heteropolymer). According to the present invention, "oligomer" refers to a molecule comprising from 2 to about 20 monomers.

In the context of the present invention, "polyester-containing material" or "polyester-containing product" refers to a product, such as a plastic product, comprising at least one polyester in crystalline, semi-crystalline or completely amorphous form. In a particular embodiment, polyester-containing material refers to any article made of at least one plastic material, such as plastic sheets, tubes, rods, profiles, shapes, films, chunks, fibers, etc., comprising at least one polyester and possibly other substances or additives, such as plasticizers, minerals or organic fillers. In another particular embodiment, polyester-containing material refers to a molten or solid plastic compound or plastic formulation suitable for making plastic products. In another particular embodiment, the polyester-containing material refers to a textile, fabric or fiber comprising at least one polyester. In another particular embodiment, polyester-containing material refers to plastic waste or fibrous waste comprising at least one polyester.

In this specification, the term "polyester" includes, but is not limited to, polyethylene terephthalate (PET), polypropylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isoparaffinate (PEIT), polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furandicarboxylate (PEF), polycaprolactone (PCL), poly (ethylene adipate) (PEA), polyethylene naphthalate (PEN), and blends/mixtures of these polymers. Polyesters may also include "polyolefin-like" polyesters, preferably "polyethylene-like" polyesters, which correspond to polyolefins (preferably polyethylenes) into which ester segments have been incorporated (typically by polycondensation of long chain alpha, omega-difunctional monomers), as defined in Lebarb e et al GREEN CHEMISTRY Issue 42014.

New esterases

The present invention provides novel esterases which have improved activity and/or improved thermostability compared to the parent esterase. More particularly, the inventors have devised novel enzymes that are particularly suitable for use in industrial processes. The esterases of the invention are particularly useful for degrading polyesters, more particularly PET, including PET-containing materials, especially PET-containing plastic products. In particular embodiments, the esterase exhibits increased activity and increased thermostability.

It is therefore an object of the present invention to provide esterases which exhibit an increased activity compared to esterases having the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2 (also referred to as parent esterases).

In particular, the inventors identified the specific amino acid residue in SEQ ID NO. 1, which is intended to be in contact with a polymeric substrate in the X-ray crystal structure (i.e., folded 3D structure) or MNR structure of an esterase, which may advantageously be modified to facilitate contact of the substrate with the esterase and which advantageously results in increased adsorption of the polymer and/or thereby increased activity of the esterase on the polymer.

In the context of the present invention, the term "increased activity" or "increased degradation activity" means that under given conditions (e.g. temperature, pH, concentration) the ability of an esterase of SEQ ID NO:1 or SEQ ID NO:2 to degrade and/or adsorb onto the same polyester is increased compared to the ability of an esterase to degrade and/or adsorb onto the same polyester under the same conditions. Such an increase in activity may be at least 10%, preferably at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130% or more higher than the polyester degrading activity of the esterase of SEQ ID NO. 1 or SEQ ID NO. 2. In particular, the degradation activity is the depolymerization activity of the monomers and/or oligomers that produce the polyester, which can be further recovered and optionally reused. In particular, the esterases of the invention have increased PET degradation activity.

The "degradation activity" of esterases can be assessed by the person skilled in the art 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 esterases. The "specific degradation activity" of the esterase on PET corresponds to the μmol/min of hydrolyzed PET or mg/hr of equivalents TA produced per milligram of esterase during the initial phase of the reaction (i.e., the first 24 hours) and is determined from the linear portion of the hydrolysis reaction curve established by multiple samplings made at different times during the first 24 hours. As another example, "degradation activity" can be assessed by measuring the rate and/or yield of oligomers and/or monomers released under suitable temperature, pH and buffer conditions after a defined period of time (e.g., after 24 hours, 48 hours, 72 hours or 96 hours) when the polymer or polymer-containing plastic product is contacted with a degrading enzyme.

The person skilled in the art can evaluate the ability of an enzyme to adsorb onto a substrate according to methods known per se in the art. For example, the ability of an enzyme to adsorb onto a substrate may be measured from a solution containing the enzyme, and wherein the enzyme has been previously incubated with the substrate under suitable conditions.

The inventors have also identified the amino acid residues of interest in the esterases of SEQ ID No. 1 or SEQ ID No.2, which can be advantageously modified to improve the stability (i.e.increased thermostability) of the corresponding esterases at elevated temperatures, and advantageously at 50℃or more and at 90℃or less, preferably at 60℃or more and at 80℃or less, more preferably at 65℃or more and at 75℃or less.

It is therefore an object of the present invention to provide novel esterases which exhibit improved thermostability compared to the thermostability of esterases having the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2 (i.e.the parent esterase).

In the context of the present invention, the term "improved thermostability" indicates an improved ability of the esterase to resist chemical and/or physical structural changes at elevated temperatures, in particular at temperatures of 50℃to 90℃compared to the esterase of SEQ ID NO. 1 or SEQ ID NO. 2. In particular embodiments, the thermostability of the esterase is improved at a temperature between 50 ℃ and 90 ℃, between 50 ℃ and 80 ℃, between 50 ℃ and 75 ℃, between 50 ℃ and 65 ℃, between 55 ℃ and 90 ℃, between 55 ℃ and 80 ℃, between 55 ℃ and 75 ℃, between 55 ℃ and 70 ℃, between 55 ℃ and 65 ℃, between 60 ℃ and 90 ℃, between 60 ℃ and 80 ℃, between 60 ℃ and 75 ℃, between 60 ℃ and 70 ℃, between 60 ℃ and 65 ℃, between 65 ℃ and 90 ℃, between 65 ℃ and 80 ℃, between 65 ℃ and 75 ℃, between 65 ℃ and 70 ℃ as compared to the thermostability of the parent esterase. Preferably, the thermostability of the esterase is improved at a temperature of at least between 50 ℃ and 65 ℃ compared to the thermostability of the parent esterase. In the context of the present invention, the temperature is given as +/-1 ℃.

In particular, thermostability may be assessed by assessing the melting temperature (Tm) of the esterase. In the context of the present invention, the "melting temperature" refers to the temperature at which half of the enzyme population under consideration unfolds or misfoldes. Typically, the esterases of the invention exhibit an increase in Tm of about 0.8 ℃,1 ℃,2 ℃,3 ℃,4 ℃,5 ℃, 10 ℃ or more compared to the Tm of the parent esterase. In particular, the esterases of the invention may have an increased half-life at temperatures of 50 ℃ to 90 ℃ compared to the parent esterase. In particular, the esterases of the invention may have an increased half-life compared to the esterases of SEQ ID NO:1 at temperatures of 50℃to 90 ℃, 50℃to 80 ℃, 50℃to 75 ℃, 50℃to 70 ℃, 50℃to 65 ℃, 55℃to 90 ℃, 55℃to 80 ℃, 55℃to 75 ℃, 55℃to 70 ℃, 55℃to 65 ℃, 60℃to 90 ℃, 60℃to 75 ℃, 60℃to 70 ℃, 60℃to 65 ℃, 65℃to 90 ℃, 65℃to 80 ℃, 65℃to 75 ℃, 65℃to 70 ℃. Preferably, the esterases of the invention have an increased half-life over the parent esterase at least at a temperature of 50 ℃ to 65 ℃.

The melting temperature (Tm) of the esterase can be measured by a person skilled in the art according to methods known per se in the art. For example, DSF can be used to quantify the change in the temperature of thermal denaturation of esterases, thereby determining their Tm. Alternatively, tm can be assessed by analyzing protein folding using circular dichroism. Preferably, tm is measured using DSF or circular dichroism as disclosed in the experimental section. In the context of the present invention, a comparison of Tm is made with Tm measured under the same conditions (e.g., pH, properties and amounts of polyester, etc.).

Alternatively, thermostability may be assessed by measuring esterase activity and/or polyester depolymerization activity of the esterase after incubation at different temperatures and comparing with the esterase activity and/or polyester depolymerization activity of the parent esterase. The ability to conduct multiple rounds of polyester depolymerization assays at different temperatures can also be assessed. A quick and valuable test may involve assessing the ability of esterases to degrade solid polyester compounds dispersed in agar plates after incubation at different temperatures by halo diameter (halo diameter) measurement.

It is an object of the present invention to provide an esterase having (I) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the full-length amino acid sequence shown in SEQ ID NO:1, (ii) at least one amino acid substitution at least one amino acid position corresponding to residues selected from the group consisting of G7, S57, T136, E141, I169, G171, V180, A184, I185, P186, Y188, E201, R234, D249, F250, R251, H77 and L191, wherein the positions are numbered with reference to the amino acid sequence shown in SEQ ID NO:1, (iii) having polyester degradation activity and/or exhibiting increased stability and/or increased thermal degradation activity compared to SEQ ID NO: 1.

In one embodiment, the esterase comprises at least one amino acid substitution selected from V219E, N204S, N243Y, L V/Q, R138K, N211F, S13L, A Y and S206N and/or one amino acid substitution at least one amino acid position corresponding to a residue selected from G7, S57, T136, I169, G171, V180, a184, I185, P186, Y188, E201, R234, D249, F250 and R251.

In specific embodiments, the esterase comprises at least one amino acid substitution selected from V219E, N204,204, 204S, N243,243Y, L V/Q, R138,138, 138K, N211,211, 211F, S13, 13L, A14Y and S206N and/or one amino acid substitution at least one amino acid position corresponding to a residue selected from V180 and F250.

In particular, the esterase comprises at least one substitution at a position selected from G7, S57, T136, E141, I169, G171, V180, a184, I185, P186, Y188, E201, R234, D249, F250 and R251, preferably selected from G7, S57, T136, I169, G171, V180, a184, I185, P186, Y188, E201, D249, F250 and R251. More preferably, the esterase comprises at least one amino acid substitution at a position selected from V180 and F250.

In one embodiment, the esterase comprises at least one amino acid substitution selected from V219E、N204S、N243Y、L15V/Q、D158C、T160C、R138K/D/E/L、E141C/K/R、G171C、N211F、S13L、A14Y、S206N、V180I/C/N/A/T/L and F250N/L/V/Y/A. Preferably, the esterase comprises at least one amino acid substitution selected from the group consisting of V219E, N204S, N243Y, L V/Q, R138K, E141C, N211F, S13L, A14Y, S206N, V I/C/N/A/T and F250N/L/V/Y/A, more preferably selected from the group consisting of V219E, N204S, N243Y, L V/Q, R138K, E141C, N211F, S13L, A Y and S206N, even more preferably selected from the group consisting of V219E, N243Y, L Q, N211F, S3213L, A14Y, S N and R138K.

For example, the esterase comprises at least one amino acid substitution selected from V219E, N, S, N243Y and L15V, more preferably from V219E, N S and L15V, even more preferably the substitution V219E. Or the esterase comprises at least one amino acid substitution selected from the group consisting of L15Q, N211F, S, L, A Y and S206N, preferably S13L.

Alternatively or additionally, the esterase may comprise at least one amino acid substitution selected from the group consisting of D158C, T160C, R K/D/E, E141C/K/R, G171C and V180C.

Alternatively or additionally, the esterase may comprise at least one amino acid substitution selected from V180I/C/N/A/T/L and F250N/L/V/Y/A, preferably selected from V180I/C/N/A/T and F250N/L/V/Y.

In one embodiment, the esterase comprises at least two substitutions selected from V219E, N204S, N243Y, L15V, D C, T C and R138K/D/E and/or at positions selected from G7, S57, T136, E141, I169, G171, V180, a184, I185, P186, Y188, E201, R234, D249, F250, R251, H77 and L191, preferably at least two substitutions selected from V219E, N204S, N243Y, L V and/or at positions selected from G7, S57, T136, I169, G171, V180, a184, I185, P186, Y188, E201, R234, D249, F250 and R251.

In particular, the esterase comprises at least two substitutions selected from the group consisting of V219E, N, S, N243Y, L15V, D, C, T160C, R K/D/E, E141C/K/R, G171C and V180C.

For example, the esterase comprises at least one substitution combination at a position selected from the group consisting of E141+D158, E141+T160, E141+R138, D158+T160 and G171+V180, preferably at least one substitution combination selected from the group consisting of 141C/K/R+D158C, E C/K/R+T160C, E C/K/R+R138K/D/E, D158 8C+T160C and G171C+V180C, more preferably selected from the group consisting of E141C+D158C, E141C+T160C, E C+R138K/D/E, D158C+T160C and G171C+V180C, even more preferably R39K+E141C.

According to the invention, the esterase may further comprise at least one substitution at least one position selected from S1、Y4、Q5、R6、N9、P10、T11、R12、S13、A14、L15、T16、A17、D18、S22、T25、Y26、T27、V28、S29、R30、L31、S32、V33、S34、G35、F36、G37、G38、G39、Y43、S48、T50、G53、I54、M56、P58、G59、Y60、T61、A62、D63、A64、S65、S66、L67、A68、W69、L70、R72、R73、L74、L82、I84、N85、T86、N87、S88、R89、F90、D91、Y92、P93、D94、S95、R96、S98、Q99、A103、L104、N105、L107、R108、S113、L119、A121、N122、L124、A125、A127、G128、H129、M131、G132、G133、G134、G135、R138、A140、N143、S145、K147、A149、V150、L152、T153、P154、W155、H156、T157、D158、K159、T160、N162、S164、V167、L168、V170、A172、E173、A174、T176、V177、A178、P179、S181、Q182、H183、F187、Q189、N190、S193、T194、P196、V198、V200、L202、D203、N204、A205、S206、F208、A209、P210、N211、S212、N213、N214、A215、A216、I217、S218、V219、Y220、T221、S223、W224、M225、N231、T233、R236、Q237、F238、L239、N241、V242、N243、D244、P245、A246、L247、S248、T252、N253、N254、R255、H256、Q258、F161 and T163, preferably at least one position selected from Y4、T50、M56、P58、G59、Y60、Y61、A62、D63、A64、I84、N85、T86、Y92、H129、M131、G132、G133、G134、G135、K147、T153、P154、W155、H156、T157、D158、K159、T160、F161、N162、T163、A172、E173、A174、T176、V177、A178、P179、Q182、H183、N190、V200、L202、D203、N204、A205、S206、F208、A209、P210、N211、S212、N213、N214、A215、M225、T233、S248、N254、H256、T11、R12、S13、A14、T16、D18、S65、S66、A68、W69、R73、R89、F90、D91、P93、D94、N105、A216、I217、S218、Y220、Q237、F238、L239、N241、V242、N243、D244、A246、L247、V167 and V170.

For example, the esterase further comprises at least one, preferably at least two, at least three or at least four substitutions at positions selected from F208, D203, S248, V170, Y92, G135, V167, Q182 and N213. In particular, the esterase further comprises at least one substitution selected from F208G/N/R/I/A/Q/L/S/M/T/E/W, D C/K/R, S248C, V170I, Y A/G/P/N/Q/T/F/C/D, G135A, V Q/T, Q182D/E and N213D/E/R/K/P, preferably selected from F208I/W, D203C, S C, V170I, Y92G, G A, V167Q, Q E and N213P.

According to the invention, the esterase may further comprise a substitution combination at position d203+s248, preferably the substitution combination d203c+s248C.

According to the invention, the esterase may further comprise at least one substitution selected from D203K/R and at least amino acid residue S248 as in the parent esterase (i.e., the esterase of SEQ ID NO: 1).

Or the esterase may further comprise a substitution combination at least at positions f208+d203+s248, preferably selected from the group consisting of f208 i+d217c+s248C or f208 w+d217c+s248C.

As an example, the esterase further comprises a combination of substitutions at least at positions f208+d203+s248 and one or two substitutions at positions selected from Y92, G135, V167, V170, Q182 and N213. In particular, the esterase comprises at least one substitution combination selected from F208 I+D21c+S248C or F208 W+D21c+S248C and one substitution selected from Y92A/G/P/N/Q/T/F/C/D, G135A, V167Q/T, V170 56182E/D and N213D/E/R/K/P, preferably selected from V170I, Y92G, G A, V167Q, V I, Q E and N213P.

In one embodiment, the esterase further comprises a substitution combination selected from at least D203C+S248C、F208G/N/R/I/A/Q/L/S/M/T/E/W+D203C+S248C、F208G/N/R/I/A/Q/L/S/M/T/E/W+D203C+S248C+V170I、F208G/N/R/I/A/Q/L/S/M/T/E/W+D203C+S248C+V170I+Y92D/E/G、F208G/N/R/I/A/Q/L/S/M/T/E/W+D203C+S248C+V170I+Y92D/E/G+N213P+Q182E、F208G/N/R/I/A/Q/L/S/M/T/E/W+D203K/R、F208G/N/R/I/A/Q/L/S/M/T/E/W+D203K/R+V170I、F208G/N/R/I/A/Q/L/S/M/T/E/W+D203K/R+V170I+Y92D/E/G and F208G/N/R/I/A/Q/L/S/M/T/E/W+D217K/R+V170I+Y92D/E/G+N219P+Q182E, preferably from D203C+S248C、F208I/W/M+D203C+S248C、F208I/W/M+D203C+S248C+V170I、F208I/W/M+D203C+S248C+V170I+Y92D/E/G、F208I/W/M+D203C+S248C+V170I+Y92D/E/G+N213P+Q182E、F208I/W+D203K/R+V170I、F208I/W/M+D203K/R+V170I+Y92D/E/G and F208I/W/M+D21K/R+V170I+Y92D/E/G+N219P+Q182E, more preferably from D203C+S248C、F208I/M+D203C+S248C、F208I/M+D203C+S248C+V170I、F208I/M+D203C+S248C+V170I+Y92G、F208I/M+D203C+S248C+V170I+Y92G+N213P+Q182E.

Alternatively or additionally, the esterase may further comprise at least one substitution at a position selected from T11、R12、S13、A14、T16、D18、G59、Y60、T61、A62、D63、S65、S66、A68、W69、R72、R73、R89、F90、D91、P93、D94、N105、H129、W155、T157、D158、T176、V177、A178、P179、L202、S206、P210、N211、S212、N214、A215、A216、I217、S218、Y220、Q237、F238、L239、N241、V242、N243、D244、A246 and L247, preferably from T11, R12, S13, a14, T16, W69, R73, a216, S218, Y220, Q237, F238, V242, N243, D244, a246 and L247. Preferably, the esterase comprises at least one substitution selected from T11Q/E/N、R12D/Q/G/E、S13D、A14D、T16E、W69E/D/M、R73E/G/M/Q、R89T/F/H/Q/L/K、N105D/E/R/K、A216Q、S218A/E、Y220F、Q237D、F238E、V242Y、N243P、D244E/C、A246E and L247T.

According to the invention, the esterase may further comprise at least one substitution selected from R89T/F/H/Q/L/K and N105D/E/R/K, preferably from R89L and N105D. In one embodiment, the esterase comprises at least one substitution combination selected from the group consisting of L15V+R89T/F/H/Q/L/K and N217S+N105D/E/R/K, preferably selected from the group consisting of L15V+R89L and N217S+N105D. In one embodiment, the esterase comprises a substitution combination selected from at least F208G/N/R/I/A/Q/L/S/M/T/E+D203C+S248C+V170I+Y92D/E/G+V219E、F208G/N/R/I/A/Q/L/S/M/T/E+D203C+S248C+V170I+Y92D/E/G+V219E+Q182D/E、F208G/N/R/I/A/Q/L/S/M/T/E+D203C+S248C+V170I+Y92D/E/G+V219E+N213P/L/D、F208G/N/R/I/A/Q/L/S/M/T/E+D203C+S248C+V170I+Y92D/E/G+V219E+R12D/N/Q/E、F208G/N/R/I/A/Q/L/S/M/T/E+D203C+S248C+V170I+Y92D/E/G+V219E+Q182D/E+R12D/N/Q/E、F208G/N/R/I/A/Q/L/S/M/T/E+D203C+S248C+V170I+Y92D/E/G+V219E+Q182D/E+N213P/L/D、F208G/N/R/I/A/Q/L/S/M/T/E+D203C+S248C+V170I+Y92D/E/G+V219E+Q182D/E+N213P/L/D+R12D/N/Q/E、F208G/N/R/I/A/Q/L/S/M/T/E+D203K/R+V170I+Y92D/E/G+V219E、F208G/N/R/I/A/Q/L/S/M/T/E+D203K/R+V170I+Y92D/E/G+V219E+Q182D/E、F208G/N/R/I/A/Q/L/S/M/T/E+D203K/R+V170I+Y92D/E/G+V219E+N213P/L/D、F208G/N/R/I/A/Q/L/S/M/T/E+D203K/R+V170I+Y92D/E/G+V219E+R12D/N/Q/E、F208G/N/R/I/A/Q/L/S/M/T/E+D203K/R+V170I+Y92D/E/G+V219E+Q182D/E+R12D/N/Q/E、F208G/N/R/I/A/Q/L/S/M/T/E+D203K/R+V170I+Y92D/E/G+V219E+Q182D/E+N213P/L/D and F208G/N/R/I/A/Q/L/S/M/T/E+D217K/R+V170I+Y92D/E/G+V217E+Q182D/E+N213P/L/D+R12D/N/Q/E, preferably from F208I/M+D203C+S248C+V170I+Y92G、F208I+D203C+S248C+V170I+Y92G+V219E、F208I/M+D203C+S248C+V170I+Y92G+V219E+Q182E、F208I/M+D203C+S248C+V170I+Y92G+V219E+N213D、F208I/M+D203C+S248C+V170I+Y92G+V219E+R12E、F208I/M+D203C+S248C+V170I+Y92G+V219E+Q182E+R12E、F208I/M+D203C+S248C+V170I+Y92G+V219E+Q182E+N213D and F208I/M+D217C+V217I+Y92G+V217E+Q217E+N217E+N7D+R 12E.

In one embodiment, the esterase comprises at least one amino acid substitution or a combination of substitutions selected from V219E、N204S、N243Y、L15V/Q、N211F、S13L、A14Y、S206N、V180L/I/C/N/A/T、F250N/L/V/Y/A、L15V+R89L、N204S+N105D、E141C+D158C、E141C+T160C、E141C+R138K/D/E/L、D158C+T160C、G171C+V180C、R138K/D/E/L and v219e+q182e+r12e, preferably at least one substitution or combination of substitutions selected from V219E、N243Y、L15Q、N211F、S13L、A14Y、S206N、V180L/I/C/N/A/T、F250N/L/V/Y/A、L15V+R89L,N204S+N105D、E141C+R138K、R138K and v219e+q182 e+r12e.

In particular, the esterase comprises at least one substitution or combination of substitutions selected from the group consisting of V219E, L V+R89T/F/H/Q/L/K and N204S+N105D/E/R/K, preferably selected from the group consisting of V219E, L V+R89L and N204 S+N105D.

In one embodiment, the esterase comprises at least substitution V219E or at least substitution combination n204s+n105d and exhibits increased polyester degrading activity at a pH between pH 6 and 9, preferably at a pH between pH 6.5 and 9, more preferably at a pH between 7 and 9, even more preferably at a pH between 7.5 and 9, for example at pH 8.

In particular, the esterase comprises at least one amino acid substitution selected from the group consisting of V219E, L15Q, N211F, S13L, A14Y, S35206N, V L/I/C/N, F250N/L/V/Y and R138K or at least a combination of substitutions selected from the group consisting of r390k+e141C and v219e+q182e+r12e and exhibits an increased specific degradation activity compared to the esterase of SEQ ID NO:1, in particular at a pH between pH 6 and 9, preferably at a pH between pH 6.5 and 9, more preferably at a pH between 7 and 9, even more preferably at a pH between 7.5 and 9, for example at pH 8.

Or the esterase comprises at least the amino acid substitution N243Y, F L/A, V180A/T or at least the substitution combination n204s+n105d and exhibits an increased depolymerization yield of PET after 24h or 96h compared to the esterase of SEQ ID NO:1, in particular at a pH between pH 6 and 9, preferably at a pH between pH 6.5 and 9, more preferably at a pH between 7 and 9, even more preferably at a pH between 7.5 and 9, for example at pH 8. According to one embodiment, the esterase comprises at least the amino acid substitution N243Y or at least the substitution combination N204S+N105D and shows an increased PET depolymerization yield after 96 hours compared to the esterase of SEQ ID NO: 1.

In another embodiment, the esterase comprises at least the substitution combination l15v+r89l and exhibits increased thermostability, particularly at a pH between pH 6 and 9, preferably at a pH between pH 6.5 and 9, more preferably at a pH between 7 and 9, even more preferably at a pH between 7.5 and 9, for example at pH 8.

In a particular embodiment, the esterase has the amino acid sequence shown in SEQ ID NO.1, having 1 to 17 substitutions at positions selected from the group consisting of G7, S57, T136, I169, G171, V180, A184, I185, P186, Y188, E201, R234, D249, F250, R251, H77 and L191, preferably having 1 to 15 substitutions at positions selected from the group consisting of G7, S57, T136, I169, G171, V180, A184, I185, P186, Y188, E201, R234, D249, F250 and R251.

In a particular embodiment, the esterase has the amino acid sequence shown in SEQ ID NO. 1, with a single amino acid substitution at a position selected from the group consisting of G7, S57, T136, I169, G171, V180, A184, I185, P186, Y188, E201, R234, D249, F250, R251, H77 and L191, preferably at a position selected from the group consisting of G7, S57, T136, I169, G171, V180, A184, I185, P186, Y188, E201, R234, D249, F250 and R251.

In one embodiment, the esterase consists of the amino acid sequence shown in SEQ ID NO.1, with one to three substitutions selected from V219E, N S and L15V, preferably with the single amino acid substitution V219E.

In another embodiment, the esterase consists of the amino acid sequence shown in SEQ ID NO.1, with a substitution combination selected from the group consisting of L15V+R89L and N204 S+N105D.

In another embodiment, the esterase consists of the amino acid sequence shown in SEQ ID NO. 1, with a substitution or combination of substitutions selected from V219E、N243Y、L15Q、N211F、S13L、A14Y、S206N、V180L/I/C/N/A/T、F250N/L/V/Y/A、L15V+R89L、N204S+N105D、E141C+R138K、R138K and V219E+Q182 E+R12E.

According to the invention, the esterase preferably comprises at least one amino acid residue selected from S130, D175, H207, C240 or C275 as in the parent esterase of SEQ ID NO.1, i.e.the esterase of the invention is not modified at one, two, three, etc. of these positions, or at all of these positions. Preferably, the esterase comprises the combination S130+D175+H2207 in the esterase as shown in SEQ ID NO. 1.

In particular, the esterase may comprise at least the amino acids S130, D175 and H207 and/or the disulfide-forming amino acids C240 and C275 as in the parent esterase, which form the catalytic site of the esterase. Preferably, the esterase comprises a combination of at least the amino acid residues selected from the group consisting of s130+d175+h207, c240+c275 and s130+d175+h207+c240+c275 as in the parent esterase.

In the context of the present invention, the amino acid sequence consisting of SEQ ID NO. 1 with the substitution combination F218I+D217C+S364C+V170I+Y92G is referred to as SEQ ID NO. 2. In contrast to SEQ ID NO. 1, all variants described by reference to SEQ ID NO. 2 must contain the substitution combination F217I+D216C+S248 C+V170I+Y92G.

It is a further object of the present invention to provide an esterase having (I) at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the full length amino acid sequence shown in SEQ ID NO:2, (ii) at least one amino acid substitution selected from the group consisting of V219E, N204S, N243Y, L V/Q, D158C, T160C, R K/D/E/L, N211F, S13L, A Y and S206N compared to SEQ ID NO:2, and/or one amino acid substitution at least one amino acid position corresponding to the residues selected from the group consisting of G7, S57, T136, E141, I169, G171, V180, A184, I185, P186, Y188, E201, R234, D249, F250, R251, H77 and L191, wherein the positions are numbered by reference to the amino acid sequence shown in SEQ ID NO:2, (iii) having polyester activity, and (iv) exhibiting improved stability and/or improved thermal degradation activity compared to SEQ ID NO: 2.

In the context of the present invention, variants of the esterase of SEQ ID NO. 2 having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the full-length amino acid sequence shown in SEQ ID NO. 2 always comprise the residue combination C203+C248+G92. That is, any sequence variation within the% identity described above does not affect this residue combination. In addition, the variant further comprises one or both of residues I208 and I170. In particular embodiments, the variant always comprises a combination of residues i208+c203+c248+i170+g92.

Preferably, the esterase exhibits increased thermostability and/or increased polyester degrading activity compared to the esterase of SEQ ID NO:2 and optionally to the esterase of SEQ ID NO:1 at a pH between pH 6 and 9, preferably at a pH between pH 6.5 and 9, more preferably at a pH between 7 and 9, even more preferably at a pH between 7.5 and 9, e.g. at pH 8.

In addition, the esterase exhibits increased thermostability and/or increased polyester degradation activity compared to the esterase of SEQ ID NO. 2 and optionally to the esterase of SEQ ID NO. 1 at a temperature of 50℃to 90 ℃, preferably 50℃to 72 ℃, more preferably 50℃to 65 ℃.

Advantageously, the esterase further exhibits increased thermostability and/or increased polyester degradation activity compared to the esterase of SEQ ID NO. 1.

In one embodiment, the esterase has at least one amino acid substitution selected from V219E, V I/C/N/A/T/L/and F250N/L/V/Y/A, preferably selected from V219E, V I/C/N/A/T and F250N/L/V/Y/A, more preferably selected from V180I/C/N/A/T and F250N/L/V/Y/A, and exhibits increased thermostability and/or increased polyester degradation activity compared to the esterase of SEQ ID NO. 2. In one embodiment, the esterase has at least one amino acid substitution selected from V180I/C/N and F250N/V/Y/L and exhibits increased specific degradation activity compared to the esterase of SEQ ID NO. 2. Or the esterase has at least one amino acid substitution selected from V180A/T and F250A/L and exhibits an increased yield of PET depolymerization compared to the esterase of SEQ ID NO. 2.

In one embodiment, the esterase has at least one amino acid substitution selected from V219E, Q D/E and R12D/N/Q/E and exhibits increased thermostability and/or increased polyester degradation activity compared to the esterase of SEQ ID NO. 2.

For example, the esterase has a substitution combination selected from at least V219E+Q182D/E+R12D/N/Q/E, preferably the combination V219E+Q182E+R12E. Advantageously, the esterase exhibits increased polyester degrading activity compared to the esterase of SEQ ID NO. 2.

According to the invention, the esterase may comprise at least one amino acid residue selected from S130, D175, H207, C240 or C275 as in the parent esterase of SEQ ID NO. 2, i.e.the esterase of the invention is not modified at one, two, three positions, etc. or at all of these positions.

Specifically, the esterase comprises at least amino acids S130, D175 and H207 forming the catalytic site of the esterase as shown in SEQ ID NO. 2 and/or amino acids C240 and C275 forming disulfide bonds and/or amino acids C203 and C248 forming disulfide bonds in the esterase as shown in SEQ ID NO. 2. Preferably, the esterase comprises a combination of at least the amino acid residues selected from the group consisting of S130+D175 +H2207, C240+C275, S130+D175 +H2207 +C240+C275 and S130+D175 +H217 +C240+C275+C203+C248 in the esterase as set forth in SEQ ID NO 2. In addition, the esterase comprises at least amino acids I208, I170 and G92 as in the parent esterase. In preferred embodiments, the esterase comprises at least the amino acids S130, D175, H207, C240, C275, C203, C248, I208, C203, C248, I170, and G92 as in the parent esterase.

In another embodiment, the esterase consists of the amino acid sequence shown in SEQ ID NO. 2, with a substitution or combination of substitutions selected from V219E, V I/C/N/A/T/L/and F250N/L/V/Y/A, preferably selected from V219E, V I/C/N/A/T and F250N/L/V/Y/A, more preferably selected from V180I/C/N/A/T and F250N/L/V/Y/A.

Advantageously, the esterase variants of the invention exhibit improved thermostability and/or improved polyester degradation activity 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, even more preferably at a pH between 7 and 9, compared to the esterase of SEQ ID NO.1 and/or SEQ ID NO. 2. In particular, the esterase variants of the invention exhibit increased thermostability and/or increased polyester degradation activity at pH 8 compared to the parent esterase (i.e., SEQ ID NO:1 or SEQ ID NO: 2).

Variant polyester degradation Activity

It is an object of the present invention to provide novel enzymes having esterase activity. In a particular embodiment, the enzyme of the invention exhibits a keratinase activity.

Advantageously, the esterases of the invention have polyester degrading activity, preferably polyethylene terephthalate (PET) degrading activity, and/or polybutylene adipate terephthalate (PBAT) degrading activity and/or Polycaprolactone (PCL) degrading activity and/or polybutylene succinate (PBS) activity, more preferably polyethylene terephthalate (PET) degrading activity, and/or polybutylene adipate terephthalate (PBAT) degrading activity. Even more preferably, the esterases of the invention have polyethylene terephthalate (PET) degrading activity.

Advantageously, the esterases of the invention exhibit polyester degrading activity in a temperature range of from 20 ℃ to 90 ℃, preferably from 30 ℃ to 90 ℃, more preferably from 40 ℃ to 90 ℃, more preferably from 50 ℃ to 90 ℃,54 ℃ to 90 ℃, even more preferably from 60 ℃ to 90 ℃,68 ℃ to 90 ℃. In particular, the esterases of the invention exhibit polyester degrading activity in the temperature range of 68 ℃ to 90 ℃,65 ℃ to 85 ℃,65 ℃ to 80 ℃,70 ℃ to 90 ℃,70 ℃ to 85 ℃,70 ℃ to 80 ℃. In particular, the esterases of the invention exhibit polyester degrading activity at temperatures between 40℃and 80℃and preferably between 50℃and 72℃and more preferably between 50℃and 65 ℃. In one embodiment, the esterases of the invention exhibit polyester degrading activity at temperatures between 55℃and 60 ℃, between 50℃and 55 ℃, between 55℃and 65 ℃, between 60℃and 72 ℃, between 60℃and 70 ℃. In particular embodiments, the esterase exhibits polyester degrading activity at least at 50 ℃,54 ℃, 60 ℃,65 ℃,68 ℃ or 70 ℃. Advantageously, the polyester degradation activity is still measurable at temperatures between 55 ℃ and 70 ℃. In the context of the present invention, the temperature is given as +/-1 ℃.

According to the invention, the esterases of the invention may have an increased polyester degrading activity at a given temperature compared to the parent esterases of SEQ ID NO. 1 and/or SEQ ID NO. 2, more particularly at a temperature of from 40℃to 90℃and more preferably at a temperature of from 50℃to 90 ℃. Advantageously, the esterases of the invention have increased polyester degrading activity compared to the esterases of SEQ ID NO. 1 and/or SEQ ID NO. 2 over the entire temperature range from 40℃to 90℃from 40℃to 80℃from 40℃to 70℃from 50℃to 70℃from 55℃to 70℃from 60℃to 70℃from 65℃to 75℃from 65℃to 80℃from 65℃to 90℃from 68℃to 80℃from 72℃to 90℃from 72℃to 85 ℃. In particular, the esterases of the invention exhibit increased polyester degrading activity at temperatures between 40 ℃ and 80 ℃, preferably between 50 ℃ and 72 ℃, more preferably between 50 ℃ and 65 ℃. In one embodiment, the esterases of the invention exhibit increased polyester degradation activity at temperatures between 55 ℃ and 60 ℃, between 50 ℃ and 55 ℃, between 55 ℃ and 65 ℃, between 60 ℃ and 72 ℃, and between 60 ℃ and 70 ℃. More particularly, the esterases of the invention exhibit increased polyester degrading activity at least 50 ℃,54 ℃,60 ℃,65 ℃ or 68 ℃, preferably at 54 ℃ and/or 60 ℃. Advantageously, the polyester degrading activity of the esterase is at least 5%, preferably at least 10%, 20%, 50%, 100% or more higher than the polyester degrading activity of the esterase of SEQ ID NO. 1 and/or SEQ ID NO. 2.

Preferably, the polyester degrading activity of the esterase at 65℃is at least 5%, preferably at least 10%, 20%, 30%, 50%, 100% or more higher than the polyester degrading activity of the esterase of SEQ ID NO:1 and/or SEQ ID NO: 2.

Advantageously, the esterases of the invention exhibit a measurable esterase activity at least in the pH range of 5-11, preferably in the pH range of 6-9, more preferably in the pH range of 6.5-9, even more preferably in the pH range of 6.5-8, even more preferably in the pH range of 7-9, in particular at pH 8.

Nucleic acids, expression cassettes, vectors, and host cells

It is a further object of the present invention to provide nucleic acids encoding esterases as defined above.

As used herein, the terms "nucleic acid," "nucleic acid sequence," "polynucleotide," "oligonucleotide," and "nucleotide sequence" refer to a sequence of deoxyribonucleotides and/or ribonucleotides. The nucleic acid may be DNA (cDNA or gDNA), RNA or a mixture thereof. It may be in single-stranded form, double-stranded form or a mixture thereof. It may be of recombinant, artificial and/or synthetic origin and may comprise modified nucleotides including, for example, modified linkages, modified purine or pyrimidine bases or modified sugars. The nucleic acids of the invention may be in isolated or purified form and may be prepared, isolated and/or manipulated by techniques known per se in the art, such as cloning and expressing cDNA libraries, amplification, enzymatic synthesis or recombinant techniques. Nucleic Acids can also be synthesized in vitro by well known chemical synthesis techniques, as described, for example, in Belousov (1997) Nucleic Acids Res.25:3440-3444.

The invention also includes nucleic acids which hybridize under stringent conditions to nucleic acids which encode esterases as defined above. Preferably, such stringent conditions include incubation of the hybridization filter in 2 XSSC/0.1% SDS at about 42℃for about 2.5 hours, followed by washing of the filter in 1 XSSC/0.1% SDS at 65℃for 15 minutes 4 times. The protocols used are described in the 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 encompasses nucleic acids encoding the esterases of the invention, wherein the sequence of the nucleic acid or at least a portion of the sequence has been engineered using optimized codon usage.

Alternatively, a nucleic acid according to the invention may be deduced from the sequence of an esterase according to the invention, and the codon usage may be adapted to the host cell in which the nucleic acid should be transcribed. These steps can be performed according to methods well known to those skilled in the art, and some of them are described in the reference handbook 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, etc., which can be used to cause or regulate expression of the polypeptide in a selected host cell or system.

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

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

The term "expression cassette" refers to a nucleic acid construct comprising a coding region (i.e., a nucleic acid of the invention) and a regulatory region (i.e., comprising one or more control sequences) operably linked.

Typically, an expression cassette comprises or consists of a nucleic acid according to the invention operably linked to a control sequence (e.g., a transcription promoter and/or a transcription terminator). The control sequences may include promoters recognized by the host cell or in vitro expression system used to express the nucleic acid encoding the esterases of the invention. Promoters contain transcriptional control sequences that mediate the expression of the enzyme. The promoter may be any polynucleotide that exhibits transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell. The control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription. The terminator is operably linked to the 3' terminus of the nucleic acid encoding the esterase. Any terminator which is functional in the host cell may be used in the present invention. Typically, an expression cassette comprises or consists of a nucleic acid according to the invention operably linked to a transcription promoter and a transcription terminator.

The invention also relates to vectors comprising a nucleic acid or an expression cassette as defined above.

As used herein, the term "vector" or "expression vector" refers to a DNA or RNA molecule comprising an expression cassette of the invention, which serves as a vehicle for the transfer of recombinant genetic material into a host cell. The main types of vectors are plasmids, phages, viruses, cosmids and artificial chromosomes. The vector itself is typically a DNA sequence consisting of an insert (heterologous nucleic acid sequence, transgene) and a larger sequence that serves as the "backbone" of the vector. The purpose of the vector for transferring genetic information to a host is typically to isolate, amplify or express the insert in the cell of interest. Vectors known as expression vectors (expression constructs) are particularly suited for expression of heterologous sequences in target cells and typically have a promoter sequence that drives expression of the heterologous sequence encoding the polypeptide. Typically, regulatory elements present in an expression vector include transcriptional promoters, ribosome binding sites, terminators and optionally operators. Preferably, the expression vector further comprises an origin of replication for autonomous replication in the host cell, a selectable marker, a limited number of useful restriction enzyme sites, and the potential for high copy number. Examples of expression vectors are cloning vectors, modified cloning vectors, specifically designed plasmids and viruses. Expression vectors providing suitable levels of polypeptide expression in different hosts are well known in the art. The choice of 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.

It is another object of the present invention to provide a host cell comprising a nucleic acid, an expression cassette or a 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 transforming, transfecting or transducing a host cell. The choice of vector will generally depend on the compatibility of the vector with the host cell into which it must be introduced.

According to the invention, host cells can be transformed, transfected or transduced in a transient or stable manner. The expression cassette or vector of the invention is introduced into a host cell such that the expression cassette or vector remains a chromosomal integrant or a self-replicating extra-chromosomal vector. The term "host cell" also includes any progeny of a parent host cell that is not identical to the parent host cell due to mutations that occur during replication. The host cell may be any cell useful for producing a variant of the invention, such as a prokaryotic cell or a eukaryotic cell. The prokaryotic host cell may be any gram-positive or gram-negative bacterium. The host cell may also be a eukaryotic cell, such as a yeast, fungal, mammalian, insect or plant cell. In particular embodiments, the host cell is selected from the group consisting of E.coli, bacillus, streptomyces, trichoderma, aspergillus, saccharomyces, pichia, vibrio, and yarrowia.

The nucleic acids, expression cassettes or expression vectors according to the invention may be introduced into host cells by any method known to the person skilled in the art, such as electroporation, conjugation, transduction, competent cell transformation, protoplast fusion, biolistic "gene gun" transformation, PEG-mediated transformation, lipid-assisted transformation or transfection, chemically-mediated transfection, lithium acetate-mediated transformation, liposome-mediated transformation.

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

In a particular embodiment, the host cell is a recombinant microorganism. The invention in fact allows engineering microorganisms with improved ability to degrade polyester-containing materials. For example, the sequences of the invention may be used to complement wild-type fungal or bacterial strains known to be capable of degrading polyesters to improve and/or enhance the ability of the strain.

Esterase production

It is a further object of the present invention to provide a method for producing the esterases of the invention comprising expressing a nucleic acid encoding the esterase and optionally recovering the esterase.

In particular, the invention relates to an in vitro method of producing an esterase of the invention comprising (a) contacting a nucleic acid, cassette or vector of the invention with an in vitro expression system; and (b) recovering the esterase produced. In vitro expression systems are well known to those skilled in the art and are commercially available.

Preferably, the method of generating comprises

(A) Culturing a host cell comprising a nucleic acid encoding an esterase of the invention under conditions suitable for expression of the nucleic acid; and optionally

(B) Recovering the esterase from the cell culture.

Advantageously, the host cell is a recombinant bacillus, a recombinant escherichia coli, a recombinant aspergillus, a recombinant trichoderma, a recombinant streptomycete, a recombinant yeast, a recombinant pichia, a recombinant vibrio or a recombinant yarrowia.

The host cells are cultured in a nutrient medium suitable for producing the polypeptide using methods known in the art. For example, the cells may be cultured by shake flask culture, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) performed in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the enzyme to be expressed and/or isolated. The culturing is performed in a suitable nutrient medium from commercial suppliers or prepared according to published compositions (e.g., in catalogues of the American type culture Collection).

If the esterase is secreted into the nutrient medium, the esterase may be recovered directly from the culture supernatant. Instead, the esterase may be recovered from the cell lysate or after permeation. The esterase may be recovered using any method known in the art. For example, the esterase may be recovered from the nutrient medium by conventional methods including, but not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. Optionally, the esterase may be partially or fully purified by a variety of methods known in the art, including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic methods (e.g., preparative isoelectric focusing), differential solubilization (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction to obtain a substantially pure polypeptide.

The esterases may be used as such, alone or in combination with additional enzymes, in purified form, to catalyze enzymatic reactions involved in the degradation and/or recycling of polyesters and/or polyester-containing materials, such as polyester-containing plastic products. The esterase may be in soluble form, or on a solid phase. In particular, it may be bound to a cell membrane or lipid vesicle, or to a synthetic support, such as glass, plastic, polymer, filter, membrane, for example in the form of beads, columns, plates, etc.

Composition and method for producing the same

It is a further object of the invention to provide a composition comprising an esterase, or a host cell of the invention, or an extract thereof containing an esterase. In the context of the present invention, the term "composition" includes any kind of composition comprising an esterase or host cell of the invention, or an extract thereof comprising an esterase.

The composition of the invention may comprise from 0.1 wt% to 99.9 wt%, preferably from 0.1 wt% to 50 wt%, more preferably from 0.1 wt% to 30 wt%, even more preferably from 0.1 wt% to 5 wt% esterase, based on the total weight of the composition. Alternatively, the composition may comprise 5 to 10% by weight of the esterase of the invention.

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

The composition may further comprise excipients and/or agents and the like. Suitable excipients include buffers commonly used in biochemistry, agents for adjusting pH, preservatives such as sodium benzoate, sodium sorbate or sodium ascorbate, preserving agents, protecting or stabilizing agents such as starch, dextrin, gum arabic, salts, sugars (e.g. sorbitol, trehalose or lactose), glycerol, polyethylene glycol, polypropylene glycol, propylene glycol, chelating agents such as EDTA, reducing agents, amino acids, carriers such as solvents or aqueous solutions, and the like. The compositions of the invention may be obtained by mixing the esterase with one or more excipients.

For example, the composition comprises from 0.1 wt% to 99.9 wt%, preferably from 50 wt% to 99.9 wt%, more preferably from 70 wt% to 99.9 wt%, even more preferably from 95 wt% to 99.9 wt% of the excipient, based on the total weight of the composition. Or the composition may comprise from 90% to 95% by weight of excipient.

The composition may further comprise an additional polypeptide exhibiting enzymatic activity. The person skilled in the art will readily adapt the amount of esterase of the invention according to, for example, the nature of the polyester to be degraded and/or the additional enzymes/polypeptides comprised in the composition.

The esterases of the invention may be dissolved in an aqueous medium together with one or more excipients, in particular excipients which are capable of stabilizing or protecting the polypeptide against degradation. For example, the esterases of the invention may be soluble in water, eventually with additional ingredients such as glycerol, sorbitol, dextrins, starches, glycols such as propylene glycol, salts, and the like. The resulting mixture was then dried to obtain a powder. Methods of drying such mixtures 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 (down-draught evaporation), thin layer evaporation, centrifugal evaporation, conveyor belt drying, fluid bed drying, drum drying, or any combination thereof.

The composition may be in powder form and may comprise esterases and stabilizing/solubilising amounts of glycerol, sorbitol or dextrins, such as maltodextrin and/or cyclodextrin, starch, glycols such as propylene glycol and/or salts.

The compositions of the invention may comprise at least one recombinant cell or extract thereof expressing an esterase of the invention. "cell extract" refers to any fraction obtained from cells, such as cell supernatant, cell debris, cell walls, DNA extract, enzyme or enzyme preparation or any preparation obtained from cells by chemical, physical and/or enzymatic treatment, which is substantially free of living cells. The preferred extract is an enzymatically active extract. The compositions of the invention may comprise one or more recombinant cells of the invention or extracts thereof, and optionally one or more additional cells.

For example, the composition consists of or comprises a medium for expressing and secreting the recombinant microorganism of the esterase of the invention. In certain embodiments, the composition comprises such a medium that is freeze-dried.

Use of esterases

It is a further object of the present invention to provide a process for degrading and/or recycling polyester or polyester-containing materials under aerobic or anaerobic conditions using the esterases of the present invention. The esterases of the invention are particularly useful in the degradation of PET and PET-containing materials.

The object of the present invention is therefore to use the esterases of the invention, or the corresponding recombinant cells having esterase activity, or extracts thereof, or compositions for the enzymatic degradation of polyesters.

Advantageously, the esterase-targeted polyester is selected from the group consisting of polyethylene terephthalate (PET), polypropylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene terephthalate (PBAT), polyethylene furandicarboxylate (PEF), polycaprolactone (PCL), poly (ethylene adipate) (PEA), polyethylene naphthalate (PEN), a "polyolefin-like" polyester, and blends/mixtures of these materials, preferably polyethylene terephthalate.

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

The invention also aims at the enzymatic degradation of at least one polyester of a polyester-containing material using the esterases of the invention, or their corresponding recombinant cells or extracts, or compositions.

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

Advantageously, the polyester is depolymerized up to monomers and/or oligomers.

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

Advantageously, at least one polyester is degraded into repolymerizable monomers and/or oligomers, which can advantageously be recovered for reuse. The recovered monomer/oligomer may be used for recycling (e.g., repolymerizing polyester) or methanation. In particular embodiments, at least one polyester is PET and monoethylene glycol, terephthalic acid, methyl-2-hydroxyethyl terephthalate (MHET), bis (2-hydroxyethyl) terephthalate (BHET), 1- (2-hydroxyethyl) 4-methyl terephthalate (HEMT), and/or dimethyl terephthalate (DMT) are recovered.

Preferably, the polyester of the polyester-containing material is completely degraded.

The time required for degradation of the polyester-containing material may vary depending on the polyester-containing material itself (i.e., the nature and source of the polyester-containing material, its composition, shape, etc.), the type and amount of esterase used, and various process parameters (i.e., temperature, pH, additional reagents, etc.). The process parameters can be readily adapted by the person skilled in the art to the polyester-containing material and the desired degradation time.

Advantageously, the degradation process is carried out at a temperature of 20 ℃ to 90 ℃, preferably 40 ℃ to 90 ℃, more preferably 50 ℃ to 70 ℃. In a particular embodiment, the degradation process is carried out at 60 ℃. In another particular embodiment, the degradation process is carried out at 65 ℃. In another particular embodiment, the degradation process is carried out at 70 ℃. More generally, the temperature is maintained below an inactivation temperature, which corresponds to the temperature at which the esterase is inactivated (i.e., the temperature at which the esterase loses more than 80% of its activity compared to its activity at the optimal temperature) and/or the temperature at which the recombinant microorganism no longer synthesizes the esterase. In particular, the temperature is maintained below the glass transition temperature (Tg) of the target polyester.

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

Advantageously, the degradation process is carried out at a pH of 5-9, preferably in the pH range of 6-9, more preferably in the pH range of 6.5-9, even more preferably in the pH range of 6.5-8, even more preferably at a pH of 7-9, in particular at a pH of 8.

The polyester-containing material may be pretreated prior to contact with the esterase to physically alter its structure, thereby increasing the contact surface between the polyester and the esterase.

It is another object of the present invention to provide a method for producing monomers and/or oligomers from polyester-containing materials comprising exposing the polyester-containing material to an esterase or corresponding recombinant cell or extract or composition of the invention, and optionally recovering the monomers and/or oligomers.

The monomers and/or oligomers resulting from the depolymerization may be recovered sequentially or continuously. A single type of monomer and/or oligomer or several different types of monomers and/or oligomers may be recovered, depending on the starting polyester-containing material.

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

The recovered monomers and/or oligomers can be further purified using all suitable purification methods and conditioned in a repolymerizable form.

The recovered re-polymerizable monomers and/or oligomers may be reused, for example, for the synthesis of polyesters. Advantageously, polyesters of the same nature are repolymerized. However, the recovered monomers and/or oligomers may be mixed with other monomers and/or oligomers, for example, to synthesize new copolymers. Or the recovered monomer may be used as a chemical intermediate to produce a new target compound.

Methods for degrading such polyester-containing materials comprising the esterases of the invention are disclosed, for example, in patent applications WO 2014/079844、WO 2015/173265、WO 2017/198786、WO 2020/094661、WO 2020/094646、WO 2021/123299、WO 2021/123301 and WO 2021/123328. The invention also relates to a method of surface hydrolysis or surface functionalization of a polyester-containing material comprising exposing the polyester-containing material to an esterase of the invention, or a corresponding recombinant cell or extract thereof, or a composition. The process of the present invention is particularly useful for increasing the hydrophilicity or water adsorptivity of polyester materials. Such increased hydrophilicity may be of particular interest for textile production, electronics and biomedical applications.

The invention also relates to a method for treating water, waste water or sewage. In wastewater or sewage treatment applications, esterases according to the invention may be used to degrade microplastic particles composed of polyesters, preferably PET, such as polymer filaments, fibers or other types of polyester-based product fragments and fractions, preferably PET-based product fragments and fractions. It is a further object of the present invention to provide a polyester-containing material comprising the esterase of the invention and/or a recombinant microorganism expressing and secreting said esterase. Methods for preparing such polyester-containing materials comprising the esterases of the invention are disclosed, for example, 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 present invention to provide a polyester-containing material comprising the esterase and/or recombinant cells of the invention and/or a composition or extract thereof and at least PET. According to one embodiment, the present invention provides a plastic product comprising PET and an esterase of the invention having polyester degrading activity.

It is therefore a further object of the present invention to provide a polyester-containing material comprising the esterase and/or recombinant cell of the invention and/or a composition or extract thereof and at least PBAT. According to one embodiment, the present invention provides a plastic product comprising PBAT and an esterase of the invention having PBAT degrading activity.

It is therefore a further object of the present invention to provide a polyester-containing material comprising the esterase and/or recombinant cells of the invention and/or a composition or extract thereof and at least PBS. According to one embodiment, the invention provides a plastic product comprising PBS and an esterase of the invention having PBS degrading activity.

It is therefore a further object of the present invention to provide a polyester-containing material comprising the esterase and/or recombinant cell of the invention and/or a composition or extract thereof and at least PCL. According to one embodiment, the present invention provides a plastic product comprising PCL and an esterase of the invention having PCL degrading activity.

Typically, the esterases of the invention are useful in detergent, food, animal feed, paper, textile and pharmaceutical applications. More specifically, the esterases of the invention are useful as components of detergent compositions. Detergent compositions include, but are not limited to, hand or machine wash detergent compositions, such as laundry additive compositions suitable for pretreatment of stained fabrics and rinse added fabric softening compositions, detergent compositions for general household hard surface cleaning operations, detergent compositions for hand or machine dishwashing operations. For example, esterases of the invention are useful as detergent additives. Accordingly, the present invention provides detergent compositions comprising the esterases of the invention. In particular, the esterases of the invention are useful as detergent additives to reduce pilling and graying effects during textile cleaning.

The invention also relates to methods of using the esterases of the invention in animal feed, and to feed compositions and feed additives comprising the esterases of the invention. The terms "feed" and "feed composition" refer to any compound, formulation, mixture or composition suitable for animal or intended for ingestion by an animal. The esterases of the invention may also be used to hydrolyze proteins and produce hydrolysates comprising peptides. Such hydrolysates may be used as feed compositions or feed additives.

It is a further object of the present invention to provide a method of using the esterases of the invention in the paper industry. More specifically, the esterases of the invention are useful for removing stickies from water lines of pulp and paper machines.

Examples

EXAMPLE 1 construction, expression and purification of esterases

-Construction

The esterases according to the invention were used to construct pET26b-LCC-His production using plasmids. The plasmid comprises cloning the gene encoding the esterase of SEQ ID NO. 1 between NdeI and XhoI restriction sites, which is optimized for E.coli expression. According to the supplier's recommendations, two site-directed mutagenesis kits were used to generate esterase variants: quikChange II site-directed mutagenesis kit and QuikChange Lightning Multi-site-directed mutagenesis kit from Agilent (SANTA CLARA, california, USA).

Expression and purification of esterases

Strains Stellar ^TM (Clontech, california, USA) and E.coli OneBL21DE3 (Life technologies, carlsbad, california, USA) was used successively for cloning and recombinant expression in 50mL LB-Miller medium or ZYM auto-induction medium (Studier et al, 2005-prot. Exp. Pur.41, 207-234). Induction in LB-Miller medium was carried out at 16℃using 0.5mM isopropyl β -D-1-thiogalactopyranoside (IPTG, euromedex, souffelweyersheim, france). The culture was stopped by centrifugation (8000 rpm, 20min at 10 ℃) in an Avanti J-26 XP centrifuge (Beckman Coulter, brea, USA). Cells were suspended in 20mL of Talon buffer (Tris-HCl 20mM,NaCl 300mM,pH 8). The cell suspension was then sonicated by FB 705 sonicator (Fisherbrand, illkirch, france) at 30% amplitude (2 second on and 1 second off cycle) for 2 minutes. Then, a centrifugation step is performed: 11000g of the mixture was placed in an Eppendorf centrifuge at 10℃for 30 minutes. The soluble fraction was collected and subjected to affinity chromatography. The purification step is carried out byMETAL AFFINITY RESIN (Clontech, calif., USA). Protein elution was performed by a procedure of supplementation with Talon buffer of imidazole. Purified proteins were dialyzed against Talon buffer and then quantified using Bio-Rad protein analysis according to manufacturer's instructions (LIFESCIENCE BIO-Rad, france) and stored at +4℃.

EXAMPLE 2 evaluation of esterase degradation Activity

The degradation activity of the esterase was determined and compared with the activity of the esterase of SEQ ID NO. 1.

Various methods of assessing specific activity were used:

(1) Specific Activity based on PET hydrolysis

(2) Degradation activity based on degradation of polyesters in solid form

(3) Degradation Activity based on PET hydrolysis in a reactor above 100mL

(4) Specific activity based on PET hydrolysis and ultraviolet light absorption (UV determination) analysis

2.1. Specific Activity based on PET hydrolysis

100Mg of amorphous PET in powder form (prepared according to WO 2017/198786, to a crystallinity of less than 20%) are weighed and added to a 100mL glass bottle. To a glass vial was added 1mL of an esterase comprising SEQ ID NO:1 (as a reference control) or an esterase preparation of the invention prepared at 0.69. Mu.M in Talon buffer (Tris-HCl 20mM,NaCl 0.3M,pH 8). Finally, 49mL of 0.1M potassium phosphate buffer pH 8 was added.

Depolymerization began by incubating each glass vial in a Max Q4450 incubator (Thermo FISHER SCIENTIFIC, INC.WALTHAM, MA, USA) at a temperature of 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, or 72 ℃ and 150 rpm.

The initial rate of the depolymerization reaction (in milligrams of equivalents TA produced/hour) was determined from samples taken at different times during the first 24 hours and analyzed by ultra-high performance liquid chromatography (UHPLC). If necessary, the sample is diluted in 0.1M potassium phosphate buffer at pH 8. Then, 150. Mu.L of methanol and 6.5. Mu.L of HCl 6N were added to 150. Mu.L of the sample or dilution. After mixing and filtration on a 0.45 μm syringe filter, samples were loaded onto a UHPLC to monitor the release of Terephthalic Acid (TA), MHET and BHET. The chromatographic system used was Ultimate UHPLC system (Thermo FISHER SCIENTIFIC, INC.WALTHAM, MA, USA) comprising a pump module, an autosampler, a column oven thermostated at 25℃and a 240nm UV detector. The column used isHS C18HPLC column (150 x 4.6mm,5 μm equipped with a pre-column, supelco, bellefonte, USA). TA, MHET and BHET were isolated at 1mL/min using a methanol gradient (30% to 90%) in 1mM H ₂SO₄. The injection was 20. Mu.L of sample. TA, MHET and BHET were measured according to standard curves prepared from commercial TA and BHET and MHET synthesized internally under the same conditions as the samples. The specific activity of PET hydrolysis (equivalent TA mg/hr/mg enzyme) was determined in the linear part of the hydrolysis curve of the reaction (i.e. at the start of the reaction), this curve being established by sampling at different times during the first 24, 48, 72, 96 hours. Equivalent TA corresponds to the sum of the measured TA and the TA contained in the measured MHET and BHET. The equivalent TA measurement can also be used to calculate the yield of a PET depolymerization assay at a given time and/or after a specific period of time (e.g., 24 hours, 48 hours, 72 hours, or 96 hours).

2.2. Activity based on degradation of polyesters in solid form

Mu.l of enzyme preparation was deposited in wells formed in PET-containing agar plates. Agar plates were prepared by dissolving 500mg of PET in hexafluoro-2-propanol (HFIP) and pouring the medium into 250mL of aqueous solution. After HFIP evaporation at 52℃and 140 mbar, the solution was mixed with 0.2M potassium phosphate buffer pH 8v/v containing 3% agar. Each plate was prepared using about 30mL of the mixture and stored at 4 ℃.

The diameter or surface area of the halos formed by degradation of the polyester by wild-type esterases and variants was measured and compared after 2 to 24 hours at 40 ℃, 45 ℃, 50 ℃, 55 ℃,60 ℃, 65 ℃ or 70 ℃.

2.3. Based on the activity of hydrolysis of polyesters in the reactor

0.69. Mu. Mol-2.07. Mu. Mol of purified esterase prepared in 80ml of 100mM potassium phosphate buffer pH 8 was mixed with 20g of amorphous polyester PET (prepared according to WO 2017/198786, crystallinity lower than 20%) in a 500mL Minibio bioreactor (Applikon Biotechnology, delft, THE NETHERLANDS). The temperature adjustment was carried out by immersing in a water bath at a temperature of 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃ or 70 ℃ and maintaining constant stirring at 250rpm using a single paddle impeller. The pH of the PET depolymerization test was adjusted to pH 8 by the addition of 6N sodium hydroxide and confirmed by my-Control bio controller system (Applikon Biotechnology, delft, THE NETHERLANDS). The alkali consumption was recorded during the assay and can be used for characterization of the PET depolymerization assay.

The final yield of the PET depolymerization assay is determined by determining the residual PET weight or by determining the equivalent TA produced or by base consumption. The gravimetric determination of residual PET was assessed by filtering the reaction volume at the end of the reaction through a 12-15 μm 11-grade ashless filter paper (Dutscher SAS, brumath, france) and drying this retentate before weighing. The equivalent TA generated was determined using the UHPLC method described in 2.1 and the percent hydrolysis was calculated based on the molar concentration at a given time (TA+MHET+BHET) relative to the total amount of TA contained in the initial sample. The acid monomer produced by depolymerization of PET is neutralized by a base to be able to maintain the pH in the reactor. The determination of the equivalent TA produced is calculated using the corresponding molar base consumption and the percent hydrolysis is calculated based on the ratio of the molar concentration of equivalent TA at a given time to the total amount of TA contained in the initial sample.

2.4 Specific Activity based on PET hydrolysis and ultraviolet light absorption (UV determination) analysis

100Mg of amorphous PET in powder form (prepared according to WO 2017/198786 to reach crystallinity lower than 20%) is weighed and added to a 100mL glass bottle. To a glass flask was added 1mL of an esterase comprising SEQ ID NO:1 (as a reference control) or an esterase preparation of the invention prepared at 0.69. Mu.M in Talon buffer (Tris-HCl 20mM,NaCl 0.3M,pH 8 or 100mM potassium phosphate buffer pH 8). Finally, 49mL of 100mM potassium phosphate buffer pH 8.0 was added.

Depolymerization was initiated by incubating each glass vial in a Max Q4450 incubator (Thermo FISHER SCIENTIFIC, INC.WALTHAM, MA, USA) at 50 ℃, 54 ℃,60 ℃ or 65 ℃ and 150 rpm.

Alternatively the reaction may be miniaturized in a deep well (deepwell) (thermo scientific, abgene, AB-0661, illkirch, france), 22mg of amorphous PET in powder form (prepared according to WO2017/198786 to achieve crystallinity below 20%) is weighed and introduced into each well of a deep well plate. Into each well of the deep well, 0.1mL of esterase preparation comprising SEQ ID NO:1 (as a reference control) or the esterase of the invention prepared in Talon buffer (Tris-HCl 20mM,NaCl 0.3M,pH 8 or 100mM potassium phosphate buffer pH 8) at 0.138. Mu.M was introduced. Finally, 0.9mL of 100mM potassium phosphate buffer pH 8.0 was added.

Depolymerization was started by incubating each well in Infors HT multitron shaking incubator (Infos HT, bottmingen, suisse) at 50 ℃, 54 ℃, 55 ℃,60 ℃ or 65 ℃ and 600 rpm.

The initial rate of the depolymerization reaction (in. Mu. Mol/hr of soluble degradation product produced) was determined by sampling at different times during the first 24 hours and analyzed by absorbance reading at 242nm using Eon Microplate Spectrophotometer (BioTek, USA). The increase in absorbance of the reaction mixture in the ultraviolet region (242 nm) of the spectrum indicates the release of soluble TA or its esters (BHET and MHET) from the insoluble PET matrix. The absorbance value at this wavelength can be used to calculate the total sum of PET hydrolysates according to Lambert-Beer law, and the enzyme-specific activity is determined as the total equivalent TA produced. If desired, the samples were diluted in 100mM potassium phosphate buffer pH 8.0. The specific activity of PET hydrolysis (μmol/hr/mg enzyme of soluble product) was determined in the linear part of the hydrolysis curve of the reaction (i.e. at the start of the reaction), which curve was established by sampling at different times during the first 24 hours. The equivalent TA measurement can also be used to calculate the yield of a PET depolymerization assay after a given time and/or a specific period of time.

Results

Specific degradation Activity based on PET hydrolysis under alkaline conditions compared to the esterase of SEQ ID NO. 1

The specific degradative activity of the esterases (variants) of the invention was measured at 65℃at pH 8 at the start of the reaction, as described in example 2.1.

The results are shown in Table 1 below. The specific degradation activity of the esterase of SEQ ID No. 1 was used as a reference and regarded as 100% specific degradation activity.

TABLE 1 specific degradation Activity of esterases of the invention compared with SEQ ID NO.1 at pH 8

Variants	Relative degradation Activity (%)
		V1:V219E	128％
V5:L15Q	110％
		V6:N211F	117％
V7:S13L	109％
		V8:A14Y	128％
V9:S206N	121％

In addition to the substitutions shown in Table 1, the variants have the exact amino acid sequence of SEQ ID NO. 1.

PET depolymerization yield under alkaline conditions compared to esterase of SEQ ID NO.1

PET depolymerization yields of esterases (variants) of the invention were measured according to example 2.1 after 96 hours at 65℃and pH 8. In the context of the present invention, PET depolymerization yields were used to evaluate degradation activity.

The results are shown in Table 2 below. The PET depolymerization yield of the esterase of SEQ ID NO:1 after 96 hours at 65℃and pH 8 was used as a reference and was considered to be 100% degradation activity.

TABLE 2 PET depolymerization yield of esterases of the invention after 96 hours

Variants	PET depolymerization yield (%)
		V2:N204S+N105D	137％
V11:N243Y	114％

In addition to the substitutions shown in Table 2, V2 and V11 have the exact amino acid sequence of SEQ ID NO. 1.

Specific degradation Activity under alkaline conditions compared to the esterase of SEQ ID NO:1

Specific degradative activity was measured at the beginning of the reaction at pH 8.0 and 65℃as described in example 2.4.

The specific degradative activity of the esterases (variants) of the invention is shown in table 3 below. The specific degradation activity of the esterase of SEQ ID No. 1 was used as a reference and regarded as 100% specific degradation activity.

Table 3: specific degradation Activity of esterases of the invention compared with SEQ ID NO. 1 at pH 8.0 and 65 ℃

Variants	Relative degradation Activity (%)
		V12:R138K	110％
V13:R138K+E141C	115％

The variants described above have the exact amino acid sequence of SEQ ID NO. 1, except for the substitutions shown in Table 3, respectively.

Specific degradation Activity based on PET hydrolysis under alkaline conditions compared to esterase of SEQ ID NO. 2

After 96 hours at 65 ℃ and pH 8, the specific degradative activity of the variants of the invention was measured as described in example 2.1. The variant is derived from SEQ ID NO.2, which has a specific degrading activity 1.4 times higher than the esterase of SEQ ID NO. 1. That is, variants of SEQ ID NO.2 which exhibit increased degradation activity compared to SEQ ID NO.2 also have increased degradation activity compared to SEQ ID NO. 1.

The results are shown in table 4 below. The specific degradative activity of the esterase of SEQ ID NO. 2 is used as a reference and is considered to be 100% specific degradative activity.

Table 4: compared with SEQ ID NO. 2, the esterase of the invention has specific degradation activity after 96 hours at 65 ℃ and pH 8

Variants	Specific degradation Activity (%)
		V4:V219E+Q182E+R12E	126％

In addition to the substitutions listed in Table 4, V4 has the exact amino acid sequence of SEQ ID NO. 2.

Specific degradation Activity based on PET hydrolysis under alkaline conditions compared to the esterase of SEQ ID NO. 2

Specific degradation activity was measured at the beginning of the reaction at pH 8.0 and 65 ℃ (variant V14-V18) or 55 ℃ (variant V19-V21) as described in example 2.4.

The specific degradative activity of the esterases (variants) of the invention is shown in table 5 below. The specific degradative activity of the esterase of SEQ ID NO.2 is used as a reference and is considered to be 100% specific degradative activity.

Table 5: specific degradation Activity of esterases of the invention

In addition to the substitutions listed in Table 5, the variants have the exact amino acid sequence of SEQ ID NO. 2.

PET depolymerization yield under alkaline conditions compared to esterase of SEQ ID NO. 2

After 24h of reaction at pH 8 and 65 ℃ (variant V15) or 50 ℃ (variant V22-V24), the PET depolymerization yield of the esterase (variant) of the invention was measured according to example 2.4. In the context of the present invention, PET depolymerization yields were used to evaluate degradation activity.

The results are shown in table 6 below. PET depolymerization yields of esterases of SEQ ID NO:2 after 24h reaction at pH 8 and at 65 ℃ (variant V15) or at 50 ℃ (variant V22-V24) were used as reference and considered as 100% degradation activity.

Table 6: PET depolymerization yield of the esterases of the invention after 24 hours.

Variants	PET depolymerization yield (%)
		V15:F250L	141％
V22:V180A	141％
		V23:V180T	125％
V24:F250A	121％

In addition to the substitutions shown in Table 6, the variants have the exact amino acid sequence of SEQ ID NO. 2.

Example 3-evaluation of the Heat stability of esterases of the invention

The thermostability of the esterases of the invention was determined and compared with that of the esterases of SEQ ID No. 1 or SEQ ID No. 2.

Different methods were used to estimate thermal stability:

(1) Circular dichroism of proteins in solution;

(2) Residual esterase activity after protein incubation at given temperature, time and buffer conditions;

(3) Depolymerization activity of residual polyester after protein incubation at given temperature, time and buffer conditions;

(4) The ability to degrade solid polyester compounds (e.g., PET or PBAT or the like) dispersed in agar plates after protein incubation at given temperature, time and buffer conditions;

(5) The ability to conduct multiple rounds of polyester depolymerization assays at given temperature, buffer, protein concentration, and polyester concentration conditions;

(6) Differential scanning fluorescence analysis (DSF);

The details of the schemes of these methods are as follows.

3.1 Circular dichroism analysis

Circular Dichroism (CD) analysis was performed using Jasco 815 equipment (Easton, USA) to compare the melting temperature (Tm) of the esterase of SEQ ID NO:1 with the Tm of the esterase of the invention. Technically, 400. Mu.L of protein samples were prepared at 0.5mg/mL in Talon buffer and used for CD. A first scan from 280 to 190nm is achieved to determine the two maximum intensities of the CD corresponding to the correct folding of the protein. A second scan was then performed from 25 ℃ to 110 ℃ and at wavelengths corresponding to these maximum intensities and providing a specific curve analyzed by the Sigmaplot version 11.0 software (sigmoid 3 parameter y=a/(1+e ((x-x 0)/b)), the Tm obtained by determination Tm. when x=x0 reflects the thermal stability of the given protein.

3.2 Residual esterase Activity

1ML of the esterase of SEQ ID NO. 1 or a 40mg/L solution of the esterase of the invention (in Talon buffer) was incubated at different temperatures (40, 50, 60, 65, 70, 75, 80 and 90 ℃) for up to 10 days. Samples were taken periodically, diluted 1-500 times in 0.1M potassium phosphate buffer pH 8.0, and analyzed for p-nitrophenol-butyrate (pNP-B). mu.L of the sample was mixed with 175. Mu.L of 0.1M potassium phosphate buffer pH 8.0 and 5. Mu.L of a solution of pNP-B in 2-methyl-2 butanol (40 mM). The enzymatic reaction was carried out with stirring at 30℃and absorbance at 405nm was obtained at 15 min and by a microplate spectrophotometer (Versamax, molecular Devices, sunnyvale, calif., USA). The activity of the pNP-B hydrolysis (initial rate expressed in. Mu. Mol/min of pNPB) was determined using a standard curve for p-nitrophenol released in the linear part of the hydrolysis curve.

3.3 Residual polyester depolymerization Activity

10ML of the esterase of SEQ ID No. 1 and a 40mg/L solution of the esterase of the invention (in Talon buffer) were incubated at different temperatures (40 ℃,50 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, 80 ℃ and 90 ℃) for up to 30 days. Periodically, 1mL of sample is taken, transferred to a bottle containing 100mg of amorphous polyester micronized at 250-500 μm (prepared according to WO 2017/198786, crystallinity lower than 20%) and 49mL of 0.1M potassium phosphate buffer pH 8.0 and incubated at 50 ℃, 55 ℃, 60 ℃, 65 ℃ or 70 ℃. 150. Mu.L of buffer was sampled periodically. When necessary, the samples were diluted in 0.1M potassium phosphate buffer pH 8. Then, 150. Mu.L of methanol and 6.5. Mu.L of HCl 6N were added to 150. Mu.L of the sample or dilution. After mixing and filtration on a 0.45 μm syringe filter, samples were loaded onto a UHPLC to monitor the release of Terephthalic Acid (TA), MHET and BHET. The chromatographic system used was Ultimate UHPLC system (Thermo FISHER SCIENTIFIC, INC.WALTHAM, MA, USA) comprising a pump module, an autosampler, a column oven thermostated at 25℃and a 240nm UV detector. The column used isHS C18 HPLC column (150 x 4.6 mm, 5 μm equipped with a pre-column, supelco, bellefonte, USA). TA, MHET and BHET were isolated at 1mL/min using a methanol gradient (30% to 90%) in 1mM H ₂SO₄. The injection was 20. Mu.L of sample. TA, MHET and BHET were measured on standard curves prepared from commercial TA and BHET, and on MHET synthesized internally under the same conditions as the samples. The activity of PET hydrolysis (μmol/min of hydrolyzed PET or mg/hr of equivalent TA produced) was determined in the linear portion of the hydrolysis curve, which was set by sampling at different times during the first 24 hours. Equivalent TA corresponds to the sum of measured TA and TA contained in measured MHET and BHET.

3.4 Degradation of polyesters in solid form

1ML of the esterase of SEQ ID No. 1, respectively, and a 40mg/L solution of the esterase of the invention (in Talon buffer) were incubated at different temperatures (40 ℃, 50 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, 80 ℃ and 90 ℃) for up to 30 days. Periodically, 20. Mu.l of enzyme preparation was placed in wells on an agar plate containing PET. PET-containing agar plates were prepared by dissolving 500mg of PET in hexafluoro-2-propanol (HFIP) and pouring the medium into 250mL of aqueous solution. After HFIP evaporation at 52℃and 140 mbar, the solution was mixed with 0.2M potassium phosphate buffer pH 8v/v containing 3% agar. Each total tray (omnitray) was prepared using about 30mL of the mixture and stored at 4 ℃.

After 2-24 hours at 50 ℃, 55 ℃, 60 ℃, 65 ℃ or 70 ℃, the diameter or surface area of the halo formed as a result of the degradation of the polyester by the wild-type esterase and the variant of the invention is measured and compared. The half-life of the enzyme at a given temperature corresponds to the time required for a 2-fold reduction in halo diameter.

3.5 Multiple rounds of polyester depolymerization

The ability of esterases to conduct successive rounds of polyester depolymerization assays was evaluated in an enzyme reactor. Minibio 500A bioreactor (Applikon Biotechnology B.V., delft, THE NETHERLANDS) was started from 3g of amorphous PET (prepared according to WO 2017/198786, crystallinity lower than 20%) and 100mL of 10mM potassium phosphate buffer pH 8 containing 3mg of esterase. The stirring speed was set to 250rpm using a paddle impeller. The bioreactor was kept at a constant temperature of 50 ℃, 55 ℃, 60 ℃,65 ℃ or 70 ℃ by immersing in an external water bath. The pH was adjusted to 8 by adding KOH at 3M. Different parameters (pH, temperature, stirring, base addition) were monitored by BioXpert software V2.95. 1.8g of amorphous PET (prepared according to WO 2017/198786, crystallinity lower than 20%) was added every 20 h. 500. Mu.L of the reaction medium was sampled periodically.

The amounts of TA, MHET and BHET were determined by HPLC as described in example 2.3. The amount of EG was measured using an Aminex HPX-87K column (Bio-Rad Laboratories, inc., hercules, california, united States) at a constant temperature of 65 ℃. The eluent was K ₂HPO₄ 5mM,0.6mL.min^-1. The injection amount was 20. Mu.L. Ethylene glycol was monitored using a refractometer.

The percent 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 initial sample, or based on the ratio of the molar concentration at a given time (EG+MHET+2xBHET) to the total amount of EG contained in the initial sample. Degradation rate is calculated as milligrams of total released TA per hour or milligrams of total EG per hour.

The half-life of the enzyme was assessed as the incubation time required to obtain a 50% degradation rate loss.

3.6 Differential scanning fluorescence analysis (DSF)

DSF was used to evaluate its thermostability by determining the melting temperature (Tm) of the wild-type protein (SEQ ID NO: 1) and its variants, which refers to the temperature at which half the protein population expands. Protein samples were prepared and stored at a concentration of 6.25. Mu.M in buffer A consisting of 100mM potassium phosphate buffer pH 8. Stock solution of SYPRO orange dye 5000x in DMSO was first diluted to 250x in water. Protein samples were loaded onto a white transparent 96-well PCR plate (Bio-Rad cat#HSP 9601) containing a final volume of 25. Mu.L per well. The final concentration of protein and SYPRO Orange dye in each well was 6. Mu.M (0.17 mg/ml) and 10X, respectively. The loading volume per well is as follows: 24. Mu.L of a 6.25. Mu.M protein solution and 1. Mu.L of a 250 XSypro Orange dilution solution. The poly PCR plate was then sealed with an optical quality sealing tape and spun at 1000rpm for 1 minute at room temperature. DSF experiments were then performed using a CFX96 real-time PCR system configured to use 450/490 excitation and 560/580 emission filters. The sample was heated from 25 ℃ to 100 ℃ at a rate of 0.3 ℃/sec. A single fluorescence measurement was performed every 0.03 seconds. Melting temperature was determined from the peak of the first derivative of the melting curve using Bio-Rad CFX Manager software. Variations in buffer type or buffer concentration can be used without affecting the Δtm between the esterase of the invention and the wild-type, provided that the same buffer is used for the wild-type esterase.

The esterase of SEQ ID NO. 1 or SEQ ID NO. 2 and the esterase of the invention are then compared based on their Tm values. Due to the high reproducibility between experiments on the same proteins from different production, Δtm at 0.8 ℃ is considered to be of great significance for comparing variants. The Tm value corresponds to the average of at least 3 measurements. The Tm of the esterase of SEQ ID No. 1 was evaluated at 84.7 ℃.

Results

Increased thermostability at pH 8 compared to the esterase of SEQ ID NO:1

The thermostability of the esterases (variants) of the invention was evaluated according to example 3.6. The results expressed as Tm values are summarized in table 7 below. The increase in Tm of the esterase compared to SEQ ID NO.1 is indicated in brackets.

Table 7 Tm of the esterases of the invention compared to SEQ ID NO.1 at pH 8.

Variants	Tm
		V3:L15V+R89L	85.5℃(+0.8℃)

In addition to the substitutions shown in Table 7, V3 has the exact amino acid sequence of SEQ ID NO. 1.

Claims

1. An esterase variant (I) having 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, (ii) having at least one amino acid substitution at least one amino acid position corresponding to a residue selected from the group consisting of V219E, N204S, N243Y, L V Q, D158C, T160C, R K/D/E/L, N211F, S13L, A Y and S206N, and/or compared to SEQ ID No. 1, at least one amino acid substitution at a position corresponding to a residue selected from the group consisting of G7, S57, T136, E141, I169, G171, V180, a184, I185, P186, Y188, E201, R234, D249, F250, R251, H77 and L191, wherein said position is numbered with reference to the amino acid sequence set forth in SEQ ID No. 1, (iii) having polyester degrading activity, and/or exhibiting increased thermostability and/or increased degrading activity compared to SEQ ID No. 1.

2. The esterase variant according to claim 1, wherein the esterase comprises at least one amino acid substitution selected from V219E、N204S、N243Y、L15V/Q、D158C、T160C、R138K/D/E/L、E141C/K/R、G171C、N211F、S13L、A14Y、S206N、V180I/C/N/A/T/L and F250N/L/V/Y/a, preferably from V219E, N204S, N243Y, L V/Q, R138K, E141C, N211F, S13L, A14Y, S N, V I/C/N/a/T and F250N/L/V/Y/a, more preferably from V219E, N S, N243Y, L V/Q, R138K, E141C, N F, S L, A Y and S206N, even more preferably from V219E, N243Y, L Q, N211F, S13L, A14Y, S N and R138K.

3. The esterase variant according to claim 1 or 2, wherein said esterase further comprises at least one substitution at least one position selected from S1、Y4、Q5、R6、N9、P10、T11、R12、S13、A14、L15、T16、A17、D18、S22、T25、Y26、T27、V28、S29、R30、L31、S32、V33、S34、G35、F36、G37、G38、G39、Y43、S48、T50、G53、I54、M56、P58、G59、Y60、T61、A62、D63、A64、S65、S66、L67、A68、W69、L70、R72、R73、L74、L82、I84、N85、T86、N87、S88、R89、F90、D91、Y92、P93、D94、S95、R96、S98、Q99、A103、L104、N105、L107、R108、S113、L119、A121、N122、L124、A125、A127、G128、H129、M131、G132、G133、G134、G135、R138、A140、N143、S145、K147、A149、V150、L152、T153、P154、W155、H156、T157、D158、K159、T160、N162、S164、V167、L168、V170、A172、E173、A174、T176、V177、A178、P179、S181、Q182、H183、F187、Q189、N190、S193、T194、P196、V198、V200、L202、D203、N204、A205、S206、F208、A209、P210、N211、S212、N213、N214、A215、A216、I217、S218、V219、Y220、T221、S223、W224、M225、N231、T233、R236、Q237、F238、L239、N241、V242、N243、D244、P245、A246、L247、S248、T252、N253、N254、R255、H256.

4. The esterase variant according to any of the preceding claims, wherein said esterase comprises at least one substitution or combination of substitutions selected from V219E、N204S、N243Y、L15V/Q、N211F、S13L、A14Y、S206N、V180L/I/C/N/A/T、F250N/L/V/Y/A、L15V+R89L、N204S+N105D、E141C+D158C、E141C+T160C、E141C+R138K/D/E/L、D158C+T160C、G171C+V180C、R138K/D/E/L and v219e+q182e+r12e, preferably at least one substitution or combination of substitutions selected from V219E、N243Y、L15Q、N211F、S13L、A14Y、S206N、V180L/I/C/N/A/T、F250N/L/V/Y/A、L15V+R89L、N204S+N105D、E141C+R138K、R138K and v219e+q182 e+r12e.

5. The esterase variant according to any of claims 1-4, wherein said esterase comprises at least one amino acid substitution selected from V219E, L Q, N211F, S L, A Y, S35206N, V L/I/C/N, F250N/L/V/Y and R138K or at least one substitution combination selected from r39k+e141C and v219e+q182e+r12e and exhibits an increased specific degradation activity compared to the esterase of SEQ ID NO: 1.

6. The esterase variant according to any of claims 1-4, wherein the esterase comprises at least the amino acid substitution N243Y, F L/A, V180A/T or at least the substitution combination n204s+n105d and shows an increased depolymerization yield of PET after 24 hours or after 96 hours compared to the esterase of SEQ ID NO: 1.

7. The esterase variant according to any of claims 1-4, wherein said esterase comprises at least the substitution combination l15v+r89L and exhibits increased thermostability.

8. The esterase variant according to any of the preceding claims, wherein said esterase further comprises at least one, preferably at least two, at least three, four substitutions at positions selected from F208, D203, S248, V170, Y92, G135, V167, Q182 and N213, preferably at least a combination of substitutions selected from D203C+S248C、F208G/N/R/I/A/Q/L/S/M/T/E/W+D203C+S248C、F208G/N/R/I/A/Q/L/S/M/T/E/W+D203C+S248C+V170I、F208G/N/R/I/A/Q/L/S/M/T/E/W+D203C+S248C+V170I+Y92D/E/G、F208G/N/R/I/A/Q/L/S/M/T/E/W+D203C+S248C+V170I+Y92D/E/G+N213P+Q182E、F208G/N/R/I/A/Q/L/S/M/T/E/W+D203K/R、F208G/N/R/I/A/Q/L/S/M/T/E/W+D203K/R+V170I、F208G/N/R/I/A/Q/L/S/M/T/E/W+D203K/R+V170I+Y92D/E/G、F208G/N/R/I/A/Q/L/S/M/T/E/W+D203K/R+V170I+Y92D/E/G+N213P+Q182E, preferably selected from D203C+S248C、F208I/W/M+D203C+S248C、F208I/W/M+D203C+S248C+V170I、F208I/W/M+D203C+S248C+V170I+Y92D/E/G、F208I/W/M+D203C+S248C+V170I+Y92D/E/G+N213P+Q182E、F208I/W/M+D203K/R+V170I、F208I/W/M+D203K/R+V170I+Y92D/E/G and a combination of substitutions of F208I/W/m+d203K/r+v170i+y92d/E/g+n21p+q182E, more preferably selected from D203C+S248C、F208I/M+D203C+S248C、F208I/M+D203C+S248C+V170I、F208I/M+D203C+S248C+V170I+Y92G、F208I/M+D203C+S248C+V170I+Y92G+N213P+Q182E.

9. The esterase variant according to any of the preceding claims, wherein said esterase comprises at least one amino acid residue selected from S130, D175 or H207 in the esterase as set forth in SEQ ID No. 1, preferably a combination s130+d175+h207 in the esterase as set forth in SEQ ID No. 1.

10. The esterase variant according to any of the preceding claims, wherein said esterase comprises at least one amino acid residue selected from S130, D175, H207, C240 or C275 in the esterase as set forth in SEQ ID No. 1, preferably a combination s130+d175+h207+c240+c275 in the esterase as set forth in SEQ ID No. 1.

11. An esterase having (I) at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the full-length amino acid sequence set forth in SEQ ID No. 2 and (ii) at least one amino acid substitution selected from V219E, N204S, N243Y, L V/Q, D158C, T160C, R K/D/L, N211F, S211L, A Y and S206N compared to SEQ ID No. 2 and/or at least one amino acid substitution at least one amino acid position corresponding to a residue selected from G7, S57, T136, E141, I169, G171, V180, a184, I185, P186, Y188, E201, R234, D249, F250, R251, H77 and L191 compared to SEQ ID No. 2, wherein said positions are numbered by reference to the amino acid sequence set forth in SEQ ID No. 2, (iii) having polyester degradation activity and (iv) exhibiting improved thermal stability and/or improved polyester degradation activity compared to SEQ ID No. 1.

12. The esterase according to claim 11, wherein the esterase further comprises the residue combination c203+c248+g92, in particular the residue combination i208+c203+c248+i170+g92, as in the parent esterase of SEQ ID No. 2.

13. The esterase according to claim 11 or 12, wherein the esterase has at least one amino acid substitution selected from V219E, V I/C/N/a/T/L/and F250N/L/V/Y/a, preferably from V219E, V I/C/N/a/T and F250N/L/V/Y/a, more preferably from V180I/C/N/a/T and F250N/L/V/Y/a.

14. The esterase according to any of claims 11-13, wherein said esterase has at least one amino acid substitution selected from V180I/C/N and F250N/V/Y/L and exhibits an increased specific degradation activity compared to the esterase of SEQ ID No. 2.

15. The esterase according to any of claims 11-13, wherein said esterase has at least one amino acid substitution selected from V180A/T and F250A/L and shows an increased depolymerization yield of PET after 24 hours compared to the esterase of SEQ ID No. 2.

16. The esterase variant according to any of claims 11-15, wherein said esterase further exhibits increased thermostability and/or increased polyester degrading activity compared to the esterase of SEQ ID No. 1.

17. The esterase variant according to any of claims 11-16, wherein said esterase comprises at least one amino acid residue selected from the group consisting of S130, D175, H207, C240, C275, C203, C248 in the esterase as set forth in SEQ ID No. 1, preferably the combination s130+d175+h207+c240+c275+c203+c248 in the esterase as set forth in SEQ ID No. 2.

18. A nucleic acid encoding an esterase as defined in any of claims 1 to 17.

19. An expression cassette or vector comprising the nucleic acid of claim 18.

20. A host cell comprising the nucleic acid of claim 18 or the expression cassette or vector of claim 19.

21. A composition comprising an esterase according to any of claims 1 to 17, or a host cell according to claim 20, or an extract thereof having esterase activity.

22. A method of degrading at least one polyester of a polyester or polyester-containing material comprising:

a. Contacting the polyester or the polyester-containing material with the esterase according to any of claims 1 to 17 or the host cell according to claim 20 or the composition according to claim 21; and optionally

B. Recovering the monomers and/or oligomers.

23. The method of claim 22, wherein the polyester is selected from the group consisting of polyethylene terephthalate (PET), polypropylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isoparaffinate (PEIT), polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate (PBSA), polybutylene terephthalate (PBAT), polyethylene furandicarboxylate (PEF), polycaprolactone (PCL), poly (ethylene adipate) (PEA), polyethylene naphthalate (PEN), a "polyolefin-like" polyester, and any blends/mixtures of at least two of these materials, preferably polyethylene terephthalate.

24. A polyester-containing material comprising an esterase according to any of claims 1 to 17 or a host cell according to claim 20 or a composition according to claim 21.

25. A detergent composition comprising the esterase according to any of claims 1 to 17 or the host cell according to claim 20 or the composition according to claim 21.