Novel esterases and uses thereof

Abstract

The present invention relates to novel esterases, and more particularly to esterase variants having improved activity and / or improved thermal stability as compared to the esterase of SEQ ID N DEG 1 and their use in degrading polyester-containing materials, such as plastic articles. The esterase provided by the invention is particularly suitable for degrading polyethylene glycol terephthalate and materials containing polyethylene glycol terephthalate.

Description

[0001] This application is a divisional application of the invention patent application with application date of July 26, 2019, application number 2019800574469, and invention name “New esterase and its use”.

[0002] Related Applications

[0003] The present invention relates to novel esterases, more particularly to esterases having improved activity and / or improved thermostability compared to a parent esterase. The present invention also relates to the use of said novel esterases for degrading polyester-containing materials (e.g., plastics). The esterases of the present invention are particularly suitable for degrading polyethylene terephthalate and materials containing polyethylene terephthalate. Background Art

[0004] Esterase can catalyze the hydrolysis of various polymers (including polyester). In this case, esterase has shown promising effects in many industrial applications, including as a detergent for dishwashing and laundry applications, as a degradation enzyme for handling biomass and food, as a biocatalyst in the detoxification of environmental pollutants or for handling polyester fabrics in the textile industry. It is particularly interesting to use esterase as a degradation enzyme to hydrolyze polyethylene terephthalate (PET). In fact, PET is used in numerous technical fields, for example, for the manufacture of clothing, carpets or for the manufacture of packaging materials or automotive plastic products in the form of thermosetting resins, so that the accumulation of PET in landfills becomes an increasing ecological problem.

[0005] The enzymatic degradation of polyesters, in particular PET, is considered an interesting solution to reduce the accumulation of this plastic waste. Indeed, enzymes can accelerate the hydrolysis of polyester-containing materials, more particularly plastic articles, even down to the monomer level. Furthermore, the hydrolysis products (i.e. monomers and oligomers) can be recycled as materials for the synthesis of new polymers.

[0006] In this context, several esterases have been identified as candidate degrading enzymes for polyesters, and some variants of such esterases have been developed. Among the esterases, cutinases, also known as cutin hydrolases (EC 3.1.1.74), are of particular interest. Cutinases have been identified from various fungi (PE Kolattukudy in "Lipases", Ed. B. Borg-stróm and H.L. Brockman, Elsevier 1984, 471-504), bacteria, and plant pollens. Recently, metagenomic approaches have identified other esterases.

[0007] However, there is still a need for esterases having improved activity and / or improved thermostability compared to known esterases in order to provide a more efficient polyester degradation process and thus be more competitive. SUMMARY OF THE INVENTION

[0009] The present invention provides novel esterases showing increased activity and / or increased thermostability compared to a parent or wild-type esterase having an amino acid sequence as shown in SEQ ID N° 1. The wild-type esterase corresponds to amino acids 36-293 of the amino acid sequence of a metagenomic derived cutinase described in Sulaiman et al., Appl Environ Microbiol. 2012 Mar and is referred to as G9BY57 in SwissProt. The esterases of the present invention are particularly useful in methods for degrading plastic products, particularly plastic products containing PET.

[0010] In this regard, an object of the present invention is to provide an esterase which (i) has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the full-length amino acid sequence shown in SEQ ID N°1, (ii) comprises at least four substitutions at positions selected from F208, D203, S248, V170, V177, T176, T61, S65 or Y92 compared to the amino acid sequence SEQ ID N°1, and (iii) exhibits increased polyester degradation activity and / or increased thermal stability compared to the esterase of SEQ ID N°1.

[0011] Another object of the present invention is to provide an esterase which (i) has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the full-length amino acid sequence shown in SEQ ID N°1, (ii) comprises at least four substitutions at positions selected from F208, D203, S248, V170, V177, T176, T61, S65, N211 or Y92 compared to the amino acid sequence SEQ ID N°1, and (iii) exhibits increased polyester degradation activity and / or increased thermal stability compared to the esterase of SEQ ID N°1.

[0012] Preferably, the esterase comprises at least substitutions at positions F208+D203+S248 and a combination of one or two substitutions at positions selected from V170, V177, T176, T61, S65, N211 or Y92.

[0013] Another object of the present invention is to provide a nucleic acid encoding the esterase of the present invention. The present invention also relates to an expression cassette or expression vector comprising the nucleic acid, and to a host cell comprising the nucleic acid, expression cassette or vector.

[0014] The present invention also provides a composition comprising the esterase of the present invention, the host cell of the present invention or an extract thereof.

[0015] A further object of the present invention is to provide a method for producing the esterase of the present invention, comprising:

[0016] (a) culturing a host cell according to the invention under conditions suitable for expression of a nucleic acid encoding an esterase; and optionally

[0017] (b) recovering the esterase from the cell culture.

[0018] A further object of the present invention is to provide a method for degrading polyester, comprising:

[0019] (a) contacting a 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

[0020] (b) recovering monomers and / or oligomers.

[0021] In particular, the present invention provides a method for degrading PET, which comprises contacting PET with at least one esterase of the present invention, and optionally recovering monomers and / or oligomers of PET.

[0022] The present invention also relates to a method for degrading at least one polyester of a polyester-containing material, comprising the following steps:

[0023] (a) contacting a polyester-containing material with an esterase or a host cell according to the present invention, thereby degrading at least one polyester of the polyester-containing material; and optionally

[0024] (b) recovering monomers and / or oligomers of the at least one polyester.

[0025] The present invention also relates to the use of the esterase of the present invention in degrading PET or plastic products containing PET.

[0026] The present invention also relates to polyester-containing materials comprising an esterase or a host cell or a composition of the present invention.

[0027] The invention further relates to a detergent composition comprising an esterase according to the invention or a host cell or a composition comprising an esterase according to the invention. DETAILED DESCRIPTION OF THE INVENTION

[0029] definition

[0030] The present disclosure will be best understood by reference to the following definitions.

[0031] As used herein, the terms "peptide", "polypeptide", "protein", "enzyme" refer to a chain of amino acids linked by peptide bonds, regardless of the number of amino acids forming the chain. As used herein, amino acids are represented 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).

[0032] The term "esterase" refers to an enzyme belonging to the class of hydrolases classified as EC 3.1.1 according to enzyme nomenclature, which catalyzes the hydrolysis of esters into acids and alcohols. The term "cutinase" or "cutin hydrolase" refers to an esterase classified as EC 3.1.1.74 according to enzyme nomenclature, which is capable of catalyzing the chemical reaction of producing cutin monomers from cutin and water.

[0033] The term "wild-type protein" or "parent protein" refers to a non-mutated form of a naturally occurring polypeptide. In the present case, the parent esterase refers to an esterase having an amino acid sequence as shown in SEQ ID N°1.

[0034] The terms "mutant" and "variant" refer to a polypeptide derived from SEQ ID N°1 and comprising at least one modification or alteration, i.e., substitution, insertion and / or deletion, at one or more (e.g., several) positions and having polyester degradation activity. Variants can be obtained by various techniques well known in the art. In particular, examples of techniques for altering the DNA sequence encoding the wild-type protein include, but are not limited to, site-directed mutagenesis, random mutagenesis, and synthetic oligonucleotide construction.

[0035] Therefore, the terms "modification" and "alteration" as used herein with respect to a specific position means that the amino acid at that specific position has been modified compared to the amino acid at that specific position in the wild-type protein.

[0036] "Substitution" refers to the replacement of an amino acid residue by another amino acid residue. Preferably, the term "substitution" refers to the replacement of an amino acid residue by another amino acid residue, wherein the other amino acid residue is selected from the 20 naturally occurring standard amino acid residues, the naturally occurring rare amino acid residues (e.g., hydroxyproline, hydroxylysine, allohydroxylysine, 6-N-methyllysine, N-ethylglycine, N-methylglycine, N-ethylasparagine, alloisoleucine, N-methylisoleucine, N-methylvaline, pyroglutamine, aminobutyric acid, ornithine, norleucine, norvaline) and usually synthetic non-naturally occurring amino acid residues (e.g., cyclohexyl-alanine). Preferably, the term "substitution" refers to the replacement of an amino acid residue by another selected from the 20 naturally occurring standard amino acid residues (G, P, A, V, L, I, M, C, F, Y, W, H, K, R, Q, N, E, D, S and T). The symbol "+" represents a combination of substitutions. In this document, the following terms are used to represent substitutions: L82A means that the 82nd amino acid residue (leucine, L) of the parent sequence is replaced by alanine (A). A121V / I / M means that the 121st amino acid residue (alanine, A) of the parent sequence is replaced by one of the following amino acids: valine (V), isoleucine (I) or methionine (M). Substitutions can be conservative or non-conservative substitutions. Examples of conservative substitutions are made within the following groups: 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).

[0037] Unless otherwise indicated, positions disclosed in this application are numbered with reference to the amino acid sequence shown in SEQ ID N°1.

[0038] As used herein, the term "sequence identity" or "identity" refers to the number (or fraction expressed as a percentage %) of matches (identical amino acid residues) between two polypeptide sequences. Sequence identity is determined by comparing the sequences when aligned so as to maximize overlap and identity while minimizing sequence gaps. In particular, sequence identity can be determined using any of a number of mathematical global or local alignment algorithms based on the length of the two sequences. It is preferred to use a global alignment algorithm that optimally aligns sequences over their entire length (e.g., the Needleman and Wunsch algorithm; Needleman and Wunsch, 1970) to align sequences of similar length, while it is preferred to use a local alignment algorithm (e.g., the Smith and Waterman algorithm (Smith and Waterman, 1981) or the Altschul algorithm (Altschul et al., 1997; Altschul et al., 2005)) to align sequences of substantially different lengths. Alignment for determining percentage of amino acid sequence identity can be achieved in various ways within the skill of the art, for example, using Internet websites such as http: / / blast.ncbi.nlm.nih.gov / or http: / / www.ebi.ac.uk / Tools / emboss / The public available computer software that can be obtained on the comparison. Those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithm required for maximum alignment on the full length of the compared sequence. For the purpose of this paper, % amino acid sequence identity value refers to the value produced using paired sequence alignment program EMBOSS Needle, which uses the Needleman-Wunsch algorithm to produce the best global alignment of two sequences, wherein all search parameters are set to default values, i.e., scoring matrix=BLOSUM62, gap open=10, gap extension=0.5, terminal gap penalty=error, terminal gap open=10 and terminal gap extension=0.5.

[0039] "Polymer" refers to a compound or mixture of compounds whose structure consists of multiple monomers (repeating units) connected by covalent chemical bonds. In the context of the present invention, the term polymer includes natural or synthetic polymers, consisting of a single type of repeating unit (i.e., homopolymer) or a mixture of different repeating units (i.e., copolymer or heteropolymer). According to the present invention, "oligomer" refers to a molecule containing 2 to about 20 monomers.

[0040] In the context of the present invention, "polyester-containing material" or "polyester-containing product" refers to a product, such as a plastic article, comprising at least one polyester in crystalline, semi-crystalline or completely amorphous form. In one embodiment, polyester-containing material refers to any article made of at least one plastic material, such as a plastic sheet, tube, rod, profile, shape, film, block, etc., which contains at least one polyester, and possibly other substances or additives, such as plasticizers, minerals or organic fillers. In another embodiment, polyester-containing material refers to a plastic compound or plastic preparation in a molten or solid state suitable for the manufacture of plastic articles. In another embodiment, polyester-containing material refers to a textile, fabric or fiber comprising at least one polyester. In another embodiment, polyester-containing material refers to plastic waste or fiber waste comprising at least one polyester.

[0041] In the present specification, the term "polyester" encompasses but is not limited to polyethylene terephthalate (PET), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polyisosorbide terephthalate (PEIT), polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), polycaprolactone (PCL), poly(ethylene adipate) (PEA), polyethylene naphthalate (PEN) and blends / mixtures of these polymers.

[0042] New esterase

[0043] The present invention provides novel esterases with improved activity and / or improved thermostability compared to the parent esterase. More particularly, the inventors have designed novel enzymes that are particularly suitable for industrial processes. The esterases of the present invention are particularly suitable for degrading polyesters, more particularly PET, including materials containing PET, particularly plastic products containing PET. In a specific embodiment, the esterases exhibit increased activity and increased thermostability.

[0044] Therefore, an object of the present invention is to provide an esterase showing increased activity compared to the esterase having the amino acid sequence shown in SEQ ID N°1.

[0045] In particular, the inventors identified specific amino acid residues in SEQ ID N°1 that are intended to contact the polymer substrate in the X-ray crystal structure of the esterase (i.e., the folded 3D structure), which X-ray crystal structure of the esterase can be advantageously modified to promote contact between the substrate and the esterase, thereby increasing the adsorption of the polymer and / or thereby increasing the activity of the esterase on the polymer.

[0046] In the context of the present invention, the term "increased activity" or "increased degradation activity" refers to an increased ability of the esterase to degrade polyester at a given temperature and / or an increased ability to adsorb on polyester compared to the ability of the esterase of SEQ ID N°1 to degrade the same polyester at the same temperature. In particular, the esterase of the present invention has an increased PET degradation activity. This increase may be at least 10% higher, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130% or more higher than the PET degradation activity of the esterase of SEQ ID N°1. In particular, the degradation activity is an activity that leads to the depolymerization of monomers and / or oligomers of the polyester, which can be further recovered and optionally reused.

[0047] According to methods known per se in the art, those skilled in the art can evaluate the "degradation activity" of esterases. For example, the degradation activity can be evaluated by measuring the depolymerization activity rate of a specific polymer, measuring the rate of degradation of a solid polymer compound dispersed in an agar plate, or measuring the depolymerization activity rate of a polymer in a reactor. In particular, the degradation activity can be evaluated by measuring the "specific degradation activity" of the esterase. The "specific degradation activity" of the esterase to PET corresponds to the equivalent TA / hour and per mg of esterase produced by μmol / min or mg of PET hydrolyzed in the initial stage of the reaction (i.e., the first 24 hours), and is determined by the linear portion of the reaction hydrolysis curve, which is established by several samplings performed at different times within the first 24 hours. As another example, the "degradation activity" can be evaluated by measuring the rate of oligomers and / or monomers released when a polymer or a plastic product containing a polymer is contacted with a degrading enzyme under suitable temperature, pH and buffer conditions after a defined period of time.

[0048] The ability of the enzyme to be adsorbed on a substrate can be assessed by a person skilled in the art according to methods known per se in the art. For example, the ability of the enzyme to be adsorbed on a substrate can be measured from an enzyme-containing solution, and wherein the enzyme has been previously incubated with the substrate under suitable conditions.

[0049] The inventors have also identified target amino acid residues in SEQ ID N° 1 which may advantageously be modified to improve the stability of the corresponding esterase at high temperatures (ie improved thermostability), and advantageously at temperatures above 50°C, preferably above 70°C.

[0050] Therefore, an object of the present invention is to provide novel esterases which show increased thermostability compared to the thermostability of the esterase having the amino acid sequence shown in SEQ ID N°1.

[0051] In the context of the present invention, the term "increased thermostability" refers to an increased ability of the esterase to resist changes in its chemical and / or physical structure at elevated temperatures, particularly at temperatures of 50°C-90°C, compared to the esterase of SEQ ID N°1.

[0052] In particular, thermal stability can be assessed by evaluating the melting temperature (Tm) of the esterase. In the context of the present invention, "melting temperature" refers to the temperature at which half of the enzyme population under consideration is unfolded or misfolded. Typically, the esterase of the present invention shows an increase in Tm of about 1°C, 2°C, 3°C, 4°C, 5°C, 10°C, 12°C or more compared to the Tm of the esterase of SEQ ID N°1. In particular, the esterase of the present invention can have an increased half-life at a temperature of 50°C-90°C compared to the esterase of SEQ ID N°1.

[0053] The melting temperature (Tm) of esterase can be measured by those skilled in the art according to methods known in the art per se. For example, DSF can be used to quantitatively measure the change in the thermal denaturation temperature of esterase, thereby determining its Tm. Alternatively, Tm can be assessed by analyzing protein folding using circular dichroism. Preferably, Tm is measured using DSF or circular dichroism described in the experimental section. In the context of the present invention, Tm comparisons are performed using Tm measured under the same conditions (e.g., pH, the nature and amount of polyester, etc.).

[0054] Alternatively, thermal stability can be assessed by measuring the esterase activity and / or polyester depolymerization activity of the esterase after incubation at different temperatures and comparing it to the esterase activity and / or polyester depolymerization activity of the parent esterase. The ability to perform multiple rounds of polyester depolymerization assays at different temperatures can also be assessed. A rapid and valuable test can include assessing the ability of the enzyme to degrade solid polyester compounds dispersed in agar plates after incubation at different temperatures by halo diameter measurements.

[0055] Therefore, one object of the present invention is to provide an esterase which (i) has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the full-length amino acid sequence shown in SEQ ID N°1, (ii) comprises at least four substitutions at positions selected from F208, D203, S248, V170, V177, T176, T61, S65, N211 or Y92 compared to the amino acid sequence SEQ ID N°1, and (iii) exhibits increased polyester degradation activity and / or increased thermal stability compared to the esterase of SEQ ID N°1.

[0056] According to the present invention, the targeted amino acid can be substituted by any of 19 other amino acids.

[0057] In a particular embodiment, the esterase variant comprises at least four substitutions selected from F208I / W, D203C, S248C, V170I, V177I, T176N, T61M, S65T or Y92G / P.

[0058] In another specific embodiment, the esterase variant comprises at least four substitutions selected from F208I / W, D203C, S248C, V170I, V177I, T176N, T61M, S65T, N211D / M or Y92G / P or Y92F.

[0059] In a preferred embodiment, the esterase of the present invention comprises at least one amino acid residue selected from S130, D175 or H207 as in the parent esterase, i.e. the esterase of the present invention is not modified at one, two or all of these positions. Preferably, the esterase comprises the combination S130+D175+H207 as in the parent esterase.

[0060] In a specific embodiment, the esterase comprises at least a combination of substitutions at positions F208+D203+S248 and one substitution at a position selected from V170, V177, T176, T61, S65, N211 or Y92. In a specific embodiment, the esterase comprises a combination of substitutions at least one position selected from F208+D203+S248+V170, F208+D203+S248+V177, F208+D203+S248+T61, F208+D203+S248+Y92, F208+D203+S248+T176, F208+D203+S248+S65, F208+D203+S248+N211 or F208+D203+S248+V170+Y92. In particular, the esterase comprises at least a combination of substitutions selected from F208I+D203C+S248C or F208W+D203C+S248C and one substitution selected from T61M, V170I, V177I, T176N, S65T, N211D / M or Y92G / P / F.

[0061] According to a specific embodiment, the esterase comprises at least a substituted combination selected from the group consisting of: F208I+D203C+S248C+V170I, F208W+D203C+S248C+V170I, F208I+D203C+S248C+V177I, F208W+D203C+S248C+V177I, F208I+D203C+S248C+Y92G, F208W+D203C+S248C+Y92G, F208I+D203C+S248C+Y92F, F208W+D203C+S248C+V177I. 8C+T61M, F208W+D203C+S248C+T61M, F208I+D203C+S248C+T176N, F208W+D203C+S248C+T176N, F208I+D203C+S248C+S65T and F208W+D203C+S248C+S65T.

[0062] According to a specific embodiment, the esterase comprises at least a combination of substitutions selected from the group consisting of: F208I+D203C+S248C+N211D, F208W+D203C+S248C+N211D, F208I+D203C+S248C+N211M and F208W+D203C+S248C+N211M.

[0063] In one embodiment, the esterase comprises at least a substitution at position F208+D203+S248 and a combination of one or two substitutions at position V170 or Y92. Preferably, the esterase comprises at least a substitution selected from F208I+D203C+S248C or F208W+D203C+S248C and a combination of one or two substitutions selected from V170I or Y92G. In particular, the esterase comprises a combination of substitutions selected from F208I+D203C+S248C+V170I, F208I+D203C+S248C+Y92G or F208I+D203C+S248C+V170I+Y92G. Alternatively, the esterase comprises a combination of substitutions selected from F208W+D203C+S248C+V170I, F208W+D203C+S248C+Y92G or F208W+D203C+S248C+V170I+Y92G.

[0064] In a specific embodiment, the variant of the invention further comprises at least one substitution at a position selected from the group consisting of: T11, R12, A14, W69, R73, A205, N214, A215, A216, 1217, F238, V242, D244, P245, A246, L247, D94, R138, D158, Q182, F187, P10, L15, D18, N87, S88, S95, Q99, K159, A174, A125, S218, S13, T16, L202, N204, S212, 252, E173, G53, A121, T157, N211, Y60, D63, or S66.

[0065] In a specific embodiment, the variant of the invention further comprises at least one substitution at a position selected from the group consisting of: N213, N211, A121, N204, S212, A125, G135, W69, N214, N241, N243, R12, P179, V242 or V167.

[0066] In one embodiment, the esterase of the present invention further comprises the substitution N213P / D, preferably N213P. In particular, the esterase comprises at least a combination of substitutions selected from the group consisting of F208I+D203C+S248C+V170I+Y92G+N213P, F208I+D203C+S248C+V170I+N213P or F208I+D203C+S248C+Y92G+N213P. Preferably, the esterase comprises a combination of substitutions consisting of F208I+D203C+S248C+V170I+Y92G+N213P.

[0067] In one embodiment, the esterase of the present invention further comprises the substitution N211D / M. In particular, the variant comprises at least a combination of substitutions selected from the group consisting of F208I / W+D203C+S248C+V170I+Y92G+N211D / M, F208I / W+D203C+S248C+V170I+N211D / M or F208I / W+D203C+S248C+Y92G+N211D / M. Preferably, the variant comprises at least a combination of substitutions selected from the group consisting of: F208I / W+D203C+S248C+V170I+Y92G+N211M, F208I / W+D203C+S248C+V170I+N211M or F208I / W+D203C+S248C+Y92G+N211M.

[0068] In one embodiment, the esterase of the invention further comprises the substitution A121 S. In particular, the variant comprises at least a combination of substitutions selected from the group consisting of F208I+D203C+S248C+V170I+Y92G+A121S, F208I+D203C+S248C+V170I+A121S or F208I+D203C+S248C+Y92G+A121S.

[0069] In one embodiment, the esterase of the present invention further comprises the substitution N204D / I / L / Y / H / F. In particular, the variant comprises at least a combination of substitutions selected from the group consisting of F208I+D203C+S248C+V170I+Y92G+N204D / I / L / Y / H / F, F208I+D203C+S248C+V170I+N204D, F208I+D203C+S248C+Y92G+N204D.

[0070] In one embodiment, the esterase of the present invention further comprises the substitution S212F / T / I / L. In particular, the variant comprises at least a combination of substitutions selected from the group consisting of F208I+D203C+S248C+V170I+Y92G+S212F / T / I / L, F208I+D203C+S248C+V170I+S212F or F208I+D203C+S248C+Y92G+S212F.

[0071] In one embodiment, the esterase of the invention comprises the substitution A125G. In particular, the variant comprises at least a combination of substitutions selected from the group consisting of F208I+D203C+S248C+V170I+Y92G+A125G, F208I+D203C+S248C+V170I+A125G or F208I+D203C+S248C+Y92G+A125G.

[0072] In one embodiment, the esterase of the invention comprises the substitution G135A. In particular, the variant comprises at least the combination of the substitutions F208I+D203C+S248C+V170I+Y92G+G135A.

[0073] In one embodiment, the esterase of the invention comprises the substitution W69R. In particular, the variant comprises at least the combination of the substitutions F208I+D203C+S248C+V170I+Y92G+W69R, F208I+D203C+S248C+V170I+W69R or F208I+D203C+S248C+Y92G+W69R.

[0074] In one embodiment, the esterase of the invention comprises the substitutions N214D / I / L / F / Y / H. In particular, the variant comprises at least the combination of the substitutions F208I+D203C+S248C+V170I+Y92G+N214D / I / L / F / Y / H.

[0075] In one embodiment, the esterase of the invention comprises the substitution N241 P. In particular, the variant comprises at least the combination of the substitutions F208I+D203C+S248C+V170I+Y92G+N241P.

[0076] In one embodiment, the esterase of the invention comprises the substitution N243P. In particular, the variant comprises at least the combination of the substitutions F208I+D203C+S248C+V170I+Y92G+N243P.

[0077] In one embodiment, the esterase of the invention comprises the substitutions R12F / Y / H. In particular, the variant comprises at least the combination of substitutions F208I+D203C+S248C+V170I+Y92G+R12F / Y / H.

[0078] In one embodiment, the esterase of the invention comprises the substitution P179E. In particular, the variant comprises at least the combination of the substitutions F208I+D203C+S248C+V170I+Y92G+P179E.

[0079] In one embodiment, the esterase of the invention comprises the substitution V242Y. In particular, the variant comprises at least the combination of the substitutions F208I+D203C+S248C+V170I+Y92G+V242Y.

[0080] In one embodiment, the esterase of the invention comprises the substitution V167Q. In particular, the variant comprises at least the combination of the substitutions F208I+D203C+S248C+V170I+Y92G+V167Q.

[0081] In one embodiment, the esterase of the invention comprises the substitution A140S.

[0082] In one embodiment, the esterase of the invention further comprises at least one substitution at a position selected from the group consisting of: T11, R12, A14, W69, R73, A205, N214, A215, A216, F238, V242, D244, P245, A246, L247, Q182, F187 or S218. More preferably, the substitution is selected from T11M / E / I / S / N / D / Q, R12Q / D / N / G / P / F / V / E / L / Y, R12H, A14E / D, W69D / M / E / R, R73I / G / M / D / E / S / C / Q / F / N / V, A205D, N214D / E / C, N214I / L / F / Y / H, A215N, A216Q, F238E, V242P / Y, D244E / C, P245D / Y / E, A246S / D / H / E, L247T, Q182D / E, F187Y / I or S218A. In one embodiment, the esterase comprises at least one substitution selected from the group consisting of T11M / I / S / N / D, R12N / G / P / V / L, A14E, W69M, R73I / G / D / S / C / Q / F / N / V, A205D, N214E / C, A215N, P245Y, or A246D / H. In another specific embodiment, the variant of the invention further comprises at least two substitutions at positions selected from the group consisting of: T11, R12, A14, W69, R73, A205, N214, A215, A216, I217, F238, V242, D244, P245, A246, L247, D94, R138, D158, Q182, F187, P10, L15, D18, N87, S88, S95, Q99, K159, A174, A125, S218, S13, T16, L202, N204, S21 2. V219, Y220, Q237, L239, N241, N243, A62, L67, D91, P93, M131, P210, A209, P179, R30, G37, R72, S98, A68, R96, H156, H183, A17, T27, S48, F90, L82, G135, A140, N143, S145, A149, S164, V167, S206, N213, T252, E173, G53, A121, T157, N211, Y60, D63 or S66.

[0083] In one embodiment, the esterase of the present invention further comprises a combination of substitutions S212F+N213P. In particular, the variant comprises at least a combination of substitutions selected from the group consisting of F208I+D203C+S248C+V170I+S212F+N213P, F208I+D203C+S248C+Y92G+S212F+N213P or F208I+D203C+S248C+V170I+Y92G+S212F+N213P.

[0084] In one embodiment, the esterase of the invention comprises at least two substitutions selected from N213P, G135A, A140S, V167Q, N241P or R12H. In particular, the variant comprises at least a combination of substitutions selected from the group consisting of F208I+D203C+S248C+V170I+Y92G+N213P+G135A, F208I+D203C+S248C+V170I+Y92G+R12H+N241P, F208I+D203C+S248C+V170I+Y92G+R12H+V167Q, F208I+D203C+S248C+V170I+Y92G+A140S+V167Q or F208I+D203C+S248C+V170I+Y92G+N241P+V167Q.

[0085] In one embodiment, the esterase of the present invention comprises at least three substitutions selected from N213P, G135A, V167Q, N241P or R12H. In particular, the variant comprises at least a combination of substitutions selected from the group consisting of F208I+D203C+S248C+V170I+Y92G+N213P+G135A+V167Q, F208I+D203C+S248C+V170I+Y92G+N213P+G135A+N241P, F208I+D203C+S248C+V170I+Y92G+N213P+G135A+R12H or F208I+D203C+S248C+V170I+Y92G+N241P+V167Q+R12H.

[0086] In one embodiment, the esterase of the invention comprises at least one substitution selected from A17T, T27S, S48T, F90L, L82I, G135A, A140S, N143I, S145T, A149G, S164P, V167Q, S206T, N213P or T252S, preferably at least two substitutions. In particular, the variant comprises at least a combination of substitutions selected from the group consisting of: F208I+D203C+S248C+Y92F+A17T+T27S+S48T+L82I+G135A+A140S+N143I+S145T+A149G+S164P+V167Q+S206T+N213P+T252S, F208I+D203C+S248C+Y92G+A17T+T27S+S48T+L82I+ F90L+G135A+A140S+N143I+S145T+A149G+S164P+V167Q+S206T+N213P+T252S or F208I+D203C+S248C+Y92F+T27S+S48T+L82I+F90L+G135A+A140S+N143I+S145T+A149G+S164P+V167Q+S206T+N213P+T252S.

[0087] In another specific embodiment, the esterase further comprises one or more substitutions or combinations of substitutions as cited in WO 2018 / 011284 and / or WO 2018 / 011281. In a further embodiment, the esterase of the present invention further comprises at least a combination of amino acids selected from C240+C257 or S130+D175+H207+C240+C257 as in the parent esterase, i.e., the esterase of the present invention is not modified at these positions compared to SEQ ID N°1.

[0088] In another specific embodiment, the esterase of the invention further comprises at least one amino acid residue selected from the group consisting of G59, Y60, T61, D63, S65, S66, N85, T86, R89, F90, H129, W155, T157, T176, V177, A178 and N211 as in the parent esterase, i.e. the esterase of the invention is not modified at one of these positions compared to SEQ ID N° 1. Preferably, the esterase comprises amino acid residue F90 as in the parent esterase.

[0089] A further object of the present invention is to provide an esterase which (i) has the amino acid sequence shown in SEQ ID N°2, (ii) has at least four substitutions at positions selected from F208, D203, S248, V170, V177, T176, S65, T61 or F92, wherein the positions are numbered with reference to the amino acid sequence shown in SEQ ID N°2, and (iii) exhibits increased polyester degradation activity and / or increased thermal stability compared to the esterase of SEQ ID N°1.

[0090] A further object of the present invention is to provide an esterase which (i) has the amino acid sequence shown in SEQ ID N°2, (ii) has at least four substitutions at positions selected from F208, D203, S248, V170, V177, T176, S65, T61, N211 or F92, wherein the positions are numbered by reference to the amino acid sequence shown in SEQ ID N°2, and (iii) exhibits increased polyester degradation activity and / or increased thermal stability compared to the esterase of SEQ ID N°1.

[0091] The amino acid sequence shown in SEQ ID N°2 corresponds to a variant of the amino acid sequence of SEQ ID N°1, which has a combination of substitutions A17T+T27S+S48T+L82I+F90L+Y92F+G135A+A140S+N143I+S145T+A149G+S164P+V167Q+S206T+N213P+T252S compared to SEQID N°1.

[0092] In a particular embodiment, the esterase variant comprises at least four substitutions selected from F208I / W, D203C, S248C, V170I, V177I, T176N, T61M, S65T or F92G / P compared to the esterase of SEQ ID N°2.

[0093] In a particular embodiment, the esterase variant comprises at least four substitutions selected from F208I / W, D203C, S248C, V170I, V177I, T176N, T61M, S65T, N211D / M or F92G / P compared to the esterase of SEQ ID N°2.

[0094] In a preferred embodiment, the esterase of the invention comprises at least one amino acid residue selected from S130, D175 or H207 as in SEQ ID N°2, i.e. the esterase of the invention is not modified at one, two or all of these positions. Preferably, the esterase comprises the combination S130+D175+H207 as in SEQ ID N°2.

[0095] In a specific embodiment, the esterase comprises at least a combination of substitutions at positions F208+D203+S248 and one substitution at a position selected from V170, V177, T176, T61 or F92 compared to the esterase of SEQ ID N° 2. In a specific embodiment, the esterase comprises at least a combination of substitutions at positions F208+D203+S248 and one substitution at a position selected from V170, V177, T176, T61, S65, N211 or F92 compared to the esterase of SEQ ID N° 2. In a specific embodiment, the esterase comprises at least one combination of substitutions at a position selected from the group consisting of F208+D203+S248+V170, F208+D203+S248+V177, F208+D203+S248+T61, F208+D203+S248+F92, F208+D203+S248+T176, F208+D203+S248+S65, F208+D203+S248+N211 or F208+D203+S248+V170+F92. In particular, the esterase comprises at least a combination of substitutions selected from F208I+D203C+S248C or F208W+D203C+S248C and one substitution selected from T61M, V170I, V177I, T176N, S65T, N211D / M or F92G / P.

[0096] According to a specific embodiment, the esterase comprises at least a combination of substitutions selected from the group consisting of the following combinations, compared to the esterase of SEQ ID N° 2: F208I+D203C+S248C+V170I, F208W+D203C+S248C+V170I, F208I+D203C+S248C+V177I, F208W+D203C+S248C+V177I, F208I+D203C+S248C+F92G, F208W+D203C+S248C+F92G, 248C+V170I+F92G, F208W+D203C+S248C+V170I+F92G, F208I+D203C+S248C+T61M, F208W+D203C+S248C+T61M, F208I+D203C+S248C+T176N, F208W+D203C+S248C+T176N, F208I+D203C+S248C+S65T and F208W+D203C+S248C+S65T.

[0097] According to a specific embodiment, the esterase comprises at least a combination of substitutions selected from the following combinations compared to the esterase of SEQ ID N°2: F208I+D203C+S248C+N211D, F208W+D203C+S248C+N211D, F208I+D203C+S248C+N211M, F208W+D203C+S248C+N211M.

[0098] In a specific embodiment, the esterase comprises at least a substitution at position F208+D203+S248 and a combination of one or two substitutions at a position selected from V170 or F92. Preferably, the esterase comprises at least a substitution at a position selected from F208I+D203C+S248C or F208W+D203C+S248C and a combination of one or two substitutions selected from V170I or F92G. In particular, the esterase comprises at least one combination of substitutions selected from F208I+D203C+S248C+V170I, F208I+D203C+S248C+F92G or F208I+D203C+S248C+V170I+F92G. Alternatively, the esterase comprises a combination of substitutions selected from the group consisting of: F208W+D203C+S248C+V170I, F208W+D203C+S248C+F92G, or F208W+D203C+S248C+V170I+F92G.

[0099] In a specific embodiment, the variant of the invention further comprises at least one substitution at a position selected from the group consisting of: T11, R12, A14, W69, R73, A205, N214, A215, A216, I217, F238, V242, D244, P245, A246, L247, D94, R138, D158, Q182, F187, P10, L15, D18, N87, S88, S95, Q99, K159, A174, A12 5, S218, S13, T16, L202, N204, S212, V219, Y220, Q237, L239, N241, N243, A62, L67, D91, P93, M131, P210, A209, P179, R30, G37, R72, S98, A68, R96, H156, H183, E173, G53, A121, T157, N211, Y60, D63 or S66, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID N°2.

[0100] In a specific embodiment, the variant of the invention further comprises at least one substitution at a position selected from the group consisting of: N211, A121, N204, S212, A125, W69, N214, N241, N243, R12, P179 or V242.

[0101] In one embodiment, the esterase of the invention comprises the substitution N211D / M compared to the esterase of SEQ ID N° 2. In particular, the variant comprises at least a combination of substitutions selected from the group consisting of F208I / W+D203C+S248C+V170I+F92G+N211D / M, F208I / W+D203C+S248C+V170I+N211D / M or F208I / W+D203C+S248C+F92G+N211D / M. Preferably, the variant comprises at least a combination of substitutions selected from the group consisting of: F208I / W+D203C+S248C+V170I+F92G+N211M, F208I / W+D203C+S248C+V170I+N211M or F208I / W+D203C+S248C+F92G+N211M.

[0102] In one embodiment, the esterase of the invention comprises the substitution A121S compared to the esterase of SEQ ID N° 2. In particular, the variant comprises at least a combination of substitutions selected from the group consisting of F208I+D203C+S248C+V170I+F92G+A121S, F208I+D203C+S248C+V170I+A121S or F208I+D203C+S248C+F92G+A121S.

[0103] In one embodiment, the esterase of the invention comprises the substitution N204D / I / L / Y / H / F compared to the esterase of SEQ ID N° 2. In particular, the variant comprises at least a combination of substitutions selected from the group consisting of F208I+D203C+S248C+V170I+F92G+N204D / I / L / Y / H / F, F208I+D203C+S248C+V170I+N204D or F208I+D203C+S248C+F92G+N204D.

[0104] In one embodiment, the esterase of the invention comprises the substitution S212F / T / I / L compared to the esterase of SEQ ID N° 2. In particular, the variant comprises at least a combination of substitutions selected from the group consisting of F208I+D203C+S248C+V170I+F92G+S212F / T / I / L, F208I+D203C+S248C+V170I+S212F or F208I+D203C+S248C+F92G+S212F.

[0105] In one embodiment, the esterase of the invention comprises the substitution A125G compared to the esterase of SEQ ID N° 2. In particular, the variant comprises at least a combination of substitutions selected from the group consisting of F208I+D203C+S248C+V170I+F92G+A125G, F208I+D203C+S248C+V170I+A125G or F208I+D203C+S248C+F92G+A125G.

[0106] In one embodiment, the esterase of the invention comprises the substitution W69R compared to the esterase of SEQ ID N° 2. In particular, the variant comprises at least a combination of substitutions selected from the group consisting of F208I+D203C+S248C+V170I+F92G+W69R, F208I+D203C+S248C+V170I+W69R or F208I+D203C+S248C+F92G+W69R.

[0107] In one embodiment, the esterase of the invention comprises the substitutions N214D / I / L / F / Y / H compared to the esterase of SEQ ID N° 2. In particular, the variant comprises at least the combination of the substitutions F208I+D203C+S248C+V170I+F92G+N214D / I / L / F / Y / H.

[0108] In one embodiment, the esterase of the invention comprises the substitution N241P compared to the esterase of SEQ ID N° 2. In particular, the variant comprises at least the combination of the substitutions F208I+D203C+S248C+V170I+F92G+N241P.

[0109] In one embodiment, the esterase of the invention comprises the substitution N243P compared to the esterase of SEQ ID N° 2. In particular, the variant comprises at least the combination of the substitutions F208I+D203C+S248C+V170I+F92G+N243P.

[0110] In one embodiment, the esterase of the invention comprises the substitutions R12F / Y / H compared to the esterase of SEQ ID N° 2. In particular, the variant comprises at least the combination of substitutions F208I+D203C+S248C+V170I+F92G+R12F / Y / H.

[0111] In one embodiment, the esterase of the invention comprises the substitution P179E compared to the esterase of SEQ ID N° 2. In particular, the variant comprises at least the combination of the substitutions F208I+D203C+S248C+V170I+F92G+P179E.

[0112] In one embodiment, the esterase of the invention comprises the substitution V242Y compared to the esterase of SEQ ID N° 2. In particular, the variant comprises at least the combination of the substitutions F208I+D203C+S248C+V170I+F92G+V242Y.

[0113] In one embodiment, the esterase of the invention further comprises at least one substitution at a position selected from the group consisting of: T11, R12, A14, W69, R73, A205, N214, A215, A216, F238, V242, D244, P245, A246, L247, Q182, F187 or S218. The substitutions are more preferably selected from T11M / E / I / S / N / D / Q, R12Q / D / N / G / P / F / V / E / L / Y, R12H, A14E / D, W69D / M / E / R, R73I / G / M / D / E / S / C / Q / F / N / V, A205D, N214D / E / C, N214I / L / F / Y / H, A215N, A216Q, F238E, V242P / Y, D244E / C, P245D / Y / E, A246S / D / H / E, L247T, Q182D / E, F187Y / I or S218A. In one embodiment, the esterase comprises at least one substitution selected from the group consisting of T11M / I / S / N / D, R12N / G / P / V / L, A14E, W69M, R73I / G / D / S / C / Q / F / N / V, A205D, N214E / C, A215N, P245Y, or A246D / H.

[0114] In one embodiment, the variant of the invention further comprises at least two substitutions at positions selected from the group consisting of: T11, R12, A14, W69, R73, A205, N214, A215, A216, I217, F238, V242, D244, P245, A246, L247, D94, R138, D158, Q182, F187, P10, L15, D18, N87, S88, S95, Q99, K159, A174, A125 , S218, S13, T16, L202, N204, S212, V219, Y220, Q237, L239, N241, N243, A62, L67, D91, P93, M131, P210, A209, P179, R30, G37, R72, S98, A68, R96, H156, H183, E173, G53, A121, T157, N211, Y60, D63 or S66, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID N°2.

[0115] In one embodiment, the esterase of the invention comprises at least the combination of substitutions N241P+R12H compared to the esterase of SEQ ID N° 2. In particular, the variant comprises at least the combination of substitutions F208I+D203C+S248C+V170I+F92G+N241P+R12H.

[0116] In a particular embodiment, the esterase further comprises one or more substitutions or combinations of substitutions as recited in WO 2018 / 011284 and / or WO 2018 / 011281.

[0117] In a further embodiment, the esterase of the present invention further comprises at least one combination of amino acid residues selected from C240+C257 or S130+D175+H207+C240+C257 as in SEQ ID N°2, i.e. the esterase of the present invention is not modified at these positions compared to SEQ ID N°2.

[0118] In another embodiment, the esterase of the present invention further comprises at least one amino acid residue selected from the group consisting of G59, Y60, T61, D63, S65, S66, N85, T86, R89, H129, W155, T157, T176, V177, A178 and N211 as in SEQ ID N°2, i.e. the esterase of the present invention is not modified at one of these positions compared to SEQ ID N°2.

[0119] In a specific embodiment, the esterase of the present invention derived from SEQ ID N°1 or SEQ ID N°2 further comprises at the N-terminus an amino acid sequence having at least 55%, 65%, 75%, 85% or 100% identity with the full-length amino acid sequence shown in SEQ ID N°3. In particular, the esterase may comprise at the N-terminus an amino acid sequence selected from the group consisting of an amino acid sequence as shown in SEQ ID N°3, SEQ ID N°4, SEQ ID N°5, SEQ ID N°6 or SEQ ID N°7. In particular, the esterase of the present invention derived from SEQ ID N°1 comprises at least a combination of substitutions selected from the group consisting of F208I+D203C+S248C+V170I+Y92G, F208I+D203C+S248C+V170I or F208I+D203C+S248C+Y92G; and comprises SEQ ID N°5 (SPSVEAQ) at the N-terminus. In particular, the esterase of the present invention derived from SEQ ID N°2 comprises at least a combination of substitutions selected from the group consisting of: F208I+D203C+S248C+V170I+F92G, F208I+D203C+S248C+V170I or F208I+D203C+S248C+F92G; and comprises SEQ ID N°5 (SPSVEAQ) at the N-terminus.

[0120] In a specific embodiment, the esterase variant comprises a combination of substitutions selected from the group consisting of F208I+D203C+S248C+V170I, F208I+D203C+S248C+Y92G, F208I+D203C+S248C+V170I+Y92G, F208I+D203C+S248C+V177I, F208W+D203C+S248C+Y92G, F208W+D203C+S248C+V177I, F208I+D203C+S248C+N211M, F208W+D203C+S248C+N211M. Compared to the esterase of SEQ ID N°1 or SEQ ID N°2, these esterases show increased polyester degradation activity and increased thermal stability.

[0121] In a specific embodiment, the esterase variant comprises a combination of substitutions selected from the group consisting of: F208I+D203C+S248C+V170I, F208I+D203C+S248C+Y92G, F208I+D203C+S248C+V170I+Y92G, F208I+D203C+S248C+V177I, F208W+D203C+S248C+Y92G, F208W+D203C+S248C +V177I、F208I+D203C+S248C+N211M、F208W+D203C+S248C+N211M、F208W+D203C+S248C+V170I、F208W +D203C+S248C+T176N, F208I+D203C+S248C+T176N, F208I+D203C+S248C+V170I+A121S, F208I+D203C+ S248C+Y92G+A121S, F208W+D203C+S248C+S65T, F208I+D203C+S248C+S65T, F208I+D203C+S248C+V17 0I+Y92G+N204H, F208I+D203C+S248C+V170I+Y92G+N241P, F208I+D203C+S248C+V170I+Y92G+N243P, F 208I+D203C+S248C+V170I+Y92G+R12F, F208I+D203C+S248C+V170I+Y92G+R12Y, F208I+D203C+S248C+V170I+Y92G+P179E, F208I+D203C+S248C+V170I+Y92G+V242Y, F208I+D203C+S248C+V170I+SEQ ID N°5 at the N-terminus, F208I+D203C+S248C+Y92G+SEQ IDN°5、F208I+D203C+S248C+V170I+Y92G+V167Q、F208I+D203C+S248C+V170I+Y92G+N213P+G 135A+V167Q, F208I+D203C+S248C+V170I+Y92G+N213P+G135A+N241P, F208I+D203C+S248C +V170I+Y92G+N213P+G135A+R12H, F208I+D203C+S248C+V170I+Y92G+R12H+N241P, F208I+D203C+S248C+V170I+Y92G+R12H+V167Q or F208I+D203C+S248C+V170I+Y92G+A140S+V167Q. Compared with the esterase of SEQ ID N°1 or SEQ ID N°2, these esterases show increased polyester degradation activity and increased thermal stability.

[0122] Preferably, the esterase variant comprises a combination of substitutions selected from the group consisting of: F208W+D203C+S248C+V177I, F208W+D203C+S248C+T176N, F208W+D203C+S248C+S65T, F208I+D203C+S248C+V170I+Y92G+N213P+G135A+N 241P, F208I+D203C+S248C+V170I+Y92G+N213P+G135A+R12H, F208I+D203C+S248C+Y92G, F208I+D203C+S248C+V170I+Y92G, F208I+D203C+S248C+Y92G+SEQ ID N°5 at the N-terminus, or F208I+D203C+S248C+V170I+Y92G+N213P+G135A+V167Q.

[0123] Polyester degradation activity of variants

[0124] It is an object of the present invention to provide novel enzymes having esterase activity. In a particular embodiment, the enzyme of the present invention exhibits cutinase activity.

[0125] In a specific embodiment, the esterase of the present invention has polyester degradation activity, preferably polyethylene terephthalate (PET) degradation activity and / or polybutylene adipate terephthalate (PBAT) degradation activity and / or polybutylene succinate (PBS) degradation activity and / or polycaprolactone (PCL) degradation activity, more preferably polyethylene terephthalate (PET) degradation activity and / or polybutylene adipate terephthalate (PBAT) degradation activity and / or polycaprolactone (PCL) degradation activity. Even more preferably. The esterase of the present invention has polyethylene terephthalate (PET) degradation activity.

[0126] Advantageously, the esterase of the present invention exhibits polyester degradation activity at least in the temperature range of 20°C-90°C, preferably 40°C-80°C, more preferably 50°C-70°C, even more preferably 60°C-70°C. In a specific embodiment, the esterase exhibits polyester degradation activity at 65°C. In a specific embodiment, the esterase exhibits polyester degradation activity at 70°C. In a specific embodiment, the polyester degradation activity can still be measured at a temperature of 60°C-90°C.

[0127] In a particular embodiment, the esterase of the present invention has increased polyester degradation activity at a given temperature, more particularly, at a temperature of 40°C-80°C, more preferably at 50°C-70°C, even more preferably at 60°C-70°C, even more preferably at 65°C, compared to the esterase of SEQ ID N°1.

[0128] In a particular embodiment, the polyester degradation activity of the esterase at 65°C is at least 5%, preferably at least 10%, 20%, 50%, 100%, 130% or more higher than the polyester degradation activity of the esterase of SEQ ID N°1.

[0129] In a particular embodiment, the esterase of the present invention exhibits 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.

[0130] Nucleic acid, expression cassette, vector, host cell

[0131] Another object of the present invention is to provide a nucleic acid encoding an esterase as defined above.

[0132] 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 can be DNA (cDNA or gDNA), RNA or a mixture thereof. It can be in single-stranded form or in double-stranded form or a mixture thereof. It can be of recombinant, artificial and / or synthetic origin, and it can contain modified nucleotides, including, for example, modified bonds, modified purine or pyrimidine bases, or modified sugars. The nucleic acids of the present invention can be in isolated or purified form and can be prepared, isolated and / or manipulated by techniques known per se in the art, such as cloning and expression of cDNA libraries, amplification, enzymatic synthesis or recombinant techniques. The nucleic acids can also be synthesized in vitro by well-known chemical synthesis techniques as described in Belousov (1997) Nucleic Acids Res. 25: 3440-3444.

[0133] The invention also encompasses nucleic acids that hybridize under stringent conditions to nucleic acids encoding esterases as defined above. Preferably, such stringent conditions comprise incubating the hybridization filter in 2×SSC / 0.1% SDS at about 42° C. for about 2.5 hours, followed by washing the filter 4 times for 15 minutes each at 65° C. in 1×SSC / 0.1% SDS. The protocols used are described in the references of Sambrook et al. (Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor NY (1988)) and Ausubel (Current Protocols in Molecular Biology (1989)).

[0134] The present invention also encompasses nucleic acids encoding the esterases of the present invention, wherein the sequence of said nucleic acid or at least a portion of said sequence has been engineered with optimized codon usage.

[0135] Alternatively, nucleic acids according to the invention can be derived from the sequences of esterases according to the invention, and codon usage can be adjusted according to the host cell in which the nucleic acid should be transcribed. These steps can be carried out according to methods well known to those skilled in the art, some of which are described in the reference manual of Sambrook et al. (Sambrook et al., 2001).

[0136] The nucleic acid of the present invention may further contain additional nucleotide sequences, such as regulatory regions, ie, promoters, enhancers, silencers, terminators, signal peptides, etc., which can be used to induce or regulate the expression of the polypeptide in a selected host cell or system.

[0137] The present invention further relates to an expression cassette comprising a nucleic acid according to the present invention operably linked to one or more control sequences which direct the expression of said nucleic acid in a suitable host cell.

[0138] As used herein, the term "expression" refers to any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0139] The term "expression cassette" denotes a nucleic acid construct comprising a coding region, ie a nucleic acid of the present invention, and a regulatory region, ie comprising one or more operably linked control sequences.

[0140] Typically, the expression cassette comprises or consists of a nucleic acid according to the present invention that is operably connected to a control sequence (e.g., a transcription promoter and / or a transcription terminator). The control sequence may include a promoter recognized by a host cell or an in vitro expression system that is used to express a nucleic acid encoding an esterase of the present invention. The promoter contains a transcription control sequence that mediates enzyme expression. The promoter may be any polynucleotide that shows transcriptional activity in a host cell, including mutations, truncations, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell. The control sequence may also be a transcription terminator that is recognized by the host cell to terminate transcription. The terminator may be operably connected to the 3' end of the nucleic acid encoding the esterase. Any terminator that is functional in a host cell may be used for the present invention. Typically, the expression cassette comprises or consists of a nucleic acid according to the present invention that is operably connected to a transcription promoter and a transcription terminator.

[0141] The invention also relates to a vector comprising a nucleic acid or an expression cassette as defined above.

[0142] As used herein, the term "vector" or "expression vector" refers to a DNA or RNA molecule containing an expression cassette of the present invention, used as a medium for transferring recombinant genetic material into a host cell. The main types of vectors are plasmids, bacteriophages, viruses, cosmids, and artificial chromosomes. The vector itself is typically a DNA sequence consisting of an insert (heterologous nucleic acid sequence, transgene) and a larger sequence that serves as the "skeleton" of the vector. The purpose of the vector that transfers genetic information to the host is typically to separate, propagate, or express the insert in the target cell. The vector referred to as an expression vector (expression construct) is particularly suitable for expressing heterologous sequences in target cells, and generally has a promoter sequence that drives the expression of heterologous sequences encoding polypeptides. Generally, the regulatory elements present in the expression vector include a transcriptional promoter, a ribosome binding site, a terminator, and an optionally present operator. Preferably, the expression vector also contains a replication origin for autonomous replication in the host cell, a selection marker, a limited number of useful restriction enzyme sites, and the possibility of a high copy number. Examples of expression vectors are cloning vectors, modified cloning vectors, specifically designed plasmids, and viruses. Expression vectors that provide suitable polypeptide expression levels in different hosts are well known in the art. The choice of vector typically depends 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.

[0143] Another object of the present invention is to provide a host cell comprising nucleic acid, expression cassette or vector as described above. Therefore, the present invention relates to the purposes of nucleic acid, expression cassette or vector according to the present invention for transformation, transfection or transduction of host cells. The selection of vector typically depends on the compatibility of the vector with the host cell into which it must be introduced.

[0144] According to the present invention, host cells can be transformed, transfected or transduced in a transient or stable manner. The expression cassette or vector of the present invention is introduced into the host cell so that the cassette or vector is maintained as a chromosome integron or a self-replicating extrachromosomal vector. The term "host cell" also encompasses any offspring of a parent host cell that is different from the parent host cell due to mutation during replication. The host cell can be any cell for producing variants of the present invention, such as a prokaryotic cell or a eukaryotic cell. The prokaryotic host cell can be any Gram-positive or Gram-negative bacteria. The host cell can also be a eukaryotic cell, such as a yeast, fungus, mammal, insect or plant cell. In a specific embodiment, the host cell is selected from the group consisting of Escherichia coli, Bacillus, Streptomyces, Trichoderma, Aspergillus, Saccharomyces, Pichia, Vibrio or Yarrowia.

[0145] The nucleic acid, expression cassette or expression vector according to the invention can be introduced into the host cell by any method known to those skilled in the art, such as electroporation, conjugation, transduction, transformation of competent cells, protoplast transformation, protoplast fusion, biolistic "gene gun" transformation, PEG-mediated transformation, lipid-assisted transformation or transfection, chemical-mediated transfection, lithium acetate-mediated transformation, liposome-mediated transformation.

[0146] Optionally, more than one copy of a nucleic acid, cassette or vector of the invention can be inserted into the host cell to increase production of the variant.

[0147] In a specific embodiment, the host cell is a recombinant microorganism. The present invention does allow engineering of microorganisms with improved ability to degrade polyester-containing materials. For example, the sequences of the present invention can be used to supplement wild-type strains of fungi or bacteria known to be able to degrade polyester to improve and / or increase strain capabilities.

[0148] Production of esterase

[0149] Another object of the present invention is to provide a method for producing the esterase of the present invention, which comprises expressing a nucleic acid encoding the esterase and optionally recovering the esterase.

[0150] In particular, the present invention relates to an in vitro method for producing an esterase of the present invention, comprising (a) contacting a nucleic acid, a cassette or a vector of the present invention with an in vitro expression system; (b) recovering the produced esterase. In vitro expression systems are well known to those skilled in the art and are commercially available.

[0151] Preferably, the production method comprises

[0152] (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

[0153] (b) recovering the esterase from the cell culture.

[0154] Advantageously, the host cell is a recombinant Bacillus, a recombinant Escherichia coli, a recombinant Aspergillus, a recombinant Trichoderma, a recombinant Streptomyces, a recombinant Saccharomyces cerevisiae, a recombinant Pichia, a recombinant Vibrio or a recombinant Yarrowia.

[0155] Host cells are cultivated in a nutrient medium suitable for producing polypeptides using methods known in the art. For example, small-scale or large-scale fermentation (including continuous, batch, batch feed or solid-state fermentation) culture cells can be carried out in a laboratory or industrial fermentor tank under suitable medium and conditions allowing expression and / or separation of enzymes. Cultivate in a suitable nutrient medium prepared from a commercial supplier or according to a disclosed composition (e.g., in the catalog of the American Type Culture Collection).

[0156] If the esterase is secreted into the nutrient medium, the esterase can be directly recovered from the culture supernatant. On the contrary, the esterase can be recovered from the cell lysate or after permeabilization. The esterase can be recovered using any method known in the art. For example, the esterase can be recovered from the nutrient medium by conventional methods, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation or precipitation. Optionally, the esterase can be partially or completely purified by various methods known in the art, including but not limited to chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing and size exclusion), electrophoresis procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE or extraction, to obtain substantially pure polypeptide.

[0157] Esterases can be used alone or in combination with other enzymes in purified form to catalyze enzymatic reactions involved in the degradation and / or recycling of polyesters and / or polyester-containing materials (e.g., polyester-containing plastics). The esterase can be in soluble form or in solid phase. In particular, it can be bound to cell membranes or lipid vesicles, or to synthetic supports (e.g., glass, plastics, polymers, filters, membranes), such as in the form of beads, columns, plates, etc.

[0158] Composition

[0159] Another object of the present invention is to provide a composition comprising the esterase or host cell of the present invention or an extract thereof.In the context of the present invention, the term "composition" encompasses any kind of composition comprising the esterase or host cell of the present invention.

[0160] Based on the total weight of the composition, the composition of the present invention may comprise 0.1 wt%-99.9 wt%, preferably 0.1 wt%-50 wt%, more preferably 0.1 wt%-30 wt%, even more preferably 0.1 wt%-5 wt% esterase. Alternatively, the composition may comprise 5-10 wt% of the esterase of the present invention.

[0161] The composition may be liquid or dry, for example in powder form. In some embodiments, the composition is a lyophilisate.

[0162] The composition may further comprise excipients and / or reagents, etc. Suitable excipients include buffers commonly used in biochemistry; reagents for adjusting pH; preservatives, such as sodium benzoate, sodium sorbate or sodium ascorbate; conservative agents, protective agents or stabilizers, such as starch, dextrin, gum arabic, salts, sugars (such as 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), etc. The composition of the present invention can be obtained by mixing the esterase with one or more excipients.

[0163] In a specific embodiment, the composition comprises 0.1 wt %-99.9 wt %, preferably 50 wt %-99.9 wt %, more preferably 70 wt %-99.9 wt %, even more preferably 95 wt %-99.9 wt % of excipients based on the total weight of the composition. Alternatively, the composition may comprise 90 wt %-95 wt % of excipients.

[0164] In a specific embodiment, the composition may further comprise other polypeptides exhibiting enzymatic activity. For example, depending on the nature of the polyester to be degraded and / or other enzymes / polypeptides contained in the composition, a person skilled in the art can easily adjust the esterase content of the present invention.

[0165] In a specific embodiment, esterase of the present invention is dissolved in an aqueous medium together with one or more excipients, and the excipient is especially an excipient that can stabilize or protect the polypeptide from degradation. For example, esterase of the present invention can eventually be dissolved in water with other components, such as glycerol, sorbitol, dextrin, starch, glycol (such as propylene glycol), salt, etc. The resulting mixture can then be dried to obtain a powder. The method of drying this mixture is well known to those skilled in the art, including but not limited to freeze drying, freeze drying, spray drying, supercritical drying, downdraft evaporation, thin layer evaporation, centrifugal evaporation, conveying drying, fluidized bed drying, drum drying or any combination thereof.

[0166] In a particular embodiment, the composition is in powder form and comprises an esterase and a stabilizing / solubilizing amount of glycerol, sorbitol or a dextrin (such as maltodextrin and / or cyclodextrin), starch, a glycol (such as propylene glycol) and / or a salt.

[0167] In a specific embodiment, the composition of the present invention comprises at least one recombinant cell expressing an esterase of the present invention or an extract thereof. "Cell extract" means any part obtained from a cell, such as a cell supernatant, cell debris, cell wall, DNA extract, enzyme or enzyme preparation, or any preparation derived from a cell by chemical, physical and / or enzymatic treatment, which is substantially free of living cells. Preferred extracts are enzyme active extracts. The composition of the present invention may comprise one or more recombinant cells of the present invention or an extract thereof, and optionally one or more additional cells.

[0168] In one embodiment, the composition comprises or consists of a culture medium of a recombinant microorganism expressing and secreting an esterase of the invention. In a specific embodiment, the composition comprises lyophilized such culture medium.

[0169] Uses of esterase

[0170] A further object of the present invention is to provide a method for degrading and / or recovering polyester or polyester-containing materials under aerobic or anaerobic conditions using the esterase of the present invention. The esterase of the present invention is particularly suitable for degrading PET and PET-containing materials.

[0171] Therefore, one object of the present invention is the use of an esterase according to the invention, or a corresponding recombinant cell or an extract thereof, or a composition for the enzymatic degradation of polyesters.

[0172] In a specific embodiment, the polyester targeted by the esterase is selected from the group consisting of polyethylene terephthalate (PET), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polyisosorbide terephthalate (PEIT), polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), polycaprolactone (PCL), poly(ethylene adipate) (PEA), polyethylene naphthalate (PEN) and blends / mixtures of these materials, preferably polyethylene terephthalate.

[0173] In a preferred embodiment, the polyester is PET and at least monomers (e.g. monoethylene glycol or terephthalic acid) and / or oligomers (e.g. 2-hydroxyethyl methyl terephthalate (MHET), bis(2-hydroxyethyl) terephthalate (BHET), 1-(2-hydroxyethyl)-4-methyl terephthalate (HEMT) and dimethyl terephthalate (DMT)) are recovered.

[0174] Another object of the present invention is the use of the esterase of the present invention, or the corresponding recombinant cells or extracts, or the compositions thereof, for the enzymatic degradation of at least one polyester in a polyester-containing material.

[0175] Another object of the present invention is 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 a host cell or a composition of the present invention, thereby degrading at least one polyester of the polyester-containing material.

[0176] Advantageously, the polyester is depolymerized into monomers and / or oligomers.

[0177] In particular, the present invention provides a method for degrading PET of a PET-containing material, wherein the PET-containing material is contacted with an esterase or a host cell or composition of the present invention, thereby degrading PET.

[0178] In one embodiment, at least one polyester is degraded into repolymerizable monomers and / or oligomers, which can be advantageously recovered for reuse. The recovered monomers / oligomers can be used for recycling (e.g., repolymerizing the polyester) or methanation. In a specific embodiment, at least one polyester is PET, and monoethylene glycol, terephthalic acid, 2-hydroxyethyl methyl terephthalate (MHET), bis(2-hydroxyethyl) terephthalate (BHET), 1-(2-hydroxyethyl) 4-methyl terephthalate (HEMT) and / or dimethyl terephthalate (DMT) are recovered.

[0179] In one embodiment, the polyester of the polyester-containing material is completely degraded.

[0180] The time required to degrade the polyester-containing material may vary depending on the polyester-containing material itself (i.e. the nature and origin of the polyester-containing material, its composition, shape, etc.), the type and amount of esterase used, and various process parameters (i.e. temperature, pH, other reagents, etc.). A person skilled in the art can easily adapt the process parameters to the polyester-containing material and the expected degradation time.

[0181] Advantageously, the degradation process is carried out at a temperature of 20°C-90°C, preferably 40°C-80°C, more preferably 50°C-70°C, more preferably 60°C-70°C. In a specific embodiment, the degradation process is carried out at 65°C. In another specific embodiment, the degradation process is carried out at 70°C. More generally, the temperature is kept below the inactivation temperature, which corresponds to a temperature at which the esterase is inactivated (i.e., a loss of activity greater than 80% compared to its activity at the optimal temperature) and / or the recombinant microorganism no longer synthesizes the esterase. In particular, the temperature is kept below the glass transition temperature (Tg) of the target polyester.

[0182] Advantageously, the process is carried out as a continuous flow process and at a temperature at which the esterase can be used several times and / or recycled.

[0183] Advantageously, the degradation process is carried out at a pH of 5-11, preferably at a pH of 6-9, more preferably at a pH of 6.5-9, even more preferably at a pH of 6.5-8.

[0184] In one embodiment, the polyester-containing material may be pretreated prior to contacting with the esterase to physically alter its structure, thereby increasing the contact surface between the polyester and the esterase.

[0185] Another object of the present invention is to provide a method for producing monomers and / or oligomers from a polyester-containing material, comprising exposing the polyester-containing material to an esterase of the present invention or its corresponding recombinant cell or extract or composition, and optionally recovering the monomers and / or oligomers.

[0186] The monomers and / or oligomers resulting from the depolymerization may be recovered sequentially or continuously.Depending on the starting polyester-containing material, a single type of monomer and / or oligomer or several different types of monomers and / or oligomers may be recovered.

[0187] The process of the present invention is particularly suitable for producing monomers selected from monoethylene glycol and terephthalic acid, and / or oligomers selected from 2-hydroxyethyl methyl terephthalate (MHET), bis(2-hydroxyethyl) terephthalate (BHET), 1-(2-hydroxyethyl)-4-methyl terephthalate (HEMT) and dimethyl terephthalate (DMT), selected from PET and / or plastic products containing PET.

[0188] The recovered monomers and / or oligomers can be further purified and conditioned in a repolymerizable form using all suitable purification methods. Examples of purification methods include steam stripping, separation by aqueous solution, selective condensation of steam, media filtration and concentration after biological processes, separation, distillation, vacuum evaporation, extraction, electrodialysis, adsorption, ion exchange, precipitation, crystallization, concentration and acid addition dehydration and precipitation, nanofiltration, acid catalyst treatment, semi-continuous mode distillation or continuous mode distillation, solvent extraction, evaporative concentration, evaporative crystallization, liquid / liquid extraction, hydrogenation, azeotropic distillation, adsorption, column chromatography, simple vacuum distillation and microfiltration, in combination or not.

[0189] The recovered repolymerizable monomers and / or oligomers can be reused, for example, in the synthesis of polyesters. Advantageously, polyesters of the same nature are repolymerized. However, the recovered monomers and / or oligomers can be mixed with other monomers and / or oligomers, for example, in order to synthesize new copolymers. Alternatively, the recovered monomers can be used as chemical intermediates to produce new target compounds.

[0190] The invention also relates to a method for surface hydrolysis or surface functionalization of a polyester-containing material, comprising exposing the polyester-containing material to an esterase of the invention, or a corresponding recombinant cell or extract thereof, or a composition. The method of the invention is particularly suitable for increasing the hydrophilicity or water absorption of the polyester material. Such increased hydrophilicity may be of particular interest in textile production, electronics and biomedical applications.

[0191] Another object of the present invention is to provide a polyester-containing material comprising an esterase of the present invention and / or a recombinant microorganism expressing and secreting the esterase. As an example, patent applications WO 2013 / 093355, WO 2016 / 198650, WO 2016 / 198652, WO 2019 / 043145 and WO 2019 / 043134 disclose methods for preparing such a polyester-containing material comprising an esterase of the present invention.

[0192] Therefore, an object of the present invention is to provide a polyester-containing material comprising an esterase and / or a recombinant cell and / or a composition or an extract thereof of the present invention and at least PET. According to one embodiment, the present invention provides a plastic article comprising PET and an esterase having PET degradation activity of the present invention.

[0193] Therefore, another object of the present invention is to provide a polyester-containing material comprising an esterase and / or a recombinant cell and / or a composition or extract thereof of the present invention and at least PBAT. According to one embodiment, the present invention provides a plastic product comprising PBAT and an esterase having PBAT degradation activity of the present invention.

[0194] Therefore, another object of the present invention is to provide a polyester-containing material comprising an esterase and / or a recombinant cell and / or a composition or extract thereof of the present invention and at least PBS. According to one embodiment, the present invention provides a plastic article comprising PBS and an esterase having PBS-degrading activity of the present invention.

[0195] Therefore, another object of the present invention is to provide a polyester-containing material comprising an esterase and / or a recombinant cell and / or a composition or extract thereof of the present invention and at least PCL. According to one embodiment, the present invention provides a plastic article comprising PCL and an esterase having PCL-degrading activity of the present invention.

[0196] Typically, esterases of the present invention can be used in detergents, food, animal feed and pharmaceutical applications. More particularly, esterases of the present invention can be used as components of detergent compositions. Detergent compositions include, but are not limited to, hand-wash or machine-washed laundry detergent compositions, for example, laundry additive compositions suitable for pre-treating dyed fabrics and rinsing added fabric softener compositions, detergent compositions for general household hard surface cleaning operations, detergent compositions for hand-wash or machine-washed dishwashing operations. In a specific embodiment, esterases of the present invention can be used as detergent additives. Therefore, the present invention provides detergent compositions comprising esterases of the present invention. Especially, esterases of the present invention can be used as detergent additives, to reduce pilling and graying effects during textile cleaning.

[0197] The present invention also relates to methods of using the esterase of the present invention in animal feeds, and to feed compositions and feed additives comprising the esterase of the present invention. The terms "feed" and "feed composition" refer to any compound, preparation, mixture or composition suitable for or intended to be ingested by an animal. In another specific embodiment, the esterase of the present invention is used to hydrolyze proteins and produce a hydrolyzate comprising peptides. This hydrolyzate can be used as a feed composition or a feed additive. DETAILED DESCRIPTION

[0198] Example 1 - Construction, expression and purification of esterase

[0199] -Build

[0200] Plasmid construction pET26b-LCC-His was used to produce esterase according to the present invention. The plasmid consists of a gene for the esterase of coding SEQ ID N ° 1 cloned between NdeI and XhoI restriction sites, which is optimized for E. coli expression. Two site-directed mutagenesis kits were used according to the supplier's advice to produce esterase variants: QuikChange II site-directed mutagenesis kit and QuikChange Lightning multi-site-directed mutagenesis kit (Santa Clara, California, USA) from Agilent.

[0201] -Expression and purification of esterase

[0202] The strain Stellar was used continuously in 50 mL LB-Miller medium or ZYM autoinduction medium (Studier et al., 2005 - Prot. Exp. Pur. 41, 207-234). TM (Clontech, California, USA) and E. coli One BL21 DE3 (Life technologies, Carlsbad, California, USA) was cloned and recombinantly expressed. Induction in LB-Miller medium was carried out using 0.5mM isopropyl β-D-1-thiogalactopyranoside (IPTG, Euromedex, Souffelweyersheim, France) at 16°C. Cultivation was terminated by centrifugation (8000rpm, 20 minutes at 10°C) in an Avanti J-26XP centrifuge (Beckman Coulter, Brea, USA). The cells were suspended in 20mL Talon buffer (Tris-HCl 20mM, NaCl 300mM, pH 8). The cell suspension was then sonicated in 2 minutes by an FB 705 sonicator (Fisherbrand, Illkirch, France), with an amplitude of 30% (2 seconds ON and 1 second OFF cycle). This was followed by a centrifugation step: 30 min in an Eppendorf centrifuge at 11000 rpm, 10°C. The soluble fraction was collected and subjected to affinity chromatography. This purification step was performed with Metal Affinity Resin (Clontech, CA, USA) was completed. Protein elution was performed with a Talon buffer step supplemented with imidazole. The purified protein was dialyzed with Talon buffer and then quantified using the Bio-Rad protein assay according to the manufacturer's instructions (Lifescience Bio-Rad, France) and stored at +4°C.

[0203] Example 2 - Evaluation of the degradation activity of esterases

[0204] The degradation activity of the esterase was determined and compared with the degradation activity of the esterase of SEQ ID N°1.

[0205] A variety of methods have been used to assess specific activity:

[0206] (1) Specific activity based on PET hydrolysis;

[0207] (2) based on the activity of polyester degradation in the solid state;

[0208] (3) Based on the activity of PET hydrolysis in a reactor larger than 100 mL.

[0209] 2.1 Specific activity based on PET hydrolysis

[0210] 100 mg of amorphous PET (in powder form and prepared according to WO 2017 / 198786 to make it less than 20% crystallinity) was weighed and introduced into a 100 mL glass bottle. 1 mL of an esterase preparation comprising SEQ ID N°1 (as a reference control) or an esterase of the present invention at 0.02 or 0.03 mg / mL prepared in Talon buffer (Tris-HCl 20 mM, NaCl 0.3 M, pH 8) was introduced into the glass bottle. Finally, 49 mL of 0.1 M potassium phosphate buffer (pH 8) was added.

[0211] Disaggregation was initiated by incubating each glass bottle in a Max Q 4450 incubator (Thermo Fisher Scientific, Inc. Waltham, MA, USA) at 60°C, 65°C, or 70°C and 150 rpm.

[0212] The initial rate of the depolymerization reaction was determined by sampling at different times during the first 24 hours and analyzed by ultra-high performance liquid chromatography (UHPLC) in mg equivalent TA produced / hour. If necessary, the samples were diluted in 0.1 M potassium phosphate buffer at pH 8. Then, 150 μL of methanol and 6.5 μL of HCl 6N were added to 150 μL of sample or dilution. After mixing and filtering on a 0.45 μm syringe filter, the samples were loaded on the UHPLC to monitor the release of terephthalic acid (TA), MHET and BHET. The chromatography system used was the Ultimate 3000 UHPLC system (Thermo Fisher Scientific, Inc. Waltham, MA, USA), which included a pump module, an autosampler, a column oven thermostated at 25°C and a UV detector at 240 nm. The column used was HSC18 HPLC column (150×4.6 mm, 5 μm, equipped with a pre-column, Supelco, Bellefonte, USA). TA, MHET and BHET were separated using a gradient of MeOH (30%-90%) in 1 mM H2SO4 at 1 mL / min. The injection volume was 20 μL of sample. TA, MHET and BHET were measured under the same conditions as the samples according to standard curves prepared based on commercial TA and BHET and MHET synthesized in-house. The specific activity of PET hydrolysis (mg equivalent TA / hour / mg enzyme) was determined in the linear part of the hydrolysis curve of the reaction, which was established by sampling at different times during the first 24 hours. Equivalent TA corresponds to the sum of the measured TA and the TA contained in the measured MHET and BHET.

[0213] 2.2 Activity based on degradation of solid-state polyester

[0214] Induced cells, semi-purified protein extracts or purified proteins can be used as compositions comprising the esterase of the present invention to assess the activity of such esterase.

[0215] The induced cells correspond to the cell culture samples obtained after cultivation in ZYM autoinduction medium or after induction by IPTG in LB-Miller medium (as described in Example 1).

[0216] Semi-purified protein extracts (as described in Example 1) were obtained after culture in ZYM auto-induction medium or after IPTG induction in LB-Miller medium under the following scheme. Cultivation was stopped by centrifugation (8000 rpm, 20 minutes at 10°C) in an Avanti J-26XP centrifuge (BeckmanCoulter, Brea, USA). The cell pellet was suspended in lysis buffer (20mM Tris-HCl, pH 8, 300mM NaCl). Cells were destroyed in a 2h freeze / thaw cycle at -80°C, followed by addition of 1 μL lysonase bioprocessing reagent (Merck Millipore, Darmstadt, Germany), and incubated at 28°C for 1h, including vortex homogenization every 15 minutes. Lysate was clarified by centrifugation (2250 × g, 15min, 4°C). To generate semi-purified fractions, lysates were treated at 70°C for 1 h and clarified by centrifugation (2250×g, 15 min, 4°C). The protein concentration of the fractions was quantified using the Bio-Rad protein assay according to the manufacturer's instructions (Lifescience Bio-Rad, France).

[0217] Purified protein was obtained as described in Example 1.

[0218] The composition samples are placed on the surface or in the wells produced in the agar omnitray containing PET or another solid polyester compound (e.g., PBAT or the like) prepared as follows. The agar plate containing PET is prepared by dissolving 500 mg PET in hexafluoro-2-propanol (HFIP), and the culture medium is poured into 250 mL of aqueous solution. After evaporating HFIP at 52° C. and 140 mbar, the solution is mixed with 0.2 M potassium phosphate buffer (pH 8) containing 3% agar in v / v. Approximately 30 mL of the mixture is used to prepare each multi-purpose plate and stored at 4° C.

[0219] After 2-24 hours, the surface area or diameter of the halos formed by degradation of polyester by wild-type esterase and the variants of the invention were measured and compared at 60°C, 65°C or 70°C.

[0220] 2.3 Activity based on PET hydrolysis in the reactor

[0221] 0.69 μmol-2.07 μmol of purified esterase prepared in 80 mL of 100 mM potassium phosphate buffer (pH 8) was mixed with 20 g of amorphous PET (prepared according to WO 2017 / 198786 to make its crystallinity less than 20%) in a 500 mL Minibio bioreactor (Applikon Biotechnology, Delft, The Netherlands). Temperature regulation was performed by immersion in a water bath at 60°C and constant stirring was maintained at 250 rpm using a single marine impeller. The pH value of the PET depolymerization assay was adjusted to 8 by 6N NaOH and confirmed by a my-Control biocontroller system (Applikon Biotechnology, Delft, The Netherlands). The base consumption was recorded during the assay and can be used for the characterization of the PET depolymerization assay.

[0222] The final yield of the PET depolymerization assay is determined by determining the weight of the residual PET or by determining the equivalent TA and EG produced or by base consumption. At the end of the reaction, the weight of the residual PET is evaluated by filtering the reaction volume through a 12-15 μm 11-grade ash-free paper filter (Dutscher SAS, Brumath, France) and drying such retentate before weighing. The equivalent TA and EG produced are determined using the UHPLC method described in 2.1, and the hydrolysis percentage is calculated based on the ratio of the molar concentration ratio (TA+MHET+BHET) at a given time to the total amount of TA contained in the initial sample. PET depolymerization produces acidic monomers, which will be neutralized with base to be able to maintain the pH in the reactor. The equivalent TA produced is calculated using the corresponding molar base consumption, and the hydrolysis percentage is calculated based on the ratio of the molar concentration of the equivalent TA at a given time to the total amount of TA contained in the initial sample.

[0223] Table 1 below shows the specific degradation activity of the esterase (variant) of the present invention. The specific degradation activity of the esterase of SEQ ID N° 1 was used as a reference and regarded as 100% of the specific degradation activity. The specific degradation activity was measured as described in Example 2.1.

[0224] Table 1: Specific degradation activity of variants of the invention

[0225]

[0226]

[0227] Apart from the combinations of substitutions listed in Table 1 respectively, variants V1-V83 have the exact amino acid sequence as shown in SEQ ID N° 1.

[0228] The comparative degradation activity of the esterase (variant) of the invention was measured in a reactor according to Example 2.3. The PET depolymerization rate after 24 hours and the time to reach 90% PET depolymerization rate of the variant were evaluated and compared with the esterase of SEQ ID N°1. The esterase of SEQ ID N°1 reached 66% PET depolymerization after 24 hours and required 43.5 hours to reach 90% PET depolymerization rate under these conditions. The results are shown in Table 2 below.

[0229] Table 2: PET depolymerization after 24 hours and time to reach 90% PET depolymerization of variants of the invention

[0230]

[0231] Example 3 - Evaluation of the thermal stability of esterases of the invention

[0232] The thermal stability of the esterase of the present invention was determined and compared with the thermal stability of the esterase of SEQ ID N°1.

[0233] Different methods are used to estimate thermal stability:

[0234] (1) Circular dichroism of proteins in solution;

[0235] (2) residual esterase activity after incubation of the protein under given temperature, time and buffer conditions;

[0236] (3) depolymerization activity of residual polyesters after incubation of the protein under given temperature, time and buffer conditions;

[0237] (4) The ability to degrade solid polyester compounds (such as PET or PBAT or the like) dispersed in an agar plate after incubation of the protein under given temperature, time, and buffer conditions;

[0238] (5) the ability to perform multiple rounds of polyester depolymerization assays under given conditions of temperature, buffer, protein concentration, and polyester concentration;

[0239] (6) Differential scanning fluorimetry (DSF);

[0240] Details of the protocols for these methods are given below.

[0241] 3.1 Circular dichroism

[0242] Circular dichroism (CD) was performed using a Jasco 815 apparatus (Easton, USA) to compare the melting temperatures (T m ) (Tm = 84.7°C) and the Tm of the esterase of the present invention.

[0243] Technically, 400 μL protein samples were prepared at 0.5 mg / mL in Talon buffer and used for CD. A first scan from 280 nm to 190 nm was performed to determine the two maximum intensities of CD corresponding to the correct folding of the protein. Then, a second scan from 25°C to 110°C was performed at a long wave corresponding to this maximum intensity and provided a specific curve (sigmoid3 parameter y=a / (1+e^((x-x0) / b))) which was analyzed by Sigmaplot version 11.0 software to determine T when x=x0. m The obtained T m Reflects the thermal stability of a given protein. m The higher it is, the more stable the variant is at high temperatures.

[0244] 3.2 Residual esterase activity

[0245] 1 mL of the esterase of SEQ ID N°1 or 40 mg / L (in Talon buffer) of the esterase of the present invention was incubated at different temperatures (65, 70, 75, 80 and 90°C) for 10 days. Samples were taken regularly and diluted 1 to 500 times in 0.1 M potassium phosphate buffer at pH 8.0 for p-nitrophenol-butyrate (pNP-B) assay. 20 μL of sample was mixed with 175 μL of 0.1 M potassium phosphate buffer at pH 8.0 and 5 μL of a solution of pNP-B in 2-methyl-2-butanol (40 mM). The enzymatic reaction was carried out at 30°C with stirring for 15 minutes and the absorbance at 405 nm was obtained by a microplate spectrophotometer (Versamax, Molecular Devices, Sunnyvale, CA, USA). The activity of pNP-B hydrolysis was determined using a standard curve of p-nitrophenol released in the linear part of the hydrolysis curve (initial rate expressed as μmol pNPB / min).

[0246] 3.3 Depolymerization activity of residual polyester

[0247] 10 mL of the esterase of SEQ ID N°1 and 40 mg / L (in Talon buffer) of the esterase of the present invention were incubated at different temperatures (65, 70, 75, 80 and 90°C) for 1 to 30 days. Take 1 mL of sample regularly and transfer to a bottle containing 100 mg of amorphous PET micronized to 250-500 μm (prepared according to WO 2017 / 198786 to make its crystallinity less than 20%) and 49 mL of 0.1 M potassium phosphate buffer at pH 8.0, and incubate at 65°C. Sample 150 μL of buffer regularly. When necessary, dilute the sample in 0.1 M potassium phosphate buffer at pH 8. Then, add 150 μL of methanol and 6.5 μL of HCl 6N to 150 μL of sample or dilution. After mixing and filtering on a 0.45 μm syringe filter, the samples were loaded on UHPLC to monitor the release of terephthalic acid (TA), MHET and BHET. The chromatography system used was an Ultimate 3000 UHPLC system (ThermoFisher Scientific, Inc. Waltham, MA, USA), which included a pump module, an autosampler, a column oven at 25°C and a UV detector at 240 nm. The column used was HSC18 HPLC column (150×4.6 mm, 5 μm, equipped with a precolumn, Supelco, Bellefonte, USA). 1 mM H 2 SO 4 TA, MHET and BHET were separated at 1 mL / min with a gradient of MeOH (30%-90%) in 4% paraformaldehyde. The injection volume was 20 μL of sample. TA, MHET and BHET were measured under the same conditions as the samples according to commercial TA and BHET and MHET synthesized in-house. The activity of PET hydrolysis (μmol hydrolyzed PET / min or mg equivalent TA produced / hour) was determined in the linear part of the hydrolysis curve, which was established by sampling at different times during the first 24 hours. The equivalent TA corresponds to the sum of the measured TA and the TA contained in the measured MHET and BHET.

[0248] 3.4 Degradation of polyester in solid form

[0249] 1 mL of the esterase of SEQ ID N°1 and 40 mg / L (in Talon buffer) of the esterase of the present invention were incubated at different temperatures (65, 70, 75, 80 and 90°C) for 1 to 30 days. 20 μL of the enzyme preparation was placed in the wells produced by the agar plate containing PET at regular intervals. The agar plate containing PET was prepared by dissolving 500 mg of PET in hexafluoro-2-propanol (HFIP) and the culture medium was poured into 250 mL of the aqueous solution. After evaporation of HFIP at 52°C and 140 mbar, the solution was mixed with 0.2 M potassium phosphate buffer (pH 8) containing 3% agar in v / v. About 30 mL of the mixture was used to prepare each multi-purpose plate and stored at 4°C.

[0250] After 2-24 hours, the diameter or surface area of ​​the halos formed by the degradation of polyester by wild-type esterase and the variants of the invention are measured and compared at 60° C., 65° C. or 70° C. The half-life of the enzyme at a given temperature corresponds to the time required for the diameter or surface area of ​​the halos to decrease by a factor of 2.

[0251] 3.5 Multi-round depolymerization of polyester

[0252] The ability of esterases to perform consecutive rounds of polyester depolymerization assays was evaluated in an enzyme reactor. A Minibio 500 bioreactor (Applikon Biotechnology BV, Delft, The Netherlands) was started with 3 g of amorphous PET (prepared according to WO 2017 / 198786 to make it less than 20% crystallinity) and 100 mL of 10 mM potassium phosphate buffer (pH 8) containing 3 mg of LC-esterase. Agitation was set to 250 rpm using a marine impeller. The bioreactor was thermostated at 60°C, 65°C or 70°C by immersion in an external water bath. The pH was adjusted to 8 by adding 3M KOH. Different parameters (pH, temperature, agitation, addition of alkali) were monitored by BioXpert software V2.95. 1.8 g of amorphous PET was added every 20 hours. 500 μL of the reaction medium was sampled regularly.

[0253] The contents of TA, MHET and BHET were determined by HPLC as described in Example 2.3. The content of EG was determined using an Aminex HPX-87K column (Bio-Rad Laboratories, Inc, Hercules, California, United States) at 65°C. The eluent was eluted at 0.6 mL / min. -1 K 2 HPO 4 5 mM. Injection volume was 20 μL. Ethylene glycol was monitored using a refractometer.

[0254] The percentage hydrolysis was 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+2x BHET) to the total amount of EG contained in the initial sample. The degradation rate was calculated as mg total released TA per hour or mg total EG per hour.

[0255] The half-life of the enzyme was estimated as the incubation time required to obtain a 50% loss in degradation rate.

[0256] 3.6 Differential Scanning Fluorescence (DSF)

[0257] DSF is used to evaluate the thermal stability of wild-type protein (SEQ ID N ° 1) and its variants by measuring its melting temperature (Tm, the temperature at which half of the protein population unfolds). Protein samples with a concentration of 14 μM (0.4 mg / mL) were prepared and stored in a buffer A consisting of 20 mM Tris HCl (pH 8.0), 300 mM NaCl. First, the SYPROorange dye 5000x stock solution in DMSO was diluted to 250x in water. The protein sample was loaded onto a white transparent 96-well PCR plate (Bio-Rad cat#HSP9601), wherein each well contained a final volume of 25 μl. The final concentrations of protein and SYPRO Orange dye in each well were 5 μM (0.14 mg / ml) and 10X, respectively. The volume loaded into each well was as follows: 15 μL buffer A, 9 μL 0.4 mg / mL protein solution, and 1 μL 250x Sypro Orange dilution solution. The PCR plate was then sealed with optical quality sealing tape and spun at 2000 rpm for 1 minute at room temperature. DSF experiments were then performed using a CFX96 real-time PCR system set to use 450 / 490 excitation and 560 / 580 emission filters. The samples were heated from 25°C to 100°C at a rate of 0.3°C / sec. Single fluorescence measurements were taken every 0.03 seconds. Melting temperatures were determined from the peak of the first derivative of the melting curve using Bio-Rad CFX Manager software.

[0258] Then, the esterase of SEQ ID N°1 and the esterase of the present invention were compared based on their Tm values. Due to the high reproducibility between experiments of the same protein from different productions, a ΔTm of 0.8°C was considered significant for comparing variants. The Tm values ​​correspond to the average of at least 3 measurements. The Tm of the esterase of SEQ ID N°1 was evaluated at 84.7°C.

[0259] The thermostability of the esterase variants of the invention is shown in Table 3 below, expressed as Tm values ​​and evaluated according to Example 2.6. The increase in Tm compared to the esterase of SEQ ID N° 1 is shown in brackets.

[0260] Table 3: Tm of the esterase of the present invention compared with SEQ ID N°1

[0261]

[0262]

[0263]

[0264]

[0265] Apart from the combinations of substitutions listed in Table 3 respectively, variants V1-V83 have the exact amino acid sequence as shown in SEQ ID N° 1.

Claims

1. An esterase, wherein the combination of substitutions in the amino acid sequence of the esterase compared to the esterase shown in SEQ ID NO: 1 is as follows:

2. A nucleic acid encoding the esterase as defined in claim 1.

3. An expression cassette or vector comprising the nucleic acid of claim 2.

4. A host cell comprising the nucleic acid of claim 2 or the expression cassette or vector of claim 3.

5. A composition comprising the esterase as defined in claim 1, or the host cell or extract thereof according to claim 4.

6. A method for degrading at least one polyethylene terephthalate of a polyethylene terephthalate-containing material, wherein include: (a) contacting the polyethylene terephthalate-containing material with the esterase according to claim 1, the host cell according to claim 4, or the composition according to claim 5.

7. A detergent composition comprising the esterase according to claim 1 or the host cell according to claim 4 or the composition according to claim 5.

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