Use of esterase

Abstract

The present invention relates to the use of certain enzymes for the degradation of polyesters or polyester-containing materials under acidic conditions at a pH of 3 to 6, more particularly to the use of certain esterases with polyester degrading activity. The present invention also relates to a process for degrading polyesters or polyester-containing materials (e.g. plastic products), comprising a step of enzymatic depolymerization carried out under acidic conditions at a pH of 3 to 6. The process of the present invention is particularly suitable for the degradation of polyethylene terephthalate and polyethylene terephthalate-containing materials.

Description

[Technical field]

[0001] The present invention relates to the use of certain enzymes, more particularly certain esterases with polyester degrading activity, for the degradation of polyesters or polyester-containing materials under acidic conditions (pH between 3 and 6). The present invention also relates to a process for degrading polyesters or polyester-containing materials (e.g. plastic products), comprising a step of enzymatic depolymerization carried out under acidic conditions (pH between 3 and 6). The process of the present invention is particularly suitable for the degradation of polyethylene terephthalate and polyethylene terephthalate-containing materials. [Background technology]

[0002] Plastics are cheap and durable materials that can be used to manufacture a variety of products with a wide range of uses (e.g., food packaging, textiles, etc.). As such, the production of plastics has increased dramatically over the past decades. Moreover, most of these are used for one-time disposable applications such as packaging, agricultural films, disposable consumer goods, or for short-lived products that are discarded within one year of production. Due to the durability of the polymers they contain, large amounts of plastics pile up in landfills and natural habitats around the world, creating serious environmental problems. For example, in recent years, polyethylene terephthalate (PET), an aromatic polyester made from terephthalic acid and ethylene glycol, has been widely used to manufacture a variety of products for human consumption, such as food and beverage packaging (e.g., bottles, convenience-size soft drinks, food pouches), or textiles, textiles, rugs, carpets, etc.

[0003] To mitigate the environmental and economic impacts associated with the accumulation of plastic waste, various solutions, from plastic decomposition to plastic recycling, are being investigated. Mechanical recycling techniques remain the most used techniques, but have several drawbacks. Indeed, they require extensive and costly sorting and lead to downgrading of applications due to the overall molecular weight loss during the process and the uncontrolled presence of additives in the recycled products. Current recycling techniques are also expensive. As a result, recycled plastic products are generally not competitive compared to virgin plastics.

[0004] In recent years, innovative processes for enzymatic recycling of plastic products have been developed and described (e.g., WO 2014 / 079844, WO 2015 / 097104, WO 2015 / 173265, WO 2017 / 198786, WO 2020 / 094661, WO 2020 / 094646, and WO 2021 / 123299). Unlike conventional recycling techniques, such enzymatic depolymerization processes eliminate expensive sorting and allow the recovery of the chemical components of the polymer (i.e., monomers and / or oligomers). The resulting monomers / oligomers can be recovered, purified, and used to remanufacture plastic products of equivalent quality to virgin plastic products, such that such processes allow infinite recycling of plastics. These processes are particularly useful for recovering terephthalic acid and ethylene glycol from plastic products, including PET. In these processes, the production of said monomers and / or oligomers (especially terephthalic acid) leads to a decrease in the pH of the reaction medium, which can adversely affect the activity of the degradative enzymes. To maintain the pH and thus the optimal enzyme activity, large amounts of bases are added to the reaction medium. In addition, the recovery of terephthalic acid by precipitation requires the use of strong acids, which leads to the production of large amounts of salts that have little value. The use of bases and acids, as well as the lack of valorization of the salts, therefore have a significant impact on the cost of these processes.

[0005] By addressing these challenges, the present inventors have identified specific enzymes capable of efficiently degrading polyesters under acidic conditions, and have discovered that said enzymes can be used under acidic conditions while maintaining depolymerization yields that are economically and industrially satisfactory, which greatly reduces the need for base during enzymatic depolymerization and results in no or very little salt formation. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2014 / 079844 [Patent Document 2] International Publication No. 2015 / 097104 [Patent Document 3] International Publication No. 2015 / 173265 [Patent Document 4] International Publication No. 2017 / 198786 [Patent Document 5] International Publication No. 2020 / 094661 [Patent Document 6] International Publication No. 2020 / 094646 [Patent Document 7] International Publication No. 2021 / 123299 [Patent Document 8] International Publication No. 1999 / 023055 [Non-patent literature]

[0007] [Non-Patent Document 1] Sulaiman et al., Appl Environ Microbiol. March 2012 [Non-Patent Document 2] http: / / blast.ncbi.nlm.nih.gov / or http: / / www.ebi.ac.uk / Tools / emboss / [Non-Patent Document 3] Lebarbe et al. Green Chemistry Issue 4 2014 [Non-Patent Document 4] Studier et al., 2005- Prot. Exp. Pur. 41, pp. 207-234 Summary of the Invention [Means for solving the problem]

[0008] The present invention relates to the use, under certain conditions, of a specific polyester degrading enzyme derived from the amino acid sequence set forth in SEQ ID NO: 1. The amino acid sequence set forth in SEQ ID NO: 1 corresponds to amino acids 36 to 293 of the amino acid sequence of a metagenomic cutinase described in Sulaiman et al., Appl Environ Microbiol. March 2012, and is referred to in SwissProt as G9BY57.

[0009] In this regard, the present invention relates to the use of an esterase for degrading polyesters or polyester-containing materials, comprising contacting the polyesters or polyester-containing materials with an enzyme at a pH of 3 to 6, the enzyme having polyester degrading activity at a pH of 3 to 6, preferably at least a pH of 5 to 5.5, the enzyme (i) having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the full-length amino acid sequence set forth in SEQ ID NO:1, and (ii) having at least one of the following amino acids: T11E / M, R12D / N / Q / E / F, S13E, A14E / D, T16E, A17T, T61S / V, A62D, F90A / Y, Y92G / R, ... D, W155A, T157S, P179D / E, Q182D / E, F187Y / I, D203C / K / R, N204D / E / G, A205D, S206D / E, F2 08M / W / G / N / R / I / A / Q / L / S / T / E, N211D / Y / E, S212F, N213P / D, N214D, A215N, S218A, V219I, Y The present invention relates to the use of an esterase comprising at least one substitution selected from 220M / F, Q237D, F238E, N241E / D, N243E / D, L247T, V170I, G135A, V167Q / T, S248C and A24R, wherein the positions are numbered with reference to the amino acid sequence set forth in SEQ ID NO:1.

[0010] Preferably, the enzyme is selected from the group consisting of A14E / D, F90A / Y, Y92G / D, Q182D / E, N204D / E / G, F208M / W / G / N / R / I / A / Q / L / S / T / E, N211D / Y / E, N213P / D, V219I, V170I, D203C / K / R, S248C, A24R, and F18 7Y / I, preferably A14E, F90A, Y92G / D, Q182D / E, N204G, F208M / G / N / R / I / A / Q / L / S / T, N211D / E, N213P, V219I, V170I, D203C / K / R, S248C, A24R, and and F187I, more preferably at least one substitution selected from F90A, Y92G, Q182D / E, N204G, F208M / G / N / R / I / A / Q / L / S / T, N211D / E, V170I, D203C / K / R, S248C, N213P, A24R, and F187I, and even more preferably at least one substitution selected from N211D / E, N204G, F208M / R / I / Q / L / S / T, Q182D / E, Y92G, V170I, D203C / K / R, S248C, N213P, A24R, and F187I.

[0011] Preferably, the esterase is selected from the group consisting of F208I + D203C + S248C + V170I + Y92G + N213P + Q182E, F208M + D203C + S248C + V170I + Y92G + N213P + Q182E, F208T + D203C + S248C + V170I + Y92G + N213 P+Q182E, F208L+D203C+S248C+V170I+Y92G+N213P+Q182E, F208M+D203C+S248 C+V170I+Y92G+N213P+Q182E+S13L+D158E, F208M+D203C+S248C+V170I+Y92G+ N213P+Q182E+S13L and F208M+D203C+S248C+V170I+Y92G+N213P+Q182E+D158E, more preferably comprising at least one combination of substitutions selected from F208M+D203C+S248C+V170I+Y92G+N213P+Q182E, F208I+D203C+S248C+V170I+Y92G+N213P+Q182E, and F208M+D203C+S248C+V170I+Y92G+N213P+Q182E+S13L+D158E.

[0012] Another object of the present invention is to provide a process for decomposing polyester or polyester-containing substances, as described above, which comprises a step of contacting the polyester or polyester-containing substances with an esterase capable of decomposing polyesters, and in which the step of contacting the polyester or polyester-containing substances with the enzyme is carried out under acidic conditions, specifically at a pH of 3 to 6, and preferably at a pH of 5 to 5.5.

[0013] The present invention also relates to detergent compositions comprising the esterase.

[0014] The present invention also relates to a polyester-containing material that includes an esterase. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] definition The present disclosure will be best understood by reference to the following definitions.

[0016] Here, the terms "peptide", "polypeptide", "protein" and "enzyme" refer to a chain of amino acids linked by peptide bonds, regardless of the number of amino acids forming said chain. Amino acids are represented herein by one-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).

[0017] The term "esterase" refers to an enzyme belonging to the class of hydrolases classified under EC 3.1.1 according to the enzyme nomenclature, which catalyzes the hydrolysis of esters to acids and alcohols. The term "cutinase" or "cutin hydrolase" refers to an esterase classified under EC 3.1.1.74 according to the enzyme nomenclature, which can catalyze the chemical reaction producing cutin monomers from cutin and water.

[0018] The term "wild-type protein" or "parent protein" refers to the non-mutated version of a polypeptide that occurs in nature. In this case, the parent esterase refers to the esterase having the amino acid sequence set forth in SEQ ID NO:1.

[0019] The terms "mutant" and "variant" refer to a polypeptide derived from SEQ ID NO: 1, which contains at least one modification or alteration (i.e., substitution, insertion, and / or deletion) at one or more (e.g., several) positions compared to SEQ ID NO: 1, and which has polyesterolytic activity. Variants can be obtained by various techniques known in the art. In particular, examples of techniques for altering a DNA sequence encoding a wild-type protein include, but are not limited to, site-directed mutagenesis, random mutagenesis, and synthetic oligonucleotide construction. Thus, the terms "modified" and "altered", when used herein with respect to a particular position, mean that the amino acid at this particular position has been altered compared to the amino acid at this particular position in the wild-type protein.

[0020] "Substitution" means that an amino acid residue is replaced by another amino acid residue. Preferably, the term "substitution" refers to the replacement of an amino acid residue by another amino acid residue selected from the 20 standard naturally occurring amino acid residues, rare naturally occurring amino acid residues (e.g., hydroxyproline, hydroxylysine, allohydroxylysine, 6-N-methyllysine, N-ethylglycine, N-methylglycine, N-ethylasparagine, allo-isoleucine, N-methylisoleucine, N-methylvaline, pyroglutamine, aminobutyric acid, ornithine, norleucine, norvaline), and non-naturally occurring amino acid residues that are often synthetically produced (e.g., cyclohexyl-alanine). Preferably, the term "substitution" refers to the replacement of an amino acid residue by another amino acid residue selected from the 20 standard naturally occurring amino acid residues (G, P, A, V, L, I, M, C, F, Y, W, H, K, R, Q, N, E, D, S, and T). The symbol "+" indicates a combination of substitutions. The following terms are used herein to designate substitutions: L82A indicates that the amino acid residue at position 82 of the parent sequence (leucine, L) is replaced by alanine (A). A121V / I / M indicates that the amino acid residue at position 121 of the parent sequence (alanine, A) is replaced by one of the following amino acids: valine (V), isoleucine (I), or methionine (M). Substitutions can be conservative or non-conservative. Examples of conservative substitutions are found within the groups of basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine, asparagine, and threonine), hydrophobic amino acids (methionine, leucine, isoleucine, cysteine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, and serine).

[0021] Unless otherwise specified, the amino acid positions disclosed in this application are numbered with reference to the amino acid sequence set forth in SEQ ID NO:1.

[0022] As used herein, the term "sequence identity" or "identity" refers to the number (or percentage) of matches (identical amino acid residues) between two polypeptide sequences. Sequence identity is determined by comparing sequences when aligned 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, depending on the length of the two sequences. Sequences of similar length are preferably aligned using a global alignment algorithm (e.g., the Needleman and Wunsch algorithm; Needleman and Wunsch, 1970), which optimally aligns sequences over their entire length, whereas sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g., the Smith and Waterman algorithm (Smith and Waterman, 1981) or the Altschul algorithm (Altschul et al., 1997; Altschul et al., 2005). Alignments to determine percent amino acid sequence identity can be performed using, for example, the following methods: This can be achieved in a variety of ways within the art using computer software available on internet websites such as http: / / www.ebi.ac.uk / Tools / emboss / . Those skilled in the art can determine the appropriate parameters for measuring alignment, such as any algorithm required to achieve maximum alignment over the entire length of the sequences being compared. For purposes herein, amino acid sequence identity values ​​% refer to values ​​generated using the pairwise sequence alignment program EMBOSS Needle, which uses the Needleman-Wunsch algorithm to generate the optimal global alignment of two sequences, with all search parameters set to default values, i.e., scoring matrix=BLOSUM62, gap open=11, gap extend=1.

[0023] "Polymer" refers to a compound or mixture of compounds having a structure composed of multiple monomers (repeating units) linked by covalent chemical bonds. In the context of the present invention, the term polymer includes natural or synthetic polymers composed of a single type of repeating unit (i.e., homopolymers) or a mixture of different repeating units (i.e., copolymers or heteropolymers). According to the present invention, "oligomer" refers to a molecule containing from 2 to about 20 monomers.

[0024] In the context of the present invention, "polyester-containing material" or "polyester-containing product" refers to a product (e.g., a plastic product or plastic product) that contains at least one polyester in crystalline, semi-crystalline, or completely amorphous form. In a particular embodiment, polyester-containing material refers to any item made of at least one plastic material, such as plastic sheets, tubes, rods, profiles, shapes, films, chunks, fibers, etc., containing at least one polyester and, optionally, other substances or additives, such as plasticizers, inorganic or organic fillers. In another particular embodiment, polyester-containing material refers to a plastic compound or plastic blend in a molten or solid state that is suitable for making plastic products. In another particular embodiment, polyester-containing material refers to a textile, fabric, or fiber that contains at least one polyester. In another particular embodiment, polyester-containing material refers to a plastic waste or fiber waste that contains at least one polyester. In particular, plastic products refer to industrial products, such as rigid or flexible packaging (bottles, trays, cups, etc.), agricultural films, bags and sacks, disposable goods, etc., carpet scraps, fabrics, textiles, etc. The plastic product may contain additional substances or additives such as plasticizers, inorganic or organic fillers or dyes. In the context of the present invention, the plastic product may contain a mixture of semi-crystalline and / or amorphous polymers and / or additives.

[0025] As used herein, the term "polyester" includes, but is not limited to, polyethylene terephthalate (PET), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polylactic acid (PLA), polyhydroxyalkanoates (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), polycaprolactone (PCL), poly(ethylene adipate) (PEA), polyethylene naphthalate (PEN), and blends / mixtures of these polymers. Polyesters may also include "polyolefin-like" polyesters (preferably "polyethylene-like" polyesters), which correspond to polyolefins (preferably polyethylenes) into which ester segments have been introduced (generally achieved by polycondensation of long-chain α,ω-difunctional monomers), as defined in Lebarbe et al. Green Chemistry Issue 4 2014.

[0026] The term "depolymerization" with respect to a polymer, or a plastic article comprising a polymer, refers to a process by which the polymer, or at least one polymer of said plastic article, is depolymerized and / or broken down into smaller molecules (e.g., monomers and / or oligomers) and / or any degradation products.

[0027] According to the present invention, "oligomer" refers to a molecule containing from 2 to about 20 monomer units. As an example, oligomers recovered from PET include methyl-2-hydroxyethyl terephthalate (MHET), and / or bis(2-hydroxyethyl) terephthalate (BHET), and / or 1-(2-hydroxyethyl), and / or 4-methyl terephthalate (HEMT), and / or dimethyl terephthalate (DMT).

[0028] Use of specific esterases By working on optimizing the enzymatic degradation process of plastic products under acidic conditions (specifically, a pH of 3 to 6, more specifically, a pH of 5 to 5.5), the inventors have identified specific esterases that exhibit efficient polyester degradation activity (specifically, PET degradation activity) and improved heat resistance under acidic conditions.

[0029] According to the present invention, "acidic conditions" refers to conditions (e.g., medium, solution, etc.) with a pH of 3 to 6. Specifically, "acidic conditions" refers to the conditions in which the present esterase is used, for example, the present esterase is contacted with a polyester in a medium with a pH of 3 to 6, more specifically, the esterase is contacted with a polyester in a medium with a pH of 5 to 5.5.

[0030] According to the present invention, the esterase exhibits higher polyester decomposition activity and / or higher thermostability under acidic conditions compared to the parent esterase or wild-type esterase having the amino acid sequence set forth in SEQ ID NO: 1. Specifically, the esterase disclosed in the present invention exhibits higher activity and / or higher thermostability compared to the parent esterase when presented at a pH of 3 to 6. Thus, this higher activity and / or higher thermostability may be observed at a specific pH of 3 to 6 and / or in a pH range of 3 to 6. Specifically, this higher activity and / or higher thermostability may be observed at least at pH 3, pH 3.5, pH 4, pH 4.5, pH 5, pH 5.2, pH 5.5, and / or pH 6. This high activity and / or high heat resistance can also be observed in the entire pH range of 3 to 6, the entire pH range of 4 to 6, the entire pH range of 4.5 to 6, the entire pH range of 5 to 6, the entire pH range of 5.5 to 6, the entire pH range of 5 to 5.5, the entire pH range of 5 to 5.2, and the entire pH range of 5.2 to 5.5.

[0031] According to the present invention, the esterase exhibits higher activity at a pH of 3 to 6, preferably at a pH of 4 to 6, more preferably at a pH of 5 to 6, even more preferably at a pH of 5 to 5.5, and even more preferably at pH 5.2, compared to the esterase having the amino acid sequence set forth in SEQ ID NO: 1 (also referred to as the parent esterase) at the same pH.

[0032] Specifically, the inventors have identified certain amino acid substitutions to be made in SEQ ID NO:1 that advantageously increase the activity of esterases towards this polymer under acidic conditions.

[0033] In the context of the present invention, the term "high activity" or "high degradation activity" refers to a higher ability of the esterase to degrade and / or adsorb the same polyester under given conditions (e.g., temperature, pH, concentration) compared to the ability of the esterase of SEQ ID NO: 1 to degrade and / or adsorb the same polyester. Such an increase may be at least 10% higher than the polyester degradation activity of the esterase of SEQ ID NO: 1, and preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130% or higher. In particular, the degradation activity refers to a depolymerization activity resulting in polyester monomers and / or oligomers, which can be further recovered and optionally reused. In particular, the esterase of the present invention has a high PET degradation activity. In the context of the present invention, the esterases exhibit a higher degradation activity at at least a pH between 3 and 6 compared to the degradation activity of the parent esterase at the same pH. Preferably, the esterases exhibit a higher activity at least at a pH between 4 and 6, preferably at a pH between 5 and 6, more preferably at a pH between 5 and 5.5, even more preferably at pH 5.2.

[0034] The "degradation activity" of an esterase may be evaluated by a person skilled in the art according to methods known in the art. For example, the degrading activity may be evaluated by measuring the depolymerization activity rate of a specific polymer, measuring the decomposition rate of a solid polymeric compound dispersed in an agar plate, or measuring the depolymerization activity rate of a polymer in a reactor. Specifically, the degrading activity may be evaluated by measuring the "specific degrading activity" of an esterase. The "specific degrading activity" of an esterase for PET corresponds to the PET μmol / min hydrolyzed or the equivalent amount of TA generated mg / hr per mg of esterase during the initial period of the reaction (i.e., the first 24 hours), and is determined from the linear portion of the hydrolysis curve of the reaction, such a curve being established by several samplings performed at various times during the first 24 hours. As another example, the "degradation activity" may be evaluated by measuring the rate and / or yield of oligomers and / or monomers released under appropriate conditions of temperature, pH, and buffer when a polymer or a plastic product containing a polymer is contacted with a degrading enzyme after a specified period of time (e.g., 24 hours, 48 ​​hours, or 72 hours).

[0035] The ability of an enzyme to adsorb to a substrate can be assessed by one of skill in the art according to methods known in the art. For example, the ability of an enzyme to adsorb to a substrate can be measured from a solution containing the enzyme, which has been pre-incubated with the substrate under suitable conditions.

[0036] The inventors have also identified target amino acid residues in SEQ ID NO:1 that can be advantageously modified to improve the stability (i.e., improve the thermostability) of the corresponding esterase under acidic conditions at high temperatures (specifically, temperatures of 50°C to 90°C, preferably 60°C to 80°C, and more preferably 65°C to 75°C).

[0037] According to the present invention, the esterase exhibits high heat resistance at a pH of 3 to 6, preferably at a pH of 4 to 6, more preferably at a pH of 5 to 6, even more preferably at a pH of 5 to 5.5, and even more preferably at pH 5.2, compared to the heat resistance of the esterase having the amino acid sequence set forth in SEQ ID NO: 1 (i.e., the parent esterase) at the same pH.

[0038] In the context of the present invention, the term "high thermostability" refers to the increased ability of the esterase to resist changes in its chemical and / or physical structure at elevated temperatures (specifically at temperatures between 50°C and 90°C) compared to the esterase of SEQ ID NO: 1. Specifically, the thermostability of the esterase is improved under acidic conditions at temperatures between 50°C and 90°C, between 50°C and 80°C, between 50°C and 75°C, between 50°C and 70°C, between 50°C and 65°C, between 55°C and 90°C, between 55°C and 80°C, between 55°C and 75°C, between 55°C and 70°C, between 55°C and 65°C, between 60°C and 90°C, between 60°C and 80°C, between 60°C and 75°C, between 60°C and 70°C, between 60°C and 65°C, between 65°C and 90°C, between 65°C and 80°C, between 65°C and 75°C, between 65°C and 70°C, compared to the thermostability of the parent esterase. Specifically, the thermostability of the present esterases is improved compared to that of the parent esterase at temperatures between 40° C. and 80° C., between 50° C. and 72° C., between 55° C. and 60° C., between 50° C. and 55° C., and between 60° C. and 72° C. Preferably, the thermostability of the present esterases is improved compared to that of the parent esterase at temperatures between at least 50° C. and 65° C. In the context of the present invention, temperatures are given to + / - 1° C.

[0039] Specifically, thermostability may be assessed by evaluation of 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 esterases of the present invention exhibit an increase in Tm of about 0.8°C, 1°C, 2°C, 3°C, 4°C, 5°C, 10°C, or more at a pH of 3-6 compared to the Tm of the esterase of SEQ ID NO:1. Specifically, at a pH of 3-6, the esterases of the present invention may have an extended half-life at temperatures between 50°C and 90°C compared to the esterase of SEQ ID NO:1. Specifically, the esterase of the present invention may have a longer half-life at temperatures of 50° C. to 90° C., 50° C. to 80° C., 50° C. to 75° C., 50° C. to 70° C., 50° C. to 65° C., 55° C. to 90° C., 55° C. to 80° C., 55° C. to 75° C., 55° C. to 70° C., 55° C. to 65° C., 60° C. to 90° C., 60° C. to 80° C., 60° C. to 75° C., 60° C. to 70° C., 60° C. to 65° C., 65° C. to 90° C., 65° C. to 80° C., 65° C. to 75° C., 65° C. to 72° C., 65° C. to 70° C., and 60° C. to 72° C., at a pH of 3 to 6. Advantageously, the esterase disclosed in the present invention has a longer half-life at temperatures of at least 50° C. to 60° C., at a pH of 3 to 6, compared to the esterase of SEQ ID NO: 1.

[0040] In the context of the present invention, the esterase disclosed in the present invention exhibits high thermostability at least at a pH of 3 to 6, as compared to an esterase having the amino acid sequence set forth in SEQ ID NO: 1. Preferably, the esterase exhibits high thermostability at least at a pH of 4 to 6, preferably at a pH of 5 to 6, more preferably at a pH of 5 to 5.5, and even more preferably at pH 5.2.

[0041] The melting temperature (Tm) of esterase can be evaluated by those skilled in the art according to methods known in the art. For example, DSF can be used to quantify the change in thermal denaturation temperature of esterase, thereby determining the Tm of this esterase. Alternatively, Tm can be evaluated by analyzing protein folding using circular dichroism. Preferably, Tm is measured using DSF or circular dichroism, as shown in the experimental section. In the context of the present invention, comparison of Tm is performed with Tm measured under the same conditions (e.g. pH, nature and amount of polyester, etc.).

[0042] Alternatively, thermostability may be assessed by measuring the esterase and / or polyester depolymerization activity of the esterase after incubation at various temperatures and comparing it to the esterase and / or polyester depolymerization activity of the parent esterase. The ability to perform multiple polyester depolymerization assays at various temperatures may also be evaluated. A rapid and informative test may consist of evaluating the ability of the esterase to degrade solid polyester compounds dispersed in agar plates after incubation at various temperatures by halo diameter measurements.

[0043] According to the invention, the esterases disclosed in the present invention further exhibit higher polyesterolytic activity and / or higher thermostability under acidic conditions, i.e. at a pH above 7, than under basic conditions, as compared to the enzyme of SEQ ID NO: 1. In the context of the present invention, "basic conditions" refers to conditions (e.g., medium, solution, etc.) at a pH above 7, preferably at a pH between 7 and 9.

[0044] In particular, these esterases are more efficient and stable compared to the parent esterases at a pH between 3 and 6, specifically between 4 and 6, 5 and 6, 5 and 5.5, compared to a pH above 7, specifically between 7 and 9. In particular, these esterases exhibit higher polyesterolytic activity and / or higher thermostability compared to the enzyme of SEQ ID NO: 1, which is higher at a pH between 3 and 6, specifically between 5 and 5.5, compared to a pH above 7, specifically between 7 and 9.

[0045] Esterases used The present invention therefore relates to the use of an esterase for degrading a polyester or polyester-containing material, the polyester or polyester-containing material being contacted with the enzyme at a pH of 3 to 6, the enzyme having (i) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the full-length amino acid sequence set forth in SEQ ID NO:1, (ii) any of the following amino acids: T11E / M, R12D / N / Q / E / F, S13E, A14E / D, T16E, A17T, T61S / V, A62D, F90A / Y, Y92G / D, W155A, T157S, P179D / E, Q182D / E, F187Y / I, D20 3C / K / R, N204D / E / G, A205D, S206D / E, F208M / W / G / N / R / I / A / Q / L / S / T / E, N211D / Y / E, S212F, N213P / D, N214D, A215N, S218A, V219I, Y220M / F, Q237D, F238E, N241E / D, N243E / D, L247T, V170I, G135A, V167Q / T, S248C and A24R, wherein the positions are numbered with reference to the amino acid sequence set forth in SEQ ID NO:1; and (iii) having polyesterolytic activity at a pH of 3 to 6.

[0046] Unless otherwise specified, the amino acid positions disclosed in this application are numbered with reference to the amino acid sequence set forth in SEQ ID NO:1.

[0047] Advantageously, the esterase has polyester degrading activity at a pH between 4 and 6, preferably at a pH between 5 and 6, more preferably at a pH between 5 and 5.5, in particular at pH 5.2.

[0048] According to the invention, the esterase is contacted with the polyester or polyester-containing material at a pH of 3 to 6, preferably at a pH of 4 to 6, more preferably at a pH of 5 to 6, even more preferably at a pH of 5 to 5.5, specifically at pH 5.2.

[0049] In certain embodiments, the esterase is F208M / W / G / N / R / I / A / Q / L / S / T / E, T11E / M, R12D / N / Q / E / F, S13E, A14E / D, T16E, T61S / V, A62D, F90A / Y, Y92G / D, W155A, T157S, Q182D / E, D203C / K, N204E / G, S206D, N211D / Y / E, S212F, N213P, V219I, Y2 20M, Q237D, N241E / D, N243E, L247T, V170I, G135A, V167Q / T, S248C, A24R and and F187I, preferably comprising at least one substitution selected from F208M / W / G / N / R / I / A / Q / L / S / T / E, T11M, R12D / E / F, A14E / D, T61S / V, A62D, F90A / Y, Y92G, W155A, T157S, Q182D / E, D203C / K, N204E / G, N211D / Y / E, S212F, N213P, V219I, Q237D, N241E / D, V170I, G135A, V167Q / T, S248C, A24R, and F187I.

[0050] In another embodiment, the esterase is selected from the group consisting of F208M / W / I / L / T / E, T11E / M, R12D / N / Q / E / F, S13E, A14E / D, T16E, T61S / V, A62D, F90A / Y, W155A, T157S, Q182D / E, N204E / G, S206D, N211D / Y, S212F, N213P, V219I, Y220M, Q237D, N241E / D, N243E, and and L247T, preferably comprising at least one substitution selected from F208M / W / I / L / T / E, T11M, R12D / E / F, A14E / D, T61S / V, A62D, F90A / Y, W155A, T157S, Q182D / E, N204E, N211D / Y, S212F, N213P, V219I, Q237D, and N241E / D.

[0051] Preferably, the esterase comprises at least one substitution selected from A14E / D, F90A / Y, Y92G / D, Q182D / E, N204D / E / G, F208M / W / G / N / R / I / A / Q / L / S / T / E, N211D / Y / E, N213P / D, V219I, V170I, D203C / K / R, S248C A24R, and F187Y / I, preferably A14E, F90A, Y92G / D, Q182D / E, N204G, F208M / G / N / R / I / A / Q / L / S / T, N211D / E, N213P, V219I, V170I, D203C / K / R, S248C and F187I, more preferably at least one substitution selected from F90A, Y92G, Q182D / E, N204G, F208M / G / N / R / I / A / Q / L / S / T, N211D / E, V170I, D203C / K / R, S248C, N213P, A24R, and F187I, and even more preferably at least one substitution selected from N211D / E, N204G, F208M / R / I / Q / L / S / T, Q182D / E, Y92G, V170I, D203C / K / R, S248C, N213P, A24R, and F187I. For example, the esterase comprises at least one substitution selected from F208T / L / S / R / G / I / M, Q182E, Y92G, V170I, D203C, S248C, and N213P.

[0052] According to the invention, the esterase may comprise at least one substitution selected from N211D, N204G, F90A, F208M / G / N / R / I / A / Q / L / S / T, Q182D / E, and A24R, and F187I, preferably at least one substitution selected from N211D, N204G, F208M / R / Q / L / S / T, Q182D / E, A24R, and F187I, more preferably at least one substitution selected from N211D, F208M / Q / L / S / T, Q182D / E, A24R, and F187I.

[0053] According to the invention, the esterase may comprise at least the substitution combination Y92G+V170I+D203C+S248C and at least one substitution selected from N211D, N204G, F90A, F208M / G / N / R / I / A / Q / L / S / T and Q182D / E, preferably selected from N211D, N204G, F208M / R / I / Q / L / S / T and Q182D / E, more preferably selected from F208M / I / / L / T and Q182E.

[0054] According to the invention, the esterase may comprise at least the substitution combination Y92G+V170I+D203C+S248C+N213P+Q182E and at least one substitution selected from N211D, N204G, F90A and F208M / G / N / R / I / A / Q / L / S / T, preferably selected from N211D, N204G and F208M / R / I / Q / L / S / T, more preferably selected from N211D, N204G and F208M / I / / L / T, even more preferably selected from F208M / I / / L / T.

[0055] According to the invention, the esterase may comprise at least one substitution selected from F208M / W / G / N / R / I / A / Q / L / S / T / E, preferably at least one substitution selected from F208M / G / N / R / I / A / Q / L / S / T, more preferably at least one substitution selected from F208M / R / I / Q / L / S / T, even more preferably at least one substitution selected from F208M / R / Q / L / S / T. In particular, the esterase comprises at least one substitution selected from F208M / I / L / T. In particular, the enzyme comprises at least the substitution F208M.

[0056] According to the invention, when the esterase comprises the substitution D203K / R, preferably the amino acid residue S248 is maintained as in the parent esterase (ie the esterase of SEQ ID NO:1).

[0057] According to the invention, the esterase is selected from A14E / D, F90A / Y, Y92G / D, Q182D / E, N204D / E / G, F208M / W / G / N / R / I / A / Q / L / S / T / E, N211D / Y / E, N213P / D, V219I, V170I, D203C / K / R, S248C A24R, and F187Y / I, preferably A14E, F90A, Y92G / D, Q182D / E, N204G, F208M / G / N / R / I / A / Q / L / S / T, N211D / E, N213P, V219I, V170I, D203C / K / R, S248C A24R, and F187I, more preferably selected from F90A, Y92G, Q182D / E, N204G, F208M / G / N / R / I / A / Q / L / S / T, N211D / E, V170I, D203C / K / R, S248C, N213P A24R, and F187I, even more preferably selected from N211D, N204G, F208M / R / I / L / S / T, Q182D / E, Y92G, V170I, D203C / K / R, S248C, N213P A24R, and F187I, and may contain at least two substitutions, preferably at least 3, 4, 5, 6, 7 substitutions.

[0058] According to the invention, the esterase may comprise at least a combination of substitutions selected from F208M / W / G / N / R / I / A / Q / L / S / T / E+D203C+S248C+V170I+Y92G / D+N213P / D+Q182D / E and F208M / W / G / N / R / I / A / Q / L / S / T / E+D203K+V170I+Y92G / D+N213P / D+Q182D / E, preferably F208M / I / L / T+D203C+S248C+V 170I+Y92G+N213P+Q182E, and F208M / I / L / T+D203K+V170I+Y92G / D+N213P / D+Q182D / E, more preferably F208M / I / L / T+D203C+S248C+V170I+Y92G+N213P+Q182E, and F208M / I / L / T+D203K+V170I+Y92G+N213P+Q182E.

[0059] For example, the esterase may comprise at least a substitution combination selected from F208M+D203C+S248C+V170I+Y92G+N213P+Q182E, and F208M+D203K+V170I+Y92G+N213P+Q182E, and preferably comprises the substitution combination F208M+D203C+S248C+V170I+Y92G+N213P+Q182E.

[0060] According to the invention, the esterase may further comprise at least one substitution selected from S13L and D158E, preferably at least two substitutions S13L and D158E.

[0061] For example, the esterase may be at least one of F208M / I / L / T+D203C+S248C+V170I+Y92G+N213P+Q182E+S13L+D158E, F208M / I / L / T+D203C+S248C+V170I+Y92G+N213P+Q182E+S13L, or F208M / I / L / T+D203C+S248C+V170I+Y92G+N213P+Q 182E+D158E, preferably the combination F208M / I / L / T+D203C+S248C+V170I+Y92G+N213P+Q182E+S13L+D158E, more preferably the combination F208M+D203C+S248C+V170I+Y92G+N213P+Q182E+S13L+D158E.

[0062] In one embodiment, the esterase is selected from the group consisting of F208M / W / G / N / R / I / A / Q / L / S / T / E, T11E / M, R12D / N / Q / E / F, S13E, A14E / D, T16E, T61S / V, A62D, F90A / Y, Y92G / D, W155A, T157S, Q182D / E, D203C, / K, N204E / G, S206D, N211D / Y / E, S212F, N213P, V219I, Y220M, Q237D, N241E / D, N243E, L247T, V170I, G135A, V167Q / T, S248C, A24R, and F187I, Preferably, the antibody has the amino acid sequence set forth in SEQ ID NO: 1, containing 1 to 25 substitutions selected from F208M / W / G / N / R / I / A / Q / L / S / T / E, T11M, R12D / E / F, A14E / D, T61S / V, A62D, F90A / Y, Y92G, W155A, T157S, Q182D / E, D203C / K, N204E / G, N211D / Y / E, S212F, N213P, V219I, Q237D, N241E / D, V170I, G135A, V167Q / T, S248C, A24R, and F187I, and is capable of detecting the amino acid sequence at a pH of 3 to 6, preferably at a pH of 5 to 5.5. It exhibits higher polyester decomposition activity and / or higher heat resistance compared to SEQ ID NO:1.

[0063] According to the invention, the esterase may comprise at least one amino acid residue selected from S130, D175, H207, C240, or C275, as in the parent esterase of SEQ ID NO:1, i.e., the enzyme of the invention is unmodified at one, two, three, etc. or all of these positions.

[0064] For example, the esterase comprises at least amino acids S130, D175, and H207, which form the catalytic site of the enzyme, and / or amino acids C240 ​​and C275, which form disulfide bonds, similar to the parent esterase. Preferably, the esterase comprises at least a combination of amino acid residues selected from S130+D175+H207, C240+C275, and S130+D175+H207+C240+C275, similar to the parent esterase of SEQ ID NO:1.

[0065] According to the present invention, the esterase disclosed in the present invention exhibits measurable polyester degradation activity at least in the pH ranges of 3-6, 4-6, 4.5-6, 5-6, 5.5-6, 5-5.5, 5-5.2, 5.2-5.5, 4-5.5, 4.5-5.5, 5-5.5, preferably in the pH range of 5-5.2, more preferably at pH 5.2.

[0066] The present esterase may further exhibit measurable polyester decomposition activity in the pH ranges of 6.5 to 10, 7 to 9.5, 7 to 9, 7.5 to 8.5, 6 to 9, 6.5 to 9, and 6.5 to 8. Preferably, the present esterase further exhibits measurable polyester decomposition activity at pH 8.

[0067] Advantageously, the esterase disclosed in the present invention exhibits higher polyester decomposition activity and / or higher thermostability in the pH range of 3 to 6, preferably at a pH of 4 to 6, more preferably at a pH of 5 to 6, and even more preferably at a pH of 5 to 5.5, compared to the esterase of SEQ ID NO: 1. Specifically, the esterase of the present invention exhibits higher polyester decomposition activity and / or higher thermostability in the pH ranges of 3 to 6, 4 to 6, 5 to 6, 5 to 5.5, 5.2 to 5.5, 5.5 to 6, and 5 to 5.2, compared to the esterase of SEQ ID NO: 1. According to the present invention, the specification of a pH range includes the lower and upper limits of said range.

[0068] The esterase disclosed in the present invention may further exhibit higher thermostability and / or higher polyester decomposition activity compared to the esterase of SEQ ID NO: 1 at a pH of 6 to 10, preferably at a pH of 6.5 to 9, more preferably at a pH of 6.5 to 8, and even more preferably at pH 8.

[0069] According to the present invention, the present esterase exhibits higher polyester decomposition activity and / or higher heat resistance at a pH of 3 to 6 compared to the esterase of SEQ ID NO: 1. Specifically, the present esterase exhibits higher polyester decomposition activity and / or higher heat resistance at a specific pH of 3 to 6 and / or in a pH range of 3 to 6 compared to the esterase of SEQ ID NO: 1. Preferably, the present esterase exhibits higher polyester decomposition activity and / or higher heat resistance at pHs of 3.5 to 6, 4 to 6, 5 to 6, 5 to 5.5, 5.2 to 5.5, 4 to 5.5, and 4.5 to 5.5 compared to the esterase of SEQ ID NO: 1. Preferably, the present esterase exhibits higher polyester decomposition activity and / or higher heat resistance at least at pH 3, pH 3.5, pH 4, pH 4.5, pH 5, pH 5.2, pH 5.5, and / or pH 6 compared to the esterase of SEQ ID NO: 1. The esterase may also exhibit higher polyester decomposition activity and / or higher thermostability compared to the esterase of SEQ ID NO: 1 over the entire pH range of 3 to 6, over the entire pH range of 4 to 6, over the entire pH range of 4.5 to 6, over the entire pH range of 5 to 6, over the entire pH range of 5.5 to 6, over the entire pH range of 5 to 5.5, over the entire pH range of 5 to 5.2, and over the entire pH range of 5.2 to 5.5.

[0070] According to the invention, the esterases exhibit a greater increase in polyester degrading activity and / or a greater increase in thermostability compared to the enzyme of SEQ ID NO: 1 at a pH between 3 and 6, particularly between 5 and 5.5, compared to a pH above 7, particularly between 7 and 9.

[0071] In one embodiment, the esterase is selected from the group consisting of N211D, N204G, F208T / M / L / S / Q / R, Q182D / E, A24R, F187I, F208M / I / T / L+D203C+S248C+V170I+Y92G+N213P+Q182E, F208M+D203C+S248C+V170I+Y92G+N213 P+Q182E+S13L+D158E, F208M+D203C+S248C+V170I+Y92G+N213P+Q182E+S13L, F208M+D203C+S248C+V170I+Y92G+N213P+Q182E+D158E.

[0072] Esterase with high polyester decomposition activity According to the invention, the esterase may comprise at least one substitution selected from N211D / Y / E, N204D / E / G, F90A / Y, F208M / W / G / N / R / I / A / Q / L / S / T / E, Q182D / E, D203C / K / R, S248C, V170I, Y92G / D, N213P / D, A24R, and F187Y / I, and preferably N211D / E, N204G, F90A, F208M / G / N / R / I / A / Q / L / S / T, Q182D / E, D203C, S248C, V170I, Y92G, N213P and F187I, more preferably at least one substitution selected from N211D / E, N204G, F90A, F208M / G / N / R / I / A / Q / L / S / T, Q182D / E, A24R, and F187I, and exhibits higher polyester degradation activity at a pH of 3 to 6, preferably at a pH of 5 to 5.5, compared to the enzyme of SEQ ID NO: 1. Advantageously, the enzyme comprises at least one substitution selected from N211D / Y / E, N204D / E / G, F208M / W / G / N / R / I / A / Q / L / S / T / E, Q182D / E, D203C / K, S248C, V170I, Y92G / D, N213P / D, A24R and F187I, preferably N211D / E, N204G, F208M / R / Q / L / S / T, Q182D / E, D203C / K, S248C, V170I, Y92G, N213P and F187I, more preferably at least one substitution selected from N211D / E, N204G, F208M / R / Q / L / S / T, Q182D / E, A24R, and F187I, and even more preferably at least one substitution selected from N211D, F208M / Q / L / S / T, Q182D / E, A24R, and F187I.

[0073] Specifically, the esterase is selected from N211D / Y / E, N204D / E / G, F208M / W / G / N / R / I / A / Q / L / S / T / E, Q182D / E, D203C / K, S248C, V170I, Y92G / D, N213P / D, A24R, and F187I, and preferably N211D / E, N204G, F208M / R / Q / L / S / T, Q182D / E, D203C / K, S248C, V170I, Y92G, N213P and F187I, more preferably selected from N211D / E, N204G, F208M / R / Q / L / S / T, Q182D / E, A24R, and F187I, and even more preferably selected from N211D, F208M / Q / L / S / T, Q182D / E, A24R, and F187I.

[0074] According to the invention, the enzyme may comprise at least a combination of substitutions selected from F208M+D203C+S248C+V170I+Y92G+N213P+Q182E, F208I+D203C+S248C+V170I+Y92G+N213P+Q182E, and F208M+D203C+S248C+V170I+Y92G+N213P+Q182E+S13L+D158E.

[0075] Indeed, the inventors have shown that these esterases exhibit a greater increase in polyesterolytic activity under acidic conditions compared to basic conditions compared to the enzyme of SEQ ID NO: 1. Specifically, the inventors have shown that the esterases are more efficient (i.e., exhibit a greater increase in polyesterolytic activity compared to the enzyme of SEQ ID NO: 1) compared to a pH above 7, specifically compared to a pH between 7 and 9, and at a pH between 3 and 6, specifically between 5 and 5.5, compared to the parent esterase.

[0076] For example, the esterase comprises at least one combination of substitutions selected from F208M / W / G / N / R / I / A / Q / L / S / T / E+D203C+S248C+V170I+Y92G / D+N213P / D+Q182D / E and F208M / W / G / N / R / I / A / Q / L / S / T / E+D203K+V170I+Y92G / D+N213P / D+Q182D / E, preferably F208M / I / L / T+ D203C + S248C + V170I + Y92G + N213P + Q182E, and F208M / I / L / T + D203K + V170I + Y92G / D + N213P / D + Q182D / E, more preferably, F208M / I / L / T + D203C + S248C + V170I + Y92G + N213P + Q182E. Specifically, the enzyme may include at least one combination of substitutions selected from F208M / I + D203C + S248C + V170I + Y92G + N213P + Q182E. Specifically, the enzyme may comprise at least one combination of substitutions selected from F208M / I + D203C + S248C + V170I + Y92G + N213P + Q182E + S13L + D158E.

[0077] In certain embodiments, the esterase consists of the amino acid sequence set forth in SEQ ID NO:1 including one combination of substitutions selected from F208M+D203C+S248C+V170I+Y92G+N213P+Q182E, F208I+D203C+S248C+V170I+Y92G+N213P+Q182E, and F208M+D203C+S248C+V170I+Y92G+N213P+Q182E+S13L+D158E.

[0078] In one embodiment, the esterase is selected from N211D / Y / E, N204D / E / G, F90A / Y, F208M / W / G / N / R / I / A / Q / L / S / T / E, Q182D / E, D203C / K, S248C, V170I, Y92G / D, N213P / D, A24R, and F187Y / I, preferably N211D / E, N204D / E / G, F90A / Y, F208M / W / G / N / R / I / A / Q / L / S / T / E, Q182D / E, D203C / K, S248C, V170I, Y92G / D, N213P / D, A24R, and F187Y / I. and the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1, containing 1 to 12 substitutions selected from among O404, O4G, F90A, F208M / G / N / R / I / A / Q / L / S / T, Q182D / E, D203C, S248C, V170I, Y92G, N213P, A24R, and F187I, and exhibits higher polyester degradation activity at a pH of 3 to 6, preferably at a pH of 5 to 5.5, compared to the amino acid sequence of SEQ ID NO: 1. Specifically, the esterase of the present invention contains 1 to 11 substitutions selected from N211D / Y / E, N204D / E / G, F208M / W / G / N / R / I / A / Q / L / S / T / E, Q182D / E, D203C / K, S248C, V170I, Y92G / D, N213P / D, A24R, and F187Y / I, and preferably contains 1 to 11 substitutions selected from N211D, N204G, F208M / R / I / Q / L / S / T, Q182D / E, D203C / K, S248C, V170I, Y92G, N213 and more preferably comprising 1 to 10 substitutions selected from N211D / E, F208M / I / Q / L / S / T, Q182E, D203C / K, S248C, V170I, Y92G, N213P, A24R, and F187I, and exhibiting a greater increase in polyesterolytic activity compared to the esterase of SEQ ID NO: 1 under acidic conditions compared to basic conditions.

[0079] In one embodiment, the esterase is selected from N211D / Y / E, N204D / E / G, F90A / Y, F208M / W / G / N / R / I / A / Q / L / S / T / E, Q182D / E, D203C / K, S248C, V170I, Y92G / D, N213P / D, A24R, and F187Y / I, preferably N211D / E, N204D / E / G, F90A / Y, F208M / W / G / N / R / I / A / Q / L / S / T / E, Q182D / E, D203C / K, S248C, V170I, Y92G / D, N213P / D, A24R, and F187Y / I. and the polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 1 containing 1 to 12 substitutions selected from among 4G, F90A, F208M / G / N / R / I / A / Q / L / S / T, Q182D / E, D203C, S248C, V170I, Y92G, N213P, A24R, and F187I, and exhibits higher polyester degradation activity at a pH of 3 to 6, preferably at a pH of 5 to 5.5, compared to the amino acid sequence of SEQ ID NO: 1. Specifically, the esterase of the present invention contains 1 to 11 substitutions selected from N211D / Y / E, N204D / E / G, F208M / W / G / N / R / I / A / Q / L / S / T / E, Q182D / E, D203C / K, S248C, V170I, Y92G / D, N213P / D, A24R, and F187Y / I, and preferably contains 1 to 11 substitutions selected from N211D, N204G, F208M / R / I / Q / L / S / T, Q182D / E, D203C / K, S248C, V170I, Y92G, N213 and more preferably, the esterase has the amino acid sequence set forth in SEQ ID NO: 1 and contains 1 to 11 substitutions selected from N211D / E, F208M / I / Q / L / S / T, Q182E, D203C / K, S248C, V170I, Y92G, N213P, A24R, and F187I, and exhibits a greater increase in polyesterolytic activity compared to the esterase of SEQ ID NO: 1 under acidic conditions compared to basic conditions.

[0080] In one embodiment, the esterase has an amino acid sequence as set forth in SEQ ID NO:1 with a single substitution selected from N211D / Y / E, N204D / E / G, F90A / Y, F208M / W / G / N / R / I / A / Q / L / S / T / E, Q182D / E, A24R, and F187Y / I, preferably selected from N211D / E, N204G, F90A, F208M / G / N / R / I / A / Q / L / S / T, Q182D / E, A24R, and F187I, and exhibits increased polyester degradation activity at a pH of 3 to 6, preferably at a pH of 5 to 5.5, compared to the amino acid sequence of SEQ ID NO:1. In particular, the esterase has the amino acid sequence as set forth in SEQ ID NO: 1, comprising a single substitution selected from N211D / E, N204G, F208M / R / Q / L / S / T, Q182E, A24R and F187I, preferably selected from F208T / L / M / S / R and Q182E. Advantageously, said esterase exhibits a greater increase in polyesterolytic activity compared to the enzyme of SEQ ID NO: 1 under acidic conditions compared to basic conditions.

[0081] Highly heat-resistant esterase According to the present invention, the esterase may contain at least one substitution selected from N211D / Y / E, N204D / E / G, F208M / W / G / N / R / I / A / Q / L / S / T / E, D203C / K / R, S248C, V170I, Y92G / D, N213P / D, and Q182D / E, preferably at least one substitution selected from N211D, N240G, F208M / I / L / T, D203C, S248C, V170I, Y92G, N213P, and Q182E, more preferably at least one substitution selected from N211D, N240G, and exhibits high thermostability at a pH of 3 to 6, preferably at a pH of 5 to 5.5, compared to the esterase of SEQ ID NO: 1.

[0082] Advantageously, the esterase comprises at least one combination of substitutions selected from F208M / W / G / N / R / I / A / Q / L / S / T / E+D203C+S248C+V170I+Y92G / D+N213P / D+Q182D / E and F208M / W / G / N / R / I / A / Q / L / S / T / E+D203K+V170I+Y92G / D+N213P / D+Q182D / E, preferably F208M / I / L / T +D203C+S248C+V170I+Y92G+N213P+Q182E, and F208M / I / L / T+D203K+V170I+Y92G / D+N213P / D+Q182D / E, more preferably comprising at least one combination of substitutions selected from F208M / I / L / T+D203C+S248C+V170I+Y92G+N213P+Q182E.

[0083] Advantageously, the esterase is selected from the group consisting of F208I + D203C + S248C + V170I + Y92G + N213P + Q182E, F208M + D203C + S248C + V170I + Y92G + N213P + Q182E, F208T + D203C + S248C + V170I + Y92G + N213P + Q182E, F208L + D203C + S248C + V170I + Y92G + N213P + Q18 2E, F208M+D203C+S248C+V170I+Y92G+N213P+Q182E+S13L+D158E, F208M+D203C+S248C+V170I+Y92G+N213P+Q182E+S13L and F208M+D203C+S248C+V170I+Y92G+N213P+Q182E+D158E.

[0084] Indeed, the inventors have shown that these esterases exhibit a large increase in thermostability under acidic conditions compared to basic conditions compared to the enzyme of SEQ ID NO: 1. Specifically, the inventors have shown that the esterases are thermostable compared to the parent esterase at a pH between 3 and 6, specifically between 5 and 5.5, compared to a pH above 7, specifically between 7 and 9.

[0085] In certain embodiments, the esterase is selected from the group consisting of F208I+D203C+S248C+V170I+Y92G+N213P+Q182E, F208M+D203C+S248C+V170I+Y92G+N213P+Q182E, F208T+D203C+S248C+V170I+Y92G+N213P+Q182E, F208L+D203C+S248C+V170I+Y92G+N213P+Q182E, F208 M+D203C+S248C+V170I+Y92G+N213P+Q182E+S13L+D158E, F208M+D203C+S248C+V170I+Y92G+N213P+Q182E+S13L and F208M+D203C+S248C+V170I+Y92G+N213P+Q182E+D158E.

[0086] Esterase with high decomposition activity and high heat resistance According to the invention, the esterase is and may comprise at least one substitution selected from N211D / Y / E, N204D / E / G, F208M / W / G / N / R / I / A / Q / L / S / T / E, D203C / K / R, S248C, V170I, Y92G / D, N213P / D, and Q182D / E, preferably may comprise at least one substitution selected from N211D, N204G, F208M / I / L / T, D203C / K, S248C, V170I, Y92G, N213P, and Q182E, more preferably may comprise at least one substitution selected from N211D and N204G, and exhibits both higher polyester degradation activity and higher heat resistance compared to the amino acid sequence of SEQ ID NO:1 at a pH of 3 to 6, preferably at a pH of 5 to 5.5.

[0087] For example, the esterase comprises at least one combination of substitutions selected from F208M / W / G / N / R / I / A / Q / L / S / T / E+D203C+S248C+V170I+Y92G / D+N213P / D+Q182D / E and F208M / W / G / N / R / I / A / Q / L / S / T / E+D203K+S248C+V170I+Y92G / D+N213P / D+Q182D / E, preferably F208M and F208M / I / L / T+D203K+S248C+V170I+Y92G / D+N213P / D+Q182D / E, more preferably comprising the substitution combination F208M / I+D203C+S248C+V170I+Y92G+N213P+Q182E.

[0088] Indeed, the inventors have shown that these esterases exhibit increased polyesterolytic activity under acidic conditions compared to basic conditions, as well as increased thermostability compared to the enzyme of SEQ ID NO: 1. Specifically, the inventors have shown that the esterases are more efficient and stable compared to the parent esterases at pHs between 3 and 6 compared to pHs above 7, specifically between 7 and 9.

[0089] In another embodiment, the esterase has the amino acid sequence as set forth in SEQ ID NO:1 containing 1 to 9 substitutions selected from N211D / Y / E, N204D / E / G, F208M / W / G / N / R / I / A / Q / L / S / T / E, D203C / K, S248C, V170I, Y92G / D, N213P / D, and Q182D / E, preferably selected from N211D, N204G, F208M / I / L / T, D203C / K, S248C, V170I, Y92G, N213P, and Q182E, and exhibits both increased polyester degradation activity and increased thermostability at a pH of 3 to 6, preferably at a pH of 5 to 5.5, compared to the amino acid sequence of SEQ ID NO:1. As an example, the esterase has the amino acid sequence set forth in SEQ ID NO:1, which contains a single substitution selected from N204D / E / G and N211D / Y / E, preferably selected from N204G and N211D.

[0090] In another example, the esterase is F208M / W / G / N / R / I / A / Q / L / S / T / E+D203C+S248C+V170I+Y92G / D+N213P / D+Q182D / E, F208M / W / G / N / R / I / A / Q / L / S / T / E+D203K+S248C+V170I+Y92G / D+N213P / D+Q182D / E, F208M / W / G / N / R / I / A / Q / L / S / T / E+D203K+S248C+V170I+Y92G / D+N213P / D+Q182D / E, / Q / L / S / T / E+D203C+S248C+V170I+Y92G+N213P+Q182E+S13L+D158E and F208M / W / G / N / R / I / A / Q / L / S / T / E+D203K+V170I+Y92G+N213P+Q182E+S13L+D158E, preferably F208M / I / L / T+D203C+S248C+V170I+Y92G / D+N213P / D+Q182D / E, F208M / I / L / T+D203K+S248C+V170I+Y92G / D+N213P / D+Q182D / E, F208M / I / L / T+D 203C+S248C+V170I+Y92G+N213P+Q182E+S13L+D158E, and F208M / I / L / T+D203K+V170I+Y92G+N213P+Q18 2E+S13L+D158E, more preferably F208M / I+D203C+S248C+V170I+Y92G+N213P+Q182E, or F208M / I+D203C+S248C+V170I+Y92G+N213P+Q182E+S13L+D158E. Specifically, said esterases exhibit a greater increase in polyesterolytic activity under acidic conditions compared to basic conditions, and a greater increase in thermostability compared to the enzyme of SEQ ID NO: 1. Specifically, the esterase has the amino acid sequence set forth in SEQ ID NO:1, comprising the substitution combination F208M / I+D203C+S248C+V170I+Y92G+N213P+Q182E, or F208M+D203C+S248C+V170I+Y92G+N213P+Q182E+S13L+D158E, and is more efficient and stable than the parent esterase at a pH greater than 7, specifically at a pH between 3 and 6, compared to a pH between 7 and 9.

[0091] Decomposition Process By working on optimizing the enzymatic degradation process of polyester or polyester-containing materials, the inventors have found that it is possible to avoid the production of by-products (salts) and improve the economic profitability of the degradation process of polyester or polyester-containing materials by reducing the consumption of base while maintaining an enzyme activity compatible with industrial performance.More specifically, the inventors have found that the enzymatic depolymerization of polyester can be carried out at acidic pH without adding any base when the reaction medium already contains a certain amount of equivalent terephthalic acid in the form of salt.The specific esterases described above are particularly suitable for degrading polyester or polyester-containing materials under acidic conditions.

[0092] In this regard, it is an object of the present invention to provide a process for degrading polyesters or polyester-containing materials, comprising a step of depolymerization of said polyesters, carried out by contacting the polyester-containing material or polyesters in a reaction medium having a pH between 3 and 6 with any of the above-described esterases capable of degrading said polyesters.

[0093] According to the invention, the depolymerization step is preferably carried out at a temperature between 40°C and 80°C, preferably between 50°C and 72°C, more preferably between 50°C and 65°C, even more preferably between 50°C and 60°C. In an embodiment, the depolymerization step is carried out at a temperature between 55°C and 60°C or between 50°C and 55°C. In another embodiment, the depolymerization step is carried out at a temperature between 55°C and 65°C. In another embodiment, the depolymerization step is carried out at a temperature between 60°C and 72°C, preferably between 60°C and 70°C. Advantageously, the temperature of the depolymerization step is kept below the Tg of the polyester of interest. In the context of the present invention, "polyester of interest" refers to the polyester that is the target of the depolymerization step. Preferably, the polyester of interest comprises at least terephthalic acid monomer (TA). Advantageously, the temperature is kept within ±1°C of the given temperature.

[0094] According to the present invention, the depolymerization step may be carried out at a pH of 4-6, 5-6, 4-5.5, 4.5-5.5, 5-5.5, 5.5-6, 5.1-5.3, 5-5.2, 3-5, 3-4, 3.5-4, 4-5, or 4.5-5.

[0095] Advantageously, the depolymerization step is carried out at a pH between 5.0 and 5.5 (for example pH 5.2) and at a temperature between 50°C and 72°C (preferably between 50°C and 55°C or between 65°C and 72°C).

[0096] Additional enzymes The process of degrading a polyester or polyester-containing material may be carried out by contacting the polyester or polyester-containing material with any one of the esterases disclosed above and at least one additional enzyme.

[0097] For example, the additional enzymes may be from Thermobifida cellulosityca, Thermobifida halotolerans, Thermobifida fusca, Thermobifida alba, Bacillus subtilis, Fusarium solani pisi, Humicola insolens, Sirococcus conigenus, Pseudomonas mendocina, Thielavia terrestris, Saccharomonospora viridis, The cutinase is derived from a microorganism selected from Thermomonospora curvata, Thermomonospora viridis, or any functional variant thereof having cutinase activity. Alternatively or in addition, the additional enzyme may be a lipase (preferably from Ideonella sakaiensis) and / or a cutinase from Humicola insolens (e.g., referenced A0A075B5G4 in Uniprot), or any functional variant thereof having cutinase activity. The additional enzyme may also be selected from commercially available enzymes (e.g., Novozym 51032) or any functional variant thereof.

[0098] Alternatively, or in addition, the process of degrading a polyester or polyester-containing material may be carried out by contacting the polyester with any one of the esterases disclosed above and an enzyme having MHET degrading activity (MHETase).

[0099] The amount of esterase in the reaction medium may be from 0.1 mg to 15 mg esterase / g of target polyester, preferably from 0.1 mg / g to 10 mg / g, more preferably from 0.1 mg / g to 5 mg / g, even more preferably from 0.5 mg / g to 4 mg / g. Preferably, the amount of esterase in the reaction medium is up to 4 mg / g of target polyester, preferably up to 3 mg / g, more preferably up to 2 mg / g of target polyester.

[0100] More generally, the amount of each enzyme in the reaction medium is preferably between 0.1 mg and 15 mg / g of target polymer.

[0101] polyester The claimed invention may be practiced with any polyester or polyester-containing material. Preferably, the polyester is selected from polyethylene terephthalate (PET), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polybutylene adipate terephthalate (PBAT), polycyclohexylene dimethylene terephthalate (PCT), glycol-modified polyethylene terephthalate (PETG), poly(butylene succinate-co-terephthalate) (PBST), poly(butylene succinate / terephthalate / isophthalate)-co-(lactate) (PBSTIL), "polyolefin-like" polyesters, and blends / mixtures of these polymers. Preferably, the polyester is polyethylene terephthalate (PET).

[0102] For example, the invention may be implemented with plastic products from plastic waste collection and / or industrial waste. More specifically, the invention may be implemented to degrade household plastic waste (e.g., plastic bottles, plastic trays, plastic bags, plastic packaging, soft plastics, and / or hard plastics) (even if contaminated with food residues, surfactants, etc.). Alternatively, or in addition, the invention may be implemented to degrade plastic fibers, such as cloth, textiles, and / or fibers obtained from industrial waste. More specifically, the invention may be used with PET plastic and / or PET fiber waste, such as cloth, textiles, and / or PET fibers obtained from tires.

[0103] The present invention is particularly suitable for degrading plastic articles that contain at least one polyester that contains at least terephthalic acid monomer (TA).

[0104] purification The process of decomposing plastic products may further comprise a step of recovering and optionally purifying the monomers and / or oligomers and / or decomposition products (preferably terephthalic acid) resulting from the depolymerization step. The monomers and / or oligomers and / or decomposition products resulting from the depolymerization may be recovered sequentially or continuously.

[0105] A single type of monomer and / or oligomer, or several different types of monomer and / or oligomer, may be recovered. The recovered monomer and / or oligomer and / or degradation products may be purified using any suitable purification method and, optionally, conditioned into a repolymerizable form. An example of purification is described in WO 1999 / 023055. For example, recovery of the TA in solid form includes separating the solid phase of the reaction medium from the liquid phase by filtration.

[0106] The recovered solid phase may be dissolved in a solvent selected from DMF, NMP, DMSO, DMAC, or any solvent known to solubilize TA, and filtered to remove impurities. The solubilized TA may then be recrystallized by any means known to one of skill in the art.

[0107] After the depolymerization step, MHETase may be added to the reaction medium prior to the purification process to hydrolyze the MHET produced during the depolymerization step to produce TA.

[0108] The repolymerizable monomers and / or oligomers can then be recycled to synthesize a polymer. A person skilled in the art can easily adapt the process parameters to the monomers / oligomers and polymers to be synthesized.

[0109] It is therefore also an object of the present invention to provide a process for recycling polyesters or polyester-containing substances by contacting said polyesters or polyester-containing substances with any one of the esterases described above at a pH between 3 and 6, and / or a method for producing monomers and / or oligomers and / or degradation products from a polyester-containing substance, comprising subjecting the polyester-containing substance to a depolymerization step carried out at a pH between 3 and 6 by using any of the esterases described above, and optionally purifying the monomers and / or oligomers.

[0110] All the embodiments described above in relation to the process for decomposing plastic products also apply to the process for producing monomers and / or oligomers, and to the recycling process.

[0111] Other uses of esterase The invention also relates to the use of any one of the above described esterases in a method for surface hydrolysis or surface functionalization of a polyester-containing material, comprising exposing the polyester-containing material to said esterase at a pH of 3 to 6. The method is particularly useful for increasing the hydrophilicity (i.e. water absorbency) of polyester materials. Such increased hydrophilicity may be of particular interest in textile manufacturing, electronics, and biomedical applications.

[0112] The present invention also relates to the use of any one of the above described esterases for treating water, wastewater or sewage at a pH between 3 and 6. In wastewater or sewage treatment applications, said esterases may be used to degrade microplastic particles made of polyester (preferably PET), such as polymer filaments, fibers, or other types of fragments and pieces of polyester-based products, preferably fragments and pieces of PET-based products.

[0113] The present invention also relates to the use of any one of the above described esterases in detergent, food, animal feed, paper, textile and pharmaceutical applications.As an example, in the paper industry, any one of the above described esterases may be used at a pH of 3 to 6.More specifically, the esterases may be used to remove stickies from paper pulp and water pipelines of paper machines. EXAMPLES

[0114] Example 1 Construction, expression, and purification of esterases - construction The esterase according to the invention was produced using the plasmid construct pET26b-LCC-His, into which the gene encoding the esterase of SEQ ID NO: 1 was cloned and optimized for Escherichia coli expression between the NdeI and XhoI restriction sites. To produce the esterase variants, two site-directed mutagenesis kits were used according to the supplier's recommendations: QuikChange II Site-Directed Mutagenesis Kit (from Santa Clara, California, USA) and QuikChange Lightning Multi Site-Directed from Agilent.

[0115] - Expression and purification of esterases Stellar™ strains (Clontech, California, USA) and E. coli BL21(DE3) (New England Biolabs, Evry, France) were employed in succession to perform cloning and recombinant expression in 50 mL of LB-Miller medium or ZYM autoinduction medium (Studier et al., 2005- Prot. Exp. Pur. 41, 207-234). Induction in LB-Miller medium was carried out at 16°C with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG, Euromedex, Souffelweyersheim, France). Cultures were stopped by centrifugation (8000 rpm, 20 min at 10°C) in an Avanti J-26 XP centrifuge (Beckman Coulter, Brea, USA). The cells were suspended in 20 mL of Talon buffer (Tris-HCl 20 mM, NaCl 300 mM, pH 8). The cell suspension was then sonicated for 2 min at 30% amplitude (2 sec ON and 1 sec OFF cycles) with an FB 705 sonicator (Fisherbrand, Illkirch, France). The following centrifugation step was then performed: 10000 g in an Eppendorf centrifuge at 10° C. for 30 min. The soluble fraction was collected and subjected to affinity chromatography. This purification step was completed with Talon® Metal Affinity Resin (Clontech, CA, USA). Elution of the protein was performed with a step of Talon buffer supplemented with imidazole. The purified proteins were dialyzed against Talon buffer or sodium acetate buffer (100-300 mM, pH 5.2), then quantified using the Bio-Rad protein assay according to the manufacturer's instructions (Lifescience Bio-Rad, France) and stored at +4°C.

[0116] Example 2 Evaluation of esterase degradation activity The decomposition activity of the esterase was determined and compared with the activity of the esterase of SEQ ID NO:1.

[0117] To assess specific activity, several methodologies were used: (1) Specific activity based on PET hydrolysis and ultra-high performance liquid chromatography (UHPLC) analysis (2) Specific activity based on PET hydrolysis and ultraviolet absorption (UV assay) analysis (3) Degradation activity based on the decomposition of polyester in solid form (4) Decomposition activity based on PET hydrolysis in a reactor of more than 100 mL

[0118] 2.1. Specific activity based on PET hydrolysis and ultra-high performance liquid chromatography (UHPLC) analysis 100 mg of powdered amorphous PET (prepared according to WO 2017 / 198786 to reach a crystallinity of less than 20%) was weighed and introduced into a 100 mL glass bottle. 1 mL of an esterase preparation containing the esterase of SEQ ID NO: 1 (as a reference control) or the esterase of the invention, prepared to 1,727 μM in sodium acetate buffer (100-300 mM, pH 5.2) for measurements in acidic conditions (or 0.69 μM in Talon buffer (Tris-HCl 20 mM, NaCl 0.3 M, pH 8) for basic conditions), was introduced into the glass bottle. Finally, 9 mL or 49 mL of the corresponding buffer (depending on the pH at which the measurements are performed) were added.

[0119] Depolymerization was initiated by incubating each vial at 50°C, 54°C, 60°C, 65°C, 68°C, or 72°C and 150 rpm in a Max Q 4450 incubator (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

[0120] The initial rate of the depolymerization reaction (mg equivalents of TA produced / h) was determined by sampling performed at various times during the first 24 h and analyzed by ultra-high performance liquid chromatography (UHPLC). If necessary, the samples were diluted with 0.1 M potassium phosphate buffer (pH 8). Then, 150 μL of methanol and 6.5 μL of 6 N HCl were added to 150 μL of sample or diluent. After mixing and filtering through a 0.45 μm syringe filter, the samples were loaded onto the UHPLC to monitor the release of terephthalic acid (TA), MHET, and BHET. The chromatographic system used was an Ultimate 3000 UHPLC system (Thermo Fisher Scientific, Inc., Waltham, MA, USA) including a pump module, an autosampler, a column oven thermostat at 25 °C, and a UV detector at 240 nm. The column used was a Discovery® HS C18 HPLC column (150×4.6 mm, 5 μm, equipped with a precolumn, Supelco, Bellefonte, USA). TA, MHET, and BHET were separated using a gradient (30%-90%) of MeOH in 1 mM H2SO4 at 1 mL / min. Injections were 20 μL of sample. TA, MHET, and BHET were measured under the same conditions as the samples according to a calibration curve made from commercially available TA and BHET, and from in-house synthesized MHET. The specific activity of PET hydrolysis (mg equivalents of TA / h / mg enzyme) was determined in the linear part of the hydrolysis curve of the reaction (i.e., at the beginning of the reaction), and such a curve was set by sampling performed at various times during the first 24, 48, or 72 hours. The equivalents of TA correspond to the sum of the measured TA and the TA contained in the measured MHET and BHET. Measurement of the equivalent TA may also be used to calculate the yield of a PET depolymerization assay at a given time and / or after a defined period of time (e.g., 24 hours or 48 hours).

[0121] 2.2. Specific Activity Based on PET Hydrolysis and Ultraviolet Absorption (UV Assay) Analysis 100 mg of powdered amorphous PET (prepared according to WO 2017 / 198786 to reach a crystallinity of less than 20%) was weighed and introduced into a 100 mL glass bottle. 1 mL of an esterase preparation containing the esterase of SEQ ID NO: 1 (as a reference control) or the esterase of the invention, prepared to 1,727 μM in sodium acetate buffer (100-300 mM, pH 5.2) for measurements in acidic conditions or 0.69 μM in Talon buffer (Tris-HCl 20 mM, NaCl 0.3 M, pH 8) for measurements in basic conditions, was introduced into the glass bottle. Finally, 9 mL or 49 mL of the corresponding buffer (depending on the pH at which the measurements are performed) were added.

[0122] Depolymerization was initiated by incubating each vial at 50°C, 54°C, 60°C, or 65°C and 150 rpm in a Max Q 4450 incubator (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

[0123] The initial rate of the depolymerization reaction (μmol soluble degradant produced / hour) was determined by sampling performed at various times during the first 24 hours and analyzed by reading the absorbance at 242 nm using an Eon Microplate Spectrophotometer (BioTek, USA). An increase in the absorbance of the reaction mixture in the ultraviolet region of the light spectrum (at 242 nm) indicates the release of soluble TA or its esters (BHET and MHET) from the insoluble PET substrate. The absorbance value at this wavelength can be used to calculate the sum of PET hydrolysis products according to the Lambert-Beer law, and the specific activity of the enzyme is determined as the total equivalent TA produced. The specific activity of PET hydrolysis (μmol soluble product / hour / mg enzyme) was determined in the linear part of the hydrolysis curve of the reaction (i.e., at the beginning of the reaction), and such a curve was set by sampling performed at various times during the first 24, 48, or 72 hours. The equivalent TA measurement can also be used to calculate the yield of the PET depolymerization assay at a given time and / or after a defined period of time (e.g., 24 hours or 48 hours). If necessary, samples were diluted with 0.1 M potassium phosphate buffer (pH 8).

[0124] 2.3. Activity based on the degradation of polyester in solid form Preparation of agar plates was achieved by dissolving 50 mg of PET in hexafluoro-2-propanol (HFIP) and pouring this medium into 250 mL of aqueous solution. After HFIP evaporation at 50° C. under 140 mbar, the solution was mixed with potassium phosphate buffer (pH 8.0), or sodium acetate buffer (pH 5.2), or sodium acetate buffer (pH 5.0) to obtain a final concentration of 1% agar with 0.5 mg / mL of PET and 0.1 M of buffer. Approximately 30 mL of this mixture was used to prepare each plate and stored at 4° C. 1 μL, 5 μL, or 20 μL of enzyme preparation (pure enzyme or cell lysate) was placed in the wells of the agar plates containing PET at pH 8.0, 5.2, or 5.0, respectively.

[0125] The diameter or surface area of ​​the halos formed due to polyester degradation by the wild-type esterase and variants was determined by measuring the diameter of the halos on agar plate photographs using the software Gimp and compared after defined periods (2 to 24 hours) at 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C.

[0126] 2.4. Activity based on PET hydrolysis in the reactor 0.69 μmol to 2.07 μmol of purified esterase prepared in 80 mL of 100 mM potassium phosphate buffer (pH 8), or 300 mM sodium acetate buffer (pH 5.0), or 300 mM sodium acetate buffer (pH 5.2), or 300 mM sodium acetate buffer (pH 6.0) was mixed with 20 g of amorphous PET (prepared according to WO 2017 / 198786 to reach a crystallinity of less than 20%) in a 500 mL Minibio bioreactor (Applikon Biotechnology, Delft, The Netherlands). Temperature regulation at 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C was performed by water bath immersion and constant agitation was maintained at 250 rpm using a single marine impeller. The pH of the PET depolymerization assay was controlled at pH 5, or pH 5.2, or pH 6, or pH 8 by addition of 6 N NaOH and was guaranteed by a my-Control bio controller system (Applikon Biotechnology, Delft, The Netherlands). Base consumption was recorded during the assay and can be used for characterization of the PET depolymerization assay.

[0127] The final yield of the PET depolymerization assay was determined either by determining the mass of the remaining PET, or by determining the equivalent amount of TA produced, or by base consumption. The mass determination of the remaining PET was evaluated at the end of the reaction by filtering the reaction volume through a 12-15 μm grade 11 ashless filter paper (Dutscher SAS, Brumath, France) and weighing such residue after drying. The determination of the equivalent amount of TA produced was realized using the UHPLC method described in 2.1, and the percentage of hydrolysis was calculated based on the ratio of the molar concentration (TA+MHET+BHET) at a given time to the total amount of TA contained in the initial sample. The PET depolymerization produced acidic monomers that were neutralized with base to be able to maintain the pH in the reactor. The determination of the equivalent amount of TA produced was calculated using the corresponding molar base consumption, and the percentage of hydrolysis was calculated based on the ratio of the molar concentration at a given time of the equivalent amount of TA to the total amount of TA contained in the initial sample.

[0128] result Activity based on the degradation of solid forms of PET under acidic conditions compared to the esterase of SEQ ID NO:1 The activity of the esterases (variants) of the invention was evaluated after 24 hours at 50° C. and pH 5.0 (V1, V3-V5, V9, V29-V35, V46-48) or pH 5.2 (V2, V20-V28, V36-42, V44-V45, V49-V68) as shown in Example 2.3. The surface area of ​​the halos of the esterases (variants) of the invention was compared with the surface area formed by the wild-type esterase of SEQ ID NO: 1. The variants with a higher surface area compared to the wild-type esterase of SEQ ID NO: 1 (i.e. the variants with a superior degradative activity compared to the esterase of SEQ ID NO: 1 after a defined period of time) are reported in Table 1 below.

[0129] [Table 1]

[0130] The variants listed in Table 1 each have the exact amino acid sequence set forth in SEQ ID NO:1, except for the substitutions listed in Table 1.

[0131] Interestingly, most of the variants exhibit a halo with a diameter greater than or equal to 110% of the halo diameter of the wild-type esterase of SEQ ID NO: 1. These variants are reported in Table 2 below.

[0132] [Table 2]

[0133] The variants listed in Table 1 each have the exact amino acid sequence set forth in SEQ ID NO:1, except for the substitutions listed in Table 1.

[0134] Specific cleavage activity under acidic conditions compared to the esterase of SEQ ID NO:1 The specific hydrolysis activity of the esterases (variants) used according to the invention was determined from the linear part of the hydrolysis curve (i.e. at the start of the reaction). The specific hydrolysis activity of the esterase of SEQ ID NO: 1 was used as a reference and considered as 100% specific hydrolysis activity. The specific hydrolysis activity was measured at 54° C. for variants V1 to B15 and at 60° C. for variants V16 to V19 and V69 to V72 at pH 5.2 as indicated in Example 2.1. An overview of the results is summarized in Table 3 below.

[0135] [Table 3A]

[0136] [Table 3B]

[0137] The variants in Table 3 each have the exact amino acid sequence set forth in SEQ ID NO:1, except for the substitution or combination of substitutions listed in Table 3.

[0138] PET depolymerization yield under acidic conditions compared to esterase of SEQ ID NO:1 The PET depolymerization yields of the esterases (variants) used according to the invention after 24 hours at pH 5.2 and 54° C. are shown in Table 4 below. The PET depolymerization yield of the esterase of SEQ ID NO: 1 is used as a reference and considered as 100% PET depolymerization yield. The PET depolymerization yield was measured after 24 hours for variant V73 as shown in Example 2.1 and for variant V74 as shown in Example 2.2.

[0139] [Table 4]

[0140] The variants in Table 4 have the exact amino acid sequence of SEQ ID NO:1, except for the substitutions listed in Table 4.

[0141] Degradation activity under alkaline conditions compared to the esterase of SEQ ID NO:1 The specific decomposition activity and / or PET depolymerization yield after 24 hours of the esterases used according to the invention were also measured at pH 8 as shown in Example 2.1 (or Example 2.2 for variants V16 and V19). The activity was measured at 65° C., except for variant V16, whose activity was measured at 68° C., and variant V74, whose activity was measured at 60° C. The specific decomposition activity and / or PET depolymerization yield after 24 hours of the esterase of SEQ ID NO: 1 was used as a reference and considered as 100% specific decomposition activity and / or 100% PET depolymerization yield after 24 hours.

[0142] Further variants showing higher specific cleavage activity at pH 8 compared to the esterase of SEQ ID NO:1 are shown in Table 5 below.

[0143] [Table 5]

[0144] The variants listed in Table 5 each have the exact amino acid sequence set forth in SEQ ID NO:1, except for the combination of substitutions listed in Table 5.

[0145] Further variants showing higher PET depolymerization yield at pH 8 compared to the esterase of SEQ ID NO:1 are shown in Table 6 below.

[0146] [Table 6]

[0147] Degradation activity under acidic and basic conditions compared to the esterase of SEQ ID NO:1 The improvement ratio between the relative specific decomposition activity at pH 5.2 and / or the PET depolymerization yield after 24 hours at pH 5.2 and the relative specific decomposition activity at pH 8 and / or the PET depolymerization yield after 24 hours at pH 8 was calculated. Variants showing an improvement ratio of more than 1.1 (i.e., variants showing a higher increase in activity under acidic conditions than under basic conditions (compared to SEQ ID NO: 1)) are listed in Table 7 below.

[0148] [Table 7]

[0149] The variants have the exact amino acid sequence set forth in SEQ ID NO:1, except for the substitutions or combinations of substitutions, respectively, listed in Table 7. The variants exhibit increased activity under acidic conditions relative to basic conditions, as compared to SEQ ID NO:1.

[0150] Example 3 Evaluation of the heat resistance of the esterase of the present invention The thermostability of the esterase of the present invention was determined and compared with that of the esterase of SEQ ID NO:1.

[0151] Heat resistance was estimated using various methodologies: (1) Circular dichroism of proteins in solution; (2) the residual esterase activity after incubation of the protein under given conditions of temperature, time, and buffer; (3) the residual polyester depolymerization activity after incubation of the protein under given conditions of temperature, time, and buffer; (4) the ability to degrade a solid polyester compound (e.g., PET, or PBAT, or similar) dispersed in an agar plate after incubation of the protein under given conditions of temperature, time, and buffer; (5) the ability to perform polyester depolymerization assays multiple times under given conditions of temperature, buffer, protein concentration, and polyester concentration; (6) Differential scanning fluorimetry (DSF).

[0152] A detailed protocol for such a method is provided below.

[0153] 3.1 Circular dichroism Circular dichroism (CD) was performed on a Jasco 815 instrument (Easton, USA) to compare the melting temperature (Tm) of the esterase of SEQ ID NO: 1 with that of the esterase of the present invention. Technically, 400 μL of protein sample was prepared at 0.5 mg / mL in defined conditions of pH (Talon buffer (pH 8), sodium acetate buffer 100 mM pH 5 or 5.2) and used for CD. A first scan from 280 to 190 nm was realized to determine the two CD intensity maxima corresponding to the correct folding of the protein. A second scan was then performed from 25 °C to 110 °C with a wave length corresponding to such maximum intensity, providing a specific curve (sigmoidal 3 parameters y=a / (1+e^((x-x0) / b))) that was analyzed by Sigmaplot version 11.0 software to determine the Tm when x=x0. The obtained Tm reflects the heat resistance of a given protein. The higher the Tm, the more stable the variant is at high temperatures.

[0154] 3.2 Residual esterase activity A 1 mL solution of 40 mg / L (in Talon buffer, or 0.2 M sodium acetate buffer (pH 5.0), or 0.2 M sodium acetate buffer (pH 5.2), or sodium acetate buffer (pH 6.0)) of the esterase of SEQ ID NO: 1 or the esterase of the invention was incubated at various temperatures (40, 50, 60, 65, 70, 75, 80, and 90 °C) for up to 10 days. Samples were taken periodically and diluted 1 to 500 times in 0.1 M potassium phosphate buffer (pH 8.0), or 0.2 M sodium acetate buffer (pH 5.0), or 0.2 M sodium acetate buffer (pH 5.2), or sodium acetate buffer (pH 6.0) to realize the paranitrophenol-butyrate (pNP-B) assay. 20 μL of sample was mixed with 175 μL of 0.1 M potassium phosphate buffer (pH 8.0), or 0.2 M sodium acetate buffer (pH 5.0), or 0.2 M sodium acetate buffer (pH 5.2), or sodium acetate buffer (pH 6.0), and 5 μL of pNP-B solution (40 mM) in 2-methyl-2-butanol. The enzyme reaction was carried out at 30° C. under stirring for 15 min, and the absorbance at 405 nm was obtained by a microplate spectrophotometer (Versamax, Molecular Devices, Sunnyvale, CA, USA). The activity of pNP-B hydrolysis (initial rate expressed in μmol pNPB / min) was determined using a calibration curve prepared in the same buffer and pH conditions as the enzyme assay for liberated paranitrophenol in the linear part of the hydrolysis curve.

[0155] 3.3 Residual polyester depolymerization activity 10 mL of a 40 mg / L solution of the esterase of SEQ ID NO:1 or the esterase of the invention (in Talon buffer, or 0.2 M sodium acetate buffer (pH 5.0), or 0.2 M sodium acetate buffer (pH 5.2), or sodium acetate buffer (pH 6.0)) was incubated at various temperatures (40° C., 50° C., 60° C., 65° C., 70° C., 75° C., 80° C., and 90° C.), respectively, for up to 30 days. Periodically, 1 mL of sample was taken and transferred to a bottle containing 100 mg of amorphous PET (prepared according to WO 2017 / 198786 to reach less than 20% crystallinity) micronized to 250-500 μm and 49 mL of 0.1 M potassium phosphate buffer (pH 8.0), or 0.2 M sodium acetate buffer (pH 5.0), or 0.2 M sodium acetate buffer (pH 5.2), or sodium acetate buffer (pH 6.0), and incubated at 50 °C, 55 °C, 60 °C, 65 °C, or 70 °C. 150 μL of buffer was sampled periodically. If necessary, the sample was diluted with 0.1 M potassium phosphate buffer (pH 8). Then, 150 μL of methanol and 6.5 μL of 6 N HCl were added to the sample or 150 μL of diluent. After mixing and filtering through a 0.45 μm syringe filter, the samples were loaded onto a UHPLC to monitor the release of terephthalic acid (TA), MHET, and BHET. The chromatographic system used was an Ultimate 3000 UHPLC system (Thermo Fisher Scientific, Inc., Waltham, MA, USA) including a pump module, an autosampler, a column oven thermostat at 25° C., and a UV detector at 240 nm. The column used was a Discovery® HS C18 HPLC column (150×4.6 mm, 5 μm, equipped with a precolumn, Supelco, Bellefonte, USA). TA, MHET, and BHET were separated using a gradient (30%-90%) of MeOH in 1 mM H2SO4 at 1 mL / min. The injection was 20 μL of sample.TA, MHET, and BHET were measured under the same conditions as the samples according to calibration curves made from commercially available TA and BHET, and in-house synthesized MHET. The activity of PET hydrolysis (μmol PET hydrolyzed / min, or mg equivalent TA produced / hr) was determined in the linear part of the hydrolysis curve, which was set up by sampling performed at various times during the first 24 h. The equivalent TA corresponds to the sum of the measured TA and the TA contained in the measured MHET and BHET.

[0156] 3.4 Degradation of polyester in solid form 1 mL of 40 mg / L solutions of the esterase of SEQ ID NO:1 and the esterase of the present invention (Talon buffer, or 0.1 M potassium phosphate buffer (pH 8.0), or 0.1 M phosphate citrate buffer (pH 6.0), or 0.1 M sodium acetate buffer (pH 5.2)) were incubated at various temperatures (40° C., 50° C., 60° C., 65° C., 70° C., 75° C., 80° C., and 90° C.) for up to 30 days. Periodically, the enzyme preparations were sampled and placed into the wells of the agar plates containing PET. The preparation of the agar plates was achieved by dissolving 50 mg of PET in hexafluoro-2-propanol (HFIP) and pouring this medium into 250 mL of the aqueous solution. After HFIP evaporation at 50° C. under 140 mbar, the solution was mixed with potassium phosphate buffer (pH 8.0), or phosphate citrate buffer (pH 6.0), or sodium acetate buffer (pH 5.2) to obtain a final concentration of 1% agar containing 0.5 mg / mL PET and 0.1 M buffer. Approximately 30 mL of this mixture is used to prepare each plate and stored at 4° C. 1 μL, 5 μL, or 20 μL of the enzyme preparation was placed in the wells of the agar plates containing PET at pH 8.0, 6.0, or 5.2, respectively.

[0157] The diameter or surface area of ​​the halos formed due to polyester degradation by wild-type esterase and the variants of the invention were measured and compared after 2 to 24 hours at 50° C., 55° C., 60° C., 65° C., or 70° C. The half-life of the enzyme at a given temperature corresponds to the time required for the diameter of the halo to decrease by a factor of two.

[0158] 3.5 Multiple depolymerization of polyester The ability of the esterases to perform successive 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 reach a crystallinity of less than 20%) and 100 mL of 100 mM sodium acetate buffer (pH 5.0), or 100 mM sodium acetate buffer (pH 5.2), or 100 mM sodium acetate buffer (pH 6.0) containing 3 mg of esterase. Agitation was set at 250 rpm using a marine impeller. The bioreactor was thermostated at 50°C, 55°C, 60°C, 65°C, or 70°C by immersion in an external water bath. The pH was adjusted to 5.0, 5.2, or 6.0 by addition of 3 M NaOH. Various parameters (pH, temperature, stirring, addition of base) were monitored by BioXpert software V2.95. 1.8 g of amorphous PET (prepared according to WO 2017 / 198786 to reach a crystallinity of less than 20%) was added every 20 hours. 500 μL of the reaction medium was sampled periodically.

[0159] The amounts of TA, MHET, and BHET were determined by HPLC as described in Example 2.3. The amount of EG was determined using an Aminex HPX-87K column (Bio-Rad Laboratories, Inc., Hercules, California, United States) thermostated at 65°C. The eluate was diluted with 0.6 mL / min. -1The solution was 5 mM K2HPO4 at 100° C. Injections were 20 μL. Ethylene glycol was monitored using a refractometer.

[0160] The percentage of hydrolysis was calculated based on the ratio of the molar concentration (TA+MHET+BHET) at a given time to the total amount of TA contained in the initial sample, or based on the ratio of the molar concentration (EG+MHET+2×BHET) at a given time to the total amount of EG contained in the initial sample. Degradation rates are calculated in mg total free TA per hour or mg total EG per hour.

[0161] The half-life of the enzyme was estimated as the incubation time required to obtain a 50% loss in degradation rate.

[0162] 3.6 Differential Scanning Fluorimetry (DSF) DSF was used to evaluate the thermal stability of wild-type protein (SEQ ID NO: 1) and its variants by determining the melting temperature (Tm), the temperature at which half of the protein population is unfolded. To estimate the Tm value at pH 8.0, the protein sample was prepared at a concentration of 6.25 μM in buffer A consisting of 20 mM Tris HCl (pH 8.0), 300 mM NaCl. To estimate the Tm value at pH 5.2, the protein sample was first prepared at a concentration of 25 μM in buffer B consisting of 100 mM potassium phosphate buffer (pH 8.0), and then 0.6 μL of the prepared protein sample was diluted with 18 μL of buffer C consisting of 100 mM sodium acetate (pH 5.09) to obtain a final pH value of 5.2 containing 6.25 μM diluted protein sample. SYPRO Orange dye 5000× stock solution in DMSO was first diluted 250 times with water. Protein samples were loaded onto a white clear 96-well PCR plate (Bio-Rad catalog # HSP9601) with each well containing a final volume of 25 μl. The final concentrations of protein and SYPRO Orange dye in each well were 6 μM (0.14 mg / ml) and 10×, respectively. The PCR plate was then sealed with optical quality sealing tape and spun at 2000 rpm for 1 min at room temperature. DSF experiments were then performed using a CFX96 Real-Time PCR System set to use 450 / 490 excitation and 560 / 580 emission filters. Samples were heated from 25 to 100°C at a rate of 0.3°C / sec. A single fluorescence measurement was taken every 0.03 sec. Melting points were determined from the peak of the first derivative of the melting curve using Bio-Rad CFX Manager software.

[0163] The esterase of SEQ ID NO: 1 and the esterase of the present invention were then compared based on their Tm values. A ΔTm of 0.8°C at pH 5.2 was considered significant for comparing variants within the same experimental set. Tm values ​​correspond to the average of at least three measurements.

[0164] result Thermostability compared to the esterase of SEQ ID NO:1 under acidic conditions The increase in Tm of the esterase variants used according to the invention compared to the esterase of SEQ ID NO:1, when evaluated according to Example 3.6 at pH 5.2, is shown in Table 8 below.

[0165] [Table 8]

[0166] The variants listed in Table 8 have the exact amino acid sequence set forth in SEQ ID NO:1, except for the substitution or combination of substitutions listed in Table 8, respectively.

[0167] Heat resistance under acidic and alkaline conditions compared to the esterase of SEQ ID NO:1 Table 9 below shows the increased thermostability of the variants used in the present invention compared to the reference esterase of SEQ ID NO: 1 at pH 5.2 and pH 8, evaluated according to Example 3.6.

[0168] [Table 9]

[0169] The variants listed in Table 9 each have the exact amino acid sequence set forth in SEQ ID NO:1, except for the combination of substitutions listed in Table 9.

[0170] Said variants exhibit increased thermostability under acidic conditions compared to basic conditions, as compared to SEQ ID NO:1.

Claims

1. 1. Use of an esterase to degrade a polyester or polyester-containing material, comprising contacting the polyester or polyester-containing material with an esterase at a pH of 3 to 6, the esterase being selected from the group consisting of: (i) an esterase having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the full-length amino acid sequence set forth in SEQ ID NO:1; and (ii) an esterase having any one of the following amino acids: F208M / W / G / N / R / I / A / Q / L / S / T / E, T11E / M, R12D / N / Q / E / F, S13E, A14E / D, T16E, A17T, T61S / V, A62D, F90A / Y, Y92G / D, W155A, T157S, 13. Use of an esterase comprising at least one substitution selected from P179D / E, Q182D / E, F187Y / I, D203C / K / R, N204D / E / G, A205D, S206D / E, N211D / Y / E, S212F, N213P / D, N214D, A215N, S218A, V219I, Y220M / F, Q237D, F238E, N241E / D, N243E / D, L247T, V170I, G135A, V167Q / T, S248C and A24R, said positions being numbered with reference to the amino acid sequence set forth in SEQ ID NO:1, and (iii) having polyesterolytic activity at a pH of at least 3 to 6.

2. 2. Use of the esterase according to claim 1, wherein the esterase has polyesterolytic activity at least at a pH of 4 to 6, preferably at a pH of 5 to 6, more preferably at a pH of 5 to 5.5, and even more preferably at pH 5.

2.

3. 3. Use of an esterase according to claim 1 or 2, wherein the esterase is contacted with the polyester or polyester-containing material at a pH of 4 to 6, preferably at a pH of 5 to 6, more preferably at a pH of 5 to 5.5, even more preferably at a pH of 5.

2.

4. The esterase is at least one selected from F208M / W / G / N / R / I / A / Q / L / S / T / E, T11E / M, R12D / N / Q / E / F, S13E, A14E / D, T16E, T61S / V, A62D, F90A / Y, Y92G / D, W155A, T157S, Q182D / E, D203C / K, N204E / G, S206D, N211D / Y / E, S212F, N213P, V219I, Y220M, Q237D, N241E / D, N243E, L247T, V170I, G135A, V167Q / T, S248C, A24R, and F187I.

4. Use of an esterase according to any one of claims 1 to 3, comprising one substitution, preferably at least one substitution selected from F208M / W / G / N / R / I / A / Q / L / S / T / E, T11M, R12D / E / F, A14E / D, T61S / V, A62D, F90A / Y, Y92G, W155A, T157S, Q182D / E, D203C / K, N204E / G, N211D / Y / E, S212F, N213P, V219I, Q237D, N241E / D, V170I, G135A, V167Q / T, S248C, A24R and F187I.

5. The esterase is selected from A14E / D, F90A / Y, Y92G / D, Q182D / E, N204D / E / G, F208M / W / G / N / R / I / A / Q / L / S / T / E, N211D / Y / E, N213P / D, V219I, V170I, D203C / K / R, S248C, A24R, and F187Y / I. and preferably at least one substitution selected from A14E, F90A, Y92G / D, Q182D / E, N204G, F208M / G / N / R / I / A / Q / L / S / T, N211D / E, N213P, V219I, V170I, D203C / K / R, S248C, A24R, and F187I.

5. Use of an esterase according to claim 1 , comprising at least one substitution, more preferably comprising at least one substitution selected from F90A, Y92G, Q182D / E, N204G, F208M / G / N / R / I / A / Q / L / S / T, N211D / E, V170I, D203C / K / R, S248C, N213P, A24R and F187I, even more preferably comprising at least one substitution selected from N211D / E, N204G, F208M / R / I / Q / L / S / T, Q182D / E, Y92G, V170I, D203C / K / R, S248C, N213P, A24R and F187I.

6. 6. Use of an esterase according to any one of claims 1 to 5, wherein the esterase comprises at least one substitution selected from F208M / W / G / N / R / I / A / Q / L / S / T / E, preferably comprises at least one substitution selected from F208M / G / N / R / I / A / Q / L / S / T, more preferably comprises at least one substitution selected from F208M / R / I / Q / L / S / T, even more preferably comprises at least one substitution selected from F208M / R / Q / L / S / T.

7. The esterase comprises at least a combination of substitutions selected from F208M / W / G / N / R / I / A / Q / L / S / T / E+D203C+S248C+V170I+Y92G / D+N213P / D+Q182D / E and F208M / W / G / N / R / I / A / Q / L / S / T / E+D203K+V170I+Y92G / D+N213P / D+Q182D / E, preferably F208M / I / L / T+D203C+S248C+V170I+Y92G+N213P+Q182E and F208M / I / L / T+D203K+V170I+Y92G / D+N213P / D+Q182D / E.

7. Use of an esterase according to claim 1 , comprising a combination of substitutions selected from the group consisting of: F208M / I / L / T+D203C+S248C+V170I+Y92G+N213P+Q182E; and F208M / I / L / T+D203K+V170I+Y92G+N213P+Q182E; and even more preferably comprising a combination of substitutions selected from the group consisting of: F208M+D203C+S248C+V170I+Y92G+N213P+Q182E; and F208M+D203K+V170I+Y92G+N213P+Q182E.

8. 8. Use of an esterase according to any one of claims 1 to 7, wherein the esterase further comprises at least one substitution selected from S13L and D158E, preferably at least two substitutions S13L and D158E.

9. The esterase is selected from at least F208M / I / L / T+D203C+S248C+V170I+Y92G+N213P+Q182E+S13L+D158E, F208M / I / L / T+D203C+S248C+V170I+Y92G+N213P+Q182E+S13L, or F208M / I / L / T+D203C+S248C+V170I+Y92G+N213P+Q182E+D158E.

9. Use of an esterase according to claim 8, comprising a combination of substitutions selected from the group consisting of F208M / I / L / T+D203C+S248C+V170I+Y92G+N213P+Q182E+S13L+D158E, preferably comprising a combination of substitutions selected from F208M / I / L / T+D203C+S248C+V170I+Y92G+N213P+Q182E+S13L+D158E, more preferably comprising the combination F208M+D203C+S248C+V170I+Y92G+N213P+Q182E+S13L+D158E.

10. 10. Use of an esterase according to any one of claims 1 to 9, wherein the esterase exhibits increased polyesterolytic activity and / or increased thermostability at a pH between 3 and 6, preferably between 4 and 6, more preferably between 5 and 6, even more preferably between 5 and 5.5, compared to the esterase of SEQ ID NO:

1.

11. 11. Use of an esterase according to any one of claims 1 to 10, wherein the esterase exhibits a greater increase in polyesterolytic activity and / or a greater increase in thermostability compared to the enzyme of SEQ ID NO: 1 at a pH of 3 to 6, in particular at a pH of 5 to 5.5, compared to a pH above 7, in particular at a pH of 7 to 9.

12. 12. Use of an esterase according to any one of claims 1 to 11, wherein the esterase comprises at least one substitution selected from N211D / E, N204G, F208M / R / Q / L / S / T, Q182D / E, A24R and F187I, preferably at least one substitution selected from N211D, F208M / Q / L / S / T, Q182D / , A24R and F187I, and exhibits a greater increase in polyesterolytic activity compared to the enzyme of SEQ ID NO: 1 at a pH of 3 to 6, in particular at a pH of 5 to 5.5, compared to a pH above 7, in particular at a pH of 7 to 9.

13. 13. Use of an esterase according to any one of claims 1 to 12, wherein the esterase comprises at least one combination of substitutions selected from F208M+D203C+S248C+V170I+Y92G+N213P+Q182E, F208I+D203C+S248C+V170I+Y92G+N213P+Q182E and F208M+D203C+S248C+V170I+Y92G+N213P+Q182E+S13L+D158E and shows a greater increase in polyesterolytic activity compared to the enzyme of SEQ ID NO: 1 at a pH of 3 to 6, in particular at a pH of 5 to 5.5, compared to a pH above 7, in particular at a pH of 7 to 9.

14. The esterase is F208I+D203C+S248C+V170I+Y92G+N213P+Q182E, F208M+D203C+S248C+V170I+Y92G+N213P+Q182E, F208T+D203C+S248C+V170I+Y92G+N213P+Q182E, F208L+D203C+S248C+V170I+Y92G+N213P+Q182E, F208M+D203C+S248C+V170I+Y92G+N213P+Q182E+S13L+D158E 14. Use of an esterase according to any one of claims 1 to 13, comprising at least one combination of substitutions selected from: F208M+D203C+S248C+V170I+Y92G+N213P+Q182E+S13L and F208M+D203C+S248C+V170I+Y92G+N213P+Q182E+D158E and exhibiting a greater increase in thermostability compared to the enzyme of SEQ ID NO: 1 at a pH of 3 to 6, in particular at a pH of 5 to 5.5, compared to a pH above 7, in particular at a pH of 7 to 9.

15. 15. Use of an esterase according to any one of claims 1 to 14, wherein the esterase comprises at least one combination of substitutions selected from F208M+D203C+S248C+V170I+Y92G+N213P+Q182E, F208I+D203C+S248C+V170I+Y92G+N213P+Q182E and F208M+D203C+S248C+V170I+Y92G+N213P+Q182E+S13L+D158E and exhibits both a greater increase in thermostability and a greater increase in polyesterolytic activity compared to the enzyme of SEQ ID NO: 1 at a pH of 3 to 6, in particular at a pH of 5 to 5.5, compared to a pH above 7, in particular at a pH of 7 to 9.

16. 16. Use of an esterase according to any one of claims 1 to 15, wherein the polyester is selected from polyethylene terephthalate (PET), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polybutylene adipate terephthalate (PBAT), polycyclohexylene dimethylene terephthalate (PCT), glycol modified polyethylene terephthalate (PETG), poly(butylene succinate-co-terephthalate) (PBST), poly(butylene succinate / terephthalate / isophthalate)-co-(lactate) (PBSTIL), "polyolefin-like" polyesters, and blends / mixtures of these polymers.

17. 17. Use of an esterase according to claim 16, wherein the polyester comprises at least a terephthalic acid monomer (TA), preferably the polyester is polyethylene terephthalate (PET).

18. 18. Use of an esterase according to any one of claims 1 to 17, wherein the esterase is used in a detergent.

19. 18. Use of an esterase according to any one of claims 1 to 17, wherein the esterase is used on a polyester-containing material.

20. A process for degrading polyesters or polyester-containing materials, the process comprising a step of depolymerizing said polyesters, carried out by contacting a plastic product or said polyester, in a reaction medium having a pH between 3 and 6, with an esterase capable of degrading said at least one polyester, said esterase having (i) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the full-length amino acid sequence set forth in SEQ ID NO:1, and (ii) an amino acid sequence Compared to SEQ ID NO: 1, F208M / W / G / N / R / I / A / Q / L / S / T / E, T11E / M, R12D / N / Q / E / F, S13E, A14E / D, T16E, A17T, T61S / V, A62D, F90A / Y, Y9 2G / D, W155A, T157S, P179D / E, Q182D / E, F187Y / I, D203C / K / R, N204D / E / G, A205D, S206D / E, N211D / Y / E, S212F, N213P / D, N214 and (iii) having polyesterolytic activity at a pH between 3 and 6, preferably between 5 and 5.

5.

21. The esterase is N211D, N204G, F208T / M / L / S / Q / R, Q182D / E, A24R, F187I, F208M / I / T / L+D203C+S248C+V170I+Y92G+N213P+Q182E, F208M+D203C+S248C+V170I+Y92G+N213P+Q182E+S13 21. The process of claim 20, comprising at least one substitution and / or combination of substitutions selected from: F208M+D203C+S248C+V170I+Y92G+N213P+Q182E+S13L, F208M+D203C+S248C+V170I+Y92G+N213P+Q182E+D158E, F208M+D203C+S248C+V170I+Y92G+N213P+Q182E+S13L, F208M+D203C+S248C+V170I+Y92G+N213P+Q182E+D158E.

22. 22. The process according to claim 21, wherein the depolymerization step is carried out at a pH between 4 and 6, preferably between 5 and 6, more preferably between 5.0 and 5.5, even more preferably at pH 5.

2.

23. 23. The process according to claim 20 or 22, wherein the depolymerization step is carried out at a temperature of from 50°C to 72°C, in particular from 50°C to 65°C or from 65°C to 72°C.

24. 24. The process of any one of claims 20 to 23, wherein the polyester is selected from polyethylene terephthalate (PET), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polybutylene adipate terephthalate (PBAT), polycyclohexylene dimethylene terephthalate (PCT), glycol modified polyethylene terephthalate (PETG), poly(butylene succinate-co-terephthalate) (PBST), poly(butylene succinate / terephthalate / isophthalate)-co-(lactate) (PBSTIL), "polyolefin-like" polyesters, and blends / mixtures of these polymers.

25. 25. The process according to any one of claims 20 to 24, wherein the polyester comprises at least terephthalic acid monomer (TA), preferably the polyester is polyethylene terephthalate (PET).

26. 26. A process according to any one of claims 20 to 25, for recycling polyesters, or polyesters of a polyester-containing material, comprising subjecting the polyesters to a depolymerisation step carried out at a pH between 3 and 6, and recovering and optionally purifying the monomers and / or oligomers.

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