Novel esterases and uses thereof

Abstract

The present invention relates to novel esterases, more specifically to esterase variants having improved thermostability compared to the esterase of SEQ ID N°1 and their use for the degradation of polyester-containing materials such as plastic articles. The esterases of the present invention are particularly suitable for the degradation of polyethylene terephthalate and materials containing polyethylene terephthalate.

Description

[0001] This application is a divisional application of the invention patent application filed on July 12, 2017, with application number 201780047657.5 and invention title "New Esterase and Its Use". Technical Field

[0002] This invention relates to novel esterases, and more particularly to esterases with improved thermal stability compared to parental esterases, and their use in degrading polyester-containing materials such as plastic articles. The esterases of this invention are particularly suitable for degrading polyethylene terephthalate (PET) and materials containing PET. Background Technology

[0003] Esterases can catalyze the hydrolysis of various polymers, including polyesters. In this context, esterases have shown promising results in numerous industrial applications, including as detergents in dishwashing and laundry applications, as degrading enzymes for treating biomass and food, as biocatalysts in the detoxification of environmental pollutants, and for treating polyester fabrics in the textile industry. Similarly, the use of esterases as degrading enzymes to hydrolyze polyethylene terephthalate (PET) is of particular interest. Indeed, PET is used in numerous technological fields, such as in the manufacture of clothing, carpets, or as a thermosetting resin in the manufacture of packaging materials, automotive plastic products, or other components, and the accumulation of PET in landfills has become a growing ecological problem.

[0004] Among esterases, keratinase, also known as keratin hydrolase (EC 3.1.1.74), is of particular interest. Keratinase has been identified from various fungi (PE Kolattukudy in "Lipases", Ed. B. Borg-stróm and HLBrockman, Elsevier 1984, 471-504), bacteria, and plant pollen. Recently, metagenomic methods have identified other esterases.

[0005] Enzymatic degradation is considered an interesting solution for reducing the accumulation of this plastic waste. In fact, enzymes can accelerate the hydrolysis of polyester-containing materials, especially plastic products, even down to the monomer level. Furthermore, the hydrolysis products (i.e., monomers and oligomers) can be recycled as materials for synthesizing new polymers.

[0006] In this context, several esterases have been identified as candidate degradative enzymes. For example, several esterase (keratinase) variants of Fusarium solani pisi have been published (Appl. Environm. Microbiol. 64, 2794-2799, 1998; Proteins: Structure, Function and Genetics 26, 442-458, 1996).

[0007] However, most of these esterases are inefficient at the industrial level because they are poorly tolerant of high temperatures. Therefore, there remains a need for esterases with improved thermal stability that can be used to degrade polyesters in high yields at the industrial level. Invention Overview

[0008] This invention provides novel esterase variants that exhibit increased thermal stability compared to parental or wild-type esterases. These esterases are particularly useful in the degradation of plastic materials and products, such as PET-containing plastic materials and products. More specifically, this invention provides variants of esterases having the amino acid sequence shown in SEQ ID NO1, which correspond to amino acids 36-293 of the metagenomically derived keratinase described by Sulaiman et al., Appl Environ Microbiol. 2012 Mar, or amino acids 36-293 of the amino acid sequence referenced in SwissProt G9BY57.

[0009] In this respect, one object of the present invention is to provide an esterase that (i) has at least 75%, 80%, 85%, 90%, 95%, or 99% identity with the full-length amino acid sequence shown in SEQ ID N°1, (ii) contains at least one amino acid modification compared to SEQ ID N°1, (iii) has polyester degradation activity, and (iv) exhibits increased thermal stability compared to the esterase of SEQ ID N°1.

[0010] More specifically, compared to SEQ ID N°1, the esterase of the present invention comprises one or more amino acid modifications at positions selected from the following: D203+S248, E173, L202, N204, F208, A172+A209, G39, A103, L82, G53, L104, L107, L119, A121, L124, I54, M56, L70, L74, A127, V150, L152, L168, V170, P196, V198, V200, V219, Y220, T221, S223, W224, M225, L239, T 252, N253, H256, S1, Y4, Q5, R6, N9, S13, T16, S22, T25, Y26, S34, Y43, S48, T50, R72, S98, N105, R108, S113, N122, S145, K147, T160, N162, S181, Q189, N190, S193, T194, N204, S212, N213, N231, T233, R236, Q237, N241, N243, N254, R255, and Q258, wherein the positions are referenced to the amino acid sequence number shown in SEQ ID N°1.

[0011] In one specific embodiment, compared to SEQ ID N°1, the variant esterase of the present invention contains one or more amino acid substitutions at positions selected from D203+S248, E173, N204, L202, F208, and V170. Preferably, compared to SEQ ID N°1, the variant esterase of the present invention contains at least one amino acid substitution at positions selected from D203+S248 and F208.

[0012] In another specific embodiment, compared to SEQ ID N°1, the variant esterase of the present invention comprises one or more amino acid substitutions at positions selected from T61, Y92, and V177, wherein said positions refer to the amino acid sequence numbers shown in SEQ ID N°1, and said substitutions are different from T61A / G, Y92A, and V177A. Preferably, compared to SEQ ID N°1, the variant esterase of the present invention comprises one or more amino acid substitutions selected from V177I, Y92G, Y92P, Y92P+F208W, and T61M.

[0013] In one specific embodiment, the variant esterase of the present invention may comprise, compared to the esterase of SEQ ID N°1:

[0014] - At least one additional disulfide bridge; and / or

[0015] - At least one additional salt bridge; and / or

[0016] - At least one mutation of an amino acid residue located in the solvent-excluded cavity of the esterase; and / or

[0017] - Inhibition of at least one N- and / or C-terminal amino acid residue.

[0018] Another object of the present invention is to provide a nucleic acid encoding the esterase of the present invention. The present invention also relates to an expression cassette or expression vector comprising said nucleic acid, and a host cell comprising said nucleic acid, expression cassette, or vector.

[0019] A further object of the present invention is to provide a method for producing esterases, comprising:

[0020] (a) Culturing host cells according to the invention under suitable conditions to express nucleic acids encoding esterases; and optionally...

[0021] (b) The esterase is recovered from the cell culture.

[0022] The present invention also relates to a method for degrading plastic articles comprising at least one polyester, comprising:

[0023] (a) Contacting the plastic article with the esterase or host cell according to the invention, thereby degrading the plastic article; and optionally...

[0024] (b) Recover monomers and / or oligomers. Invention Details

[0026] definition

[0027] This disclosure will be best understood by referring to the following definitions.

[0028] In this document, 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 the chain. Amino acids are represented by their single-letter or three-letter codes according to the following nomenclature: A: Alanine (Ala); C: Cysteine ​​(Cys); D: Aspartic acid (Asp); E: Glutamic acid (Glu); F: Phenylalanine (Phe); G: Glycine (Gly); H: Histidine (His); I: Isoleucine (Ile); K: Lysine (Lys); L: Leucine (Leu); M: Methionine (Met); N: Asparagine (Asn); P: Proline (Pro); Q: Glutamine (Gln); R: Arginine (arginine); S: Serine (Ser); T: Threonine (Thr); V: Valine (Val); W: Tryptophan (Trp); and Y: Tyrosine (Tyr).

[0029] The term "esterase" refers to an enzyme classified under EC 3.1.1 according to enzyme nomenclature, which catalyzes the hydrolysis of esters into acids and alcohols. The term "keratinase" or "keratin hydrolase" refers to an esterase classified under EC 3.1.1.74 according to enzyme nomenclature, which catalyzes the chemical reaction that produces keratin monomers from keratin and water.

[0030] The terms "wild-type protein" and "parental protein" are used interchangeably and refer to the non-mutated form of a naturally occurring polypeptide. In this case, parental esterase refers to an esterase having the amino acid sequence shown in SEQ ID N°1.

[0031] Therefore, the terms "mutant" and "variant" are used interchangeably, referring to a polypeptide derived from SEQ ID N°1 that contains modifications or alterations, i.e., substitutions, insertions, and / or deletions, at one or more (e.g., several) positions and has polyester degradation activity. Variants can be obtained using various techniques well known in the art. In particular, examples of techniques for altering the DNA sequence encoding wild-type proteins include, but are not limited to, site-directed mutagenesis, random mutagenesis, and synthetic oligonucleotide construction.

[0032] As used in this article, the terms “modification” or “alteration” related to position or amino acids refer to the modification of amino acids at a specific position compared to the wild-type protein.

[0033] "Substitution" refers to the replacement of one amino acid residue with another amino acid residue. Preferably, the term "substitution" refers to the replacement of one amino acid residue with another amino acid residue selected from the 20 naturally occurring standard amino acid residues, naturally occurring rare amino acid residues (e.g., hydroxyproline, hydroxylysine, allohydroxylysine, 6-N-methyllysine, N-ethylglycine, N-methylglycine, N-ethylasparagine, alloleucine, N-methylisoleucine, N-methylvaline, pyroglutamine, GABA, ornithine, leucine, valine), and normally synthetic non-natural amino acid residues (e.g., cyclohexyl-alanine). Preferably, the term "substitution" refers to the replacement of one amino acid residue with another selected from the 20 naturally occurring standard amino acid residues (G, P, A, V, L, I, M, C, F, Y, W, H, K, R, Q, N, E, D, S, and T). The symbol "+" indicates a combination of substitutions. In this document, the following terms are used to indicate substitution: L82A indicates that the 82nd amino acid residue (leucine, L) of the parental sequence is replaced by alanine (A). A121V / I / M indicates that the 121st amino acid residue (alanine, A) of the parental sequence is substituted by one of the following amino acids: valine (V), isoleucine (I), or methionine (M). Substitution can be conserved or non-conserved. Examples of conserved substitution are those occurring within the following group: 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).

[0034] The term "deficiency" in the context of amino acids refers to the removal or absence of an amino acid.

[0035] The term "insertion" refers to the addition of one or more amino acids.

[0036] Unless otherwise stated, the location disclosed in this application refers to the amino acid sequence number shown in SEQ ID N°1.

[0037] As used herein, the term “sequence identity” or “identity” refers to the number (or a fraction expressed as a percentage %) of matches (identical amino acid residues) between two polypeptide sequences. Sequence identity is determined by comparing sequences during alignment to maximize overlap and identity while minimizing sequence gaps. Specifically, sequence identity can be determined based on the lengths of the two sequences using any of a number of mathematical global or local alignment algorithms. Global alignment algorithms that optimally align sequences over their full length (e.g., the Needleman and Wunsch algorithm; Needleman and Wunsch, 1970) are preferred for aligning sequences of similar lengths, while local alignment algorithms (e.g., the Smith and Waterman algorithm (Smith and Waterman, 1981) or the Altschul algorithm (Altschul et al., 1997; Altschul et al., 2005)) are preferred for aligning sequences of substantially different lengths. Alignment used to determine the percentage of amino acid sequence identity can be achieved in various ways within the scope of the art, for example, using publicly available computer software available on internet websites such as http: / / blast.ncbi.nlm.nih.gov / or http: / / www.ebi.ac.uk / Tools / emboss / . Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment across the full length of the sequences being compared. For the purposes of this document, the % amino acid sequence identity value refers to the value generated using the pairwise sequence alignment procedure EMBOSS Needle, which uses the Needleman-Wunsch algorithm to produce the optimal global alignment of two sequences, where all retrieval parameters are set to default values: score matrix = BLOSUM62, vacancy open = 10, vacancy extension = 0.5, terminal vacancy penalty = error, terminal vacancy open = 10, and terminal vacancy extension = 0.5.

[0038] The terms "disulfide bridge," "disulfide bond," and "SS bond" are used interchangeably and refer to a covalent bond between two sulfur atoms of cysteine.

[0039] The term "salt bridge" or "ion pair" refers to a non-covalent electrostatic interaction between two residues with opposite charges in a protein. Salt bridges are typically formed between the anionic carboxylates of aspartic acid or glutamate (RCOO). - ) and lysine cationic ammonium (RNH3) + ) or guanidine salt of arginine (RNHC(NH2)2 + It forms between the parts. Other amino acid residues with ionizable side chains, such as histidine, tyrosine, serine, threonine, and cysteine, can also be part of the salt bridge.

[0040] Regarding peptides, the term "glycosylation" refers to the linkage of one or more glycans to at least one amino acid residue of the peptide. In the context of this invention, glycosylation includes N-linked glycans linked to the amide nitrogen of asparagine residues, O-linked glycans linked to the hydroxyl oxygen of serine or tyrosine residues, and C-linked glycans linked to the carbon of tryptophan residues.

[0041] "Protein conformation" or "crystal structure" refers to the three-dimensional structure of a protein.

[0042] The term "recombination" refers to nucleic acid constructs, vectors, polypeptides, or cells produced through genetic engineering.

[0043] As used herein, the term “expression” refers to any step involved in the production of a polypeptide, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0044] The term "expression cassette" refers to a nucleic acid construct that contains a coding region, namely the nucleic acid of the present invention, and a regulatory region, namely containing one or more operatively linked control sequences.

[0045] As used herein, the term "expression vector" refers to a DNA or RNA molecule containing the expression cassette of the present invention. Preferably, the expression vector is a linear or circular double-stranded DNA molecule.

[0046] "Polymer" refers to a compound or mixture of compounds whose structure consists of a plurality of monomers (repeating units) linked by covalent chemical bonds. In the context of this invention, the term polymer includes natural or synthetic polymers composed of a single type of repeating unit (i.e., homopolymer) or a mixture of different repeating units (i.e., copolymer or hybrid). According to this invention, "oligomer" refers to a molecule containing 2 to about 20 monomers.

[0047] In the context of this invention, "polyester-containing material" or "polyester-containing product" refers to a product comprising at least one crystalline, semi-crystalline, or completely amorphous form of polyester, such as plastic articles. In one embodiment, polyester-containing material refers to any article made of at least one plastic material, such as plastic sheets, tubes, rods, profiles, shapes, films, blocks, etc., comprising at least one polyester and possible other substances or additives, such as plasticizers, mineral or organic fillers. In another embodiment, polyester-containing material refers to a plastic compound or plastic formulation in a molten or solid state suitable for manufacturing plastic articles.

[0048] In this specification, "polyester" includes, but is not limited to, polyethylene terephthalate (PET), polypropylene terephthalate (PTT), polybutylene terephthalate (PBT), polyisosorbate terephthalate (PEIT), polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene adipate succinate (PBSA), polybutylene adipate terephthalate (PBAT), polyfuran acetate (PEF), polycaprolactone (PCL), polyethylene adipate (PEA), polyethylene naphthalate (PEN), and blends / mixtures of these polymers.

[0049] New esterases with improved thermal stability

[0050] This invention provides novel esterases with improved thermal stability. More specifically, the inventors have developed various methods to improve the stability of esterases at high temperatures, and advantageously at temperatures above 50°C, which allows for the design of novel enzymes with excellent properties for industrial applications.

[0051] To improve the stability and / or activity of esterases under conditions suitable for the industrial degradation of plastic products, the inventors have developed novel esterases derived from SEQ ID NO. 1, which exhibit high temperature resistance. The esterases of this invention are particularly suitable for degrading PET-containing plastic products.

[0052] This invention demonstrates that novel proteins with improved thermal stability and polyester degradation activity are obtained by generating new disulfide bridges and / or salt bridges in the crystal structure of proteins; by reducing protein fluidity and / or the volume of solvent removal from the lumen; and / or by reducing the N-terminus or C-terminus.

[0053] Therefore, one object of the present invention is to provide an esterase that (i) has at least 75%, 80%, 85%, 90%, 95%, or 99% identity with the full-length amino acid sequence shown in SEQ ID N°1, (ii) contains at least one amino acid modification compared to SEQ ID N°1, (iii) has polyester degradation activity, and (iv) exhibits increased thermal stability compared to the esterase of SEQ ID N°1.

[0054] In the context of this invention, the term "increased thermal stability" refers to an increased ability of an enzyme to resist changes in its chemical and / or physical structure at high temperatures, particularly at temperatures between 50°C and 90°C, compared to the esterase of SEQ ID N°1. This increase is typically about 1, 2, 3, 4, 5, or more times. In particular, the esterases of this invention can exhibit an increased melting temperature (Tm) compared to the esterase of SEQ ID N°1. In the context of this invention, melting temperature refers to the temperature at which half of the protein / enzyme population under consideration unfolds or misfolds. Typically, the esterases of this invention show an increase in Tm of about 1°C, 2°C, 3°C, 4°C, 5°C, 10°C, or higher compared to the Tm of the esterase of SEQ ID N°1.

[0055] In particular, compared with the esterase of SEQ ID N°1, the esterase of the present invention has an increased half-life at temperatures of 50°C-90°C. Furthermore, at such temperatures, the esterase of the present invention exhibits higher degradation activity compared with the esterase of SEQ ID N°1.

[0056] Those skilled in the art can assess the thermal stability of proteins using methods known in the art itself. For example, thermal stability can be assessed by using circular dichroism analysis to analyze protein folding. Alternatively, thermal stability can be assessed by measuring the residual esterase activity and / or residual polyester depolymerization activity of the enzyme after incubation at different temperatures. The ability to perform multiple rounds of polyester depolymerization assays at different temperatures can also be assessed. Rapid and valuable tests may include assessing the ability of an enzyme to degrade solid polyester compounds dispersed in an agar plate after incubation at different temperatures by measuring the halo diameter. Preferably, differential scanning fluorometry (DSF) is performed to assess the thermal stability of the protein / enzyme. More specifically, DSF can be used to quantify changes in the thermal denaturation temperature of a protein, thereby determining its melting temperature (Tm). In the context of this invention, unless specifically indicated, Tm is measured using the DSF described in the experimental section. In the context of this invention, Tm is compared using Tm measured under the same conditions (e.g., pH, nature and amount of polyester, etc.).

[0057] In one specific embodiment, the variant of the present invention has improved thermal stability and increased polyester degradation activity compared to the esterase of SEQ ID N°1.

[0058] In the context of this invention, the terms "increased activity" or "increased degradation activity" refer to an increased ability of the enzyme to degrade plastic articles or materials (more particularly polyester-containing plastic articles or materials) compared to the esterase of SEQ ID N°1. This increase is typically about 1, 2, 3, 4, 5, or more times. In particular, the polyester degradation activity of the esterase variant is at least 10%, preferably at least 20%, 50%, 100%, 200%, 300%, or higher than that of the esterase of SEQ ID N°1.

[0059] Protein activity can be assessed by methods known in the art itself. For example, activity can be assessed by measuring the activity rate of a specific esterase, the depolymerization activity rate of a specific polyester, the rate of degradation of a solid polyester compound dispersed in an agar plate, or the depolymerization activity rate of a specific polyester in a reactor.

[0060] In the context of this invention, the term "specific activity" or "degradation specific activity" refers to the initial rate of release of oligomers and / or monomers when a polyester-containing plastic article is contacted with a degrading enzyme, such as the esterase according to the invention, under suitable temperature, pH, and buffer conditions. For example, the specific activity of PET hydrolysis corresponds to μmol / min of hydrolyzed PET or mg / h of equivalent TA produced per mg of enzyme, as determined in the linear portion of the hydrolysis curve.

[0061] Those skilled in the art can assess the ability of proteins to adsorb onto a matrix using methods known in the art itself. For example, protein content or residual esterase activity, residual depolymerization activity of polyesters, residual degradation of solid polyester compounds dispersed in an agar plate, or depolymerization activity of residual polyesters in a reactor can be measured from a solution containing the esterase of the present invention, wherein the esterase is pre-incubated with the substrate under suitable conditions in which no enzymatic reaction occurs.

[0062] The esterases of the present invention may include one or more of the modifications disclosed below.

[0063] In one embodiment, the esterase of the present invention has at least 75%, 80%, 85%, 90%, 95%, or 99% identity with the full-length amino acid sequence shown in SEQ ID N°1, and contains at least one additional disulfide bridge compared to the esterase of SEQ ID N°1.

[0064] In one specific embodiment, the esterase variant contains a substitution at position A172+A209, wherein the position refers to the amino acid sequence number shown in SEQ ID N°1.

[0065] In another specific embodiment, the esterase variant contains at least one mutation at a position selected from V28 to G39, L82 and A103, wherein the position refers to the amino acid sequence number shown in SEQ ID NO1.

[0066] In particular, the esterase variants show deletions at amino acid residues V28 to S34 of SEQ ID N°1, and substitutions at positions selected from G35, F36, G37, G38, G39, L82 and / or A103 compared to SEQ ID N°1, more particularly substitutions consisting of G35E / A+F36G+G37P+G38S+G39C, L82A and / or A103C.

[0067] Alternatively, the esterase variant shows the deletion of amino acids V33 to G39 in SEQ ID N°1, and the substitution of L82A and / or A103C consisting of V28E+S29G+R30P+L31S+S32C or V28A+S29G+R30P+L31S+S32C compared to SEQ ID N°1.

[0068] Alternatively, the esterase variant comprises replacing amino acids V28 to G39 of SEQ IDN°1 with an amino acid sequence consisting of EGPSC or AGPSC, and finally replacing L82A and / or A103C.

[0069] Alternatively, the esterase variants show the deletion of amino acids V33 to G39 in SEQ ID N°1, and the substitutions consisting of V28E+S29G+R30P+L31S+S32C or V28A+S29G+R30P+L31S+S32C, and the substitutions of L82A, A103C, A172C, and A209C.

[0070] In another specific embodiment, the esterase variant contains a substitution at position D203+S248, wherein the position refers to the amino acid sequence number shown in SEQ ID NO1. Preferably, the substitution consists of D203C+S248C. In one specific embodiment, such an esterase variant having a substitution at position D203+S248 further contains at least one substitution at a position selected from E173, L202, N204, and F208. Preferably, the additional substitution is selected from E173R, E173A, F208W, or F208I. More specifically, the esterase variant contains a substitution selected from D203C+S248C+E173R, D203C+S248C+E173A, D203C+S248C+F208W, and D203C+S248C+F208I. In one specific embodiment, the esterase variant having a substitution at position D203+S248 further comprises at least two substitutions at positions selected from E173, L202, N204, and F208. For example, the variant comprises at least the substitutions D203C+S248C+E173R+N204D+L202R, F208W+D203C+S248C+E173A, and F208I+D203C+S248C+E173A.

[0071] One object of the present invention is to provide an esterase having polyester degradation activity, which has at least 75%, 80%, 85%, 90%, 95%, or 99% identity with the full-length amino acid sequence shown in SEQ ID N°1, and, compared with SEQ ID N°1, includes at least one mutation of an amino acid residue located in the solvent-removing cavity of the protein. In particular, such mutation is capable of reducing the solvent removal volume of the cavity.

[0072] In one specific embodiment, referring to SEQ ID N°1, the esterase variant contains at least one substitution at a position selected from G53, L104, L107, L119, A121, L124, I54, M56, L70, L74, A127, V150, L152, L168, V170, P196, V198, V200, V219, Y220, T221, S223, W224, M225, L239, T252, N253, and H256. Advantageously, the esterase variant contains at least two, three, four, five, or more amino acid substitutions at said positions.

[0073] In one embodiment, at least one of the amino acids is replaced by a larger (i.e., a larger volume) amino acid.

[0074] Advantageously, the esterase variant comprises at least one substitution selected from G53A / I, I54L, M56I, L70M / I, L74M, L104M, L107M, L119M / A, A121V / I / M / Y, L124I / R / Q, A127V / I, V150I, L152I, L168I, V170I, V198I, V219I, Y220F / P / M, T221A / V / L / I / M, S223A, W224I / M, and T252S / D.

[0075] In one specific embodiment, the esterase variant contains a substitution at the V170+F208 position. Advantageously, the esterase variant contains the substituted V170I+F208W.

[0076] In one specific embodiment, the esterase variant contains amino acid substitutions at at least two positions belonging to the following groups (i)-(v). Advantageously, the esterase variant contains substitutions at at least one position in each group (i)-(v).

[0077] (i)G53, L104, L107, L119, A121, L124;

[0078] (ii) I54, M56, L70, L74, A127, V150, L152, T221, M225;

[0079] (iii)V150, L152, L168, V170, V200, T221;

[0080] (iv)P196, V198, W224, T252, N253, H256;

[0081] (v)V219, Y220, S223, L239.

[0082] One object of the present invention is to provide an esterase having polyester degradation activity, having at least 75%, 80%, 85%, 90%, 95%, or 99% identity with the full-length amino acid sequence shown in SEQ ID N°1, and containing at least one additional salt bridge compared to SEQ ID N°1. Preferably, the esterase contains at least one additional surface salt bridge located on the outer surface of the protein structure.

[0083] For this purpose, the esterase variant advantageously contains at least one amino acid substitution at a position selected from S1, Y4, Q5, R6, N9, S13, T16, S22, T25, Y26, S34, Y43, S48, T50, R72, S98, N105, R108, S113, N122, S145, K147, T160, N162, E173, S181, Q189, N190, S193, T194, D203, N204, S212, N213, N231, T233, R236, Q237, N241, N243, N254, R255, and Q258.

[0084] Advantageously, the esterase variant has at least one salt bridge between the two amino acids at the stated position.

[0085] Typically, salt bridges are formed through the interaction between the anionic charge of aspartic acid (D) or glutamic acid (E) and the cationic charge of lysine (K) or arginine (R). Therefore, depending on the nature of the target amino acid pair under consideration, the salt bridges in the esterases of the present invention are advantageously obtained by using D or E and / or K or R to represent at least one amino acid of at least one target amino acid pair listed in 1.

[0086] Table 1: Combinations of amino acid positions (first and second) targeting to form salt bridges

[0087]

[0088]

[0089] Advantageously, when the amino acid residue of the targeted amino acid pair is R or K, only the second amino acid of the targeted pair is substituted with D or E. For example, the esterase variant of the present invention may contain the amino acid substitution Y4D and thus can display a salt bridge between the mutated amino acid residue and R6. Similarly, when the amino acid residue of the targeted amino acid pair is D or E, only the second amino acid of the targeted pair is substituted with R or K. For example, the esterase variant of the present invention may contain the amino acid substitution N204R and thus can display a salt bridge between the mutated amino acid residue and E173.

[0090] Another object of the present invention is to provide an esterase having polyester degradation activity, having at least 75%, 80%, 85%, 90%, 95% or 99% identity with the full-length amino acid sequence shown in SEQ ID N°1, and having, compared with SEQ ID N°1, contained at least one N- and / or C-terminal amino acid residue inhibition, and preferably at least one N-terminal amino acid residue inhibition.

[0091] In one specific embodiment, compared to SEQ ID N°1, the variant esterase of the present invention contains one or more amino acid substitutions at positions selected from T61, Y92, and V177, wherein said positions refer to the amino acid sequence numbers shown in SEQ ID N°1, and said substitutions are different from T61A / G, Y92A, and V177A. Preferably, compared to SEQ ID N°1, the variant esterase of the present invention contains one or more amino acid substitutions selected from V177I, Y92G / P, and T61M.

[0092] In another specific embodiment, compared to SEQ ID N°1, the variant esterase of the present invention contains at least one substitution at position F208. According to the present invention, F208 can be substituted by any of the 19 other amino acids. Preferably, the substitution is F208W.

[0093] In another specific embodiment, compared with SEQ ID N°1, the variant esterase of the present invention contains at least two substitutions at positions selected from T61, Y92, V177 and F208, preferably at positions F208 and Y92.

[0094] In one specific implementation, the variant includes at least a replacement for Y92P+F208W.

[0095] In another specific implementation, the variant includes at least a replacement for V170I+F208W.

[0096] According to the present invention, compared with the enzyme of SEQ ID NO1, the esterase variant can be further glycosylated to further increase the thermostability of the enzyme.

[0097] In one specific embodiment, the esterase variant includes a glycosylated moiety on at least one asparagine residue of the enzyme, said asparagine residue preferably located at positions selected from N9, N143, N162, N204, and N231 of reference SEQ ID N°1, more preferably at positions selected from N9, N162, and N231. In one embodiment, the esterase variant includes glycosylated moieties on N9, N162, and N231.

[0098] For example, the N-linked polysaccharide moiety is nitrogen-linked to at least one of the asparagine residues.

[0099] Alternatively, the esterase variants of the present invention may further comprise one or more proline residues inserted and / or one or more glycine residues deleted.

[0100] In one specific embodiment, the esterase variant of the present invention comprises one or more modifications and / or mutations as listed above.

[0101] New esterases with improved thermal stability and activity

[0102] Another object of the present invention is to provide a novel esterase that exhibits increased thermal stability and increased polyester degradation activity compared to the esterase of SEQ ID NO1.

[0103] Therefore, another object of the present invention is to provide an esterase that (i) has at least 75%, 80%, 85%, 90%, 95% or 99% identity with the full-length amino acid sequence shown in SEQ ID N°1, (ii) contains at least one amino acid modification compared with SEQ ID N°1, and (iii) exhibits increased thermal stability and increased activity compared with the esterase of SEQ ID N°1.

[0104] In one specific embodiment, referring to SEQ ID N°1, the esterase variant comprises at least one mutation as disclosed above and at least one additional substitution selected from F208I or F208W.

[0105] In another specific embodiment, the variant comprises at least one substitution selected from T61M, Y92G / P, F208W, Y92P+F208W and F208W+V170I, and exhibits increased thermostability and increased activity compared to the esterase of SEQ ID N°1.

[0106] In one specific embodiment, the variant comprises at least a substitution selected from F208W+D203C+S248C and F208I+D203C+S248C, and exhibits increased thermal stability and increased activity compared to the esterase of SEQ ID N°1.

[0107] Polyester degradation activity

[0108] One object of the present invention is to provide novel enzymes with esterase activity. In one specific embodiment, the enzyme of the present invention further exhibits keratinase activity.

[0109] In one specific embodiment, the esterase of the present invention has polyester degradation activity, preferably polyethylene terephthalate (PET) degradation activity.

[0110] In another specific embodiment, the esterase of the present invention also has PBAT degradation activity.

[0111] Advantageously, the esterase variants of the present invention exhibit polyester degradation activity at least in the temperature range of 20°C-90°C, preferably 40°C-80°C, more preferably 50°C-70°C, even more preferably 60°C-70°C, and even more preferably 65°C. In one specific embodiment, the esterase variants of the present invention exhibit polyester degradation activity at 70°C. In another specific embodiment, the polyester degradation activity can still be measured at a temperature of 60°C-90°C.

[0112] In one specific embodiment, compared with the esterase of SEQ ID N°1, the esterase variant of the present invention has an increased half-life at a given temperature, more particularly, at 40°C-80°C, more preferably at 50°C-70°C, even more preferably at 60°C-70°C, and even more preferably at 65°C. In one specific embodiment, the half-life of the esterase variant at 65°C is at least 5%, preferably at least 10%, 20%, 50%, 100%, 200%, 300%, or more longer than the half-life of the esterase of SEQ ID N°1.

[0113] In another specific embodiment, the esterase variant of the present invention has an increased melting temperature (Tm) compared to the esterase of SEQ ID N°1. Advantageously, the esterase variant of the present invention has a melting temperature (Tm) increased by about 1°C, 2°C, 3°C, 4°C, 5°C, 10°C or higher compared to the esterase of SEQ ID N°1.

[0114] In one specific embodiment, the esterase variant of the present invention exhibits measurable esterase activity at least in a pH range of 5-11, preferably in a pH range of 6-9, more preferably in a pH range of 6.5-9, and even more preferably in a pH range of 6.5-8.

[0115] Nucleic acid, expression cassette, vector

[0116] Another object of the present invention is to provide a nucleic acid encoding an esterase as defined above.

[0117] As used herein, the terms “nucleic acid,” “nucleic acid sequence,” “polynucleotide,” “oligonucleotide,” and “nucleotide sequence” are used interchangeably and refer to a sequence of deoxyribonucleotides and / or ribonucleotides. Nucleic acid can be DNA (cDNA or gDNA), RNA, or a mixture of both. It can be in single-stranded or double-stranded form, or a mixture of both. It can be of recombinant, artificial, and / or synthetic origin, and it can contain modified nucleotides, including, for example, modified bonds, modified purine or pyrimidine bases, or modified sugars. The nucleic acids of the present invention can be in isolated or purified form and can be prepared, isolated, and / or manipulated by techniques known in the art itself, such as cloning and expression of cDNA libraries, amplification, enzymatic synthesis, or recombinant techniques. Nucleic acids can also be synthesized in vitro using well-known chemical synthesis techniques as described in Belousov (1997) Nucleic Acids Res. 25:3440-3444.

[0118] The present invention also includes nucleic acids that hybridize with nucleic acids encoding esterases as defined above under stringent conditions. Preferably, such stringent conditions consist of incubating the hybridization filter at about 42°C for about 2.5 hours in 2×SSC / 0.1% SDS, followed by washing the filter four times at 65°C for 15 minutes each time in 1×SSC / 0.1% SDS. The protocols used are described in the references of Sambrook et al. (Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor NY (1988)) and Ausubel (Current Protocols in Molecular Biology (1989)).

[0119] The present invention also includes a nucleic acid encoding the esterase of the present invention, wherein the sequence of said nucleic acid or at least a portion thereof has been engineered using optimized codons.

[0120] Alternatively, the nucleic acid according to the invention can be deduced from the sequence of the esterase according to the invention, and codon usage can be adjusted according to the host cell in which the nucleic acid should be transcribed. These steps can be performed according to methods well known to those skilled in the art, some of which are described in the reference manual of Sambrook et al. (Sambrook et al., 2001).

[0121] The nucleic acids of the present invention may further include additional nucleotide sequences, such as regulatory regions, i.e. promoters, enhancers, silencers, terminators, signal peptides, etc., which can be used to induce or regulate polypeptide expression in selected host cells or systems.

[0122] The present invention further relates to an expression cassette comprising, or consisting of, a nucleic acid according to the invention operably linked to one or more control sequences, said control sequences directing the expression of said nucleic acid in a suitable host cell. Typically, the expression cassette comprises, or consists of, a nucleic acid according to the invention operably linked to, a control sequence such as a transcription promoter and / or a transcription terminator. The control sequence may include a promoter recognized by a host cell or in vitro expression system used to express a nucleic acid encoding an esterase of the present invention. The promoter contains a transcriptional control sequence mediating enzyme expression. The promoter may be any polynucleotide exhibiting transcriptional activity in a host cell, including mutant, truncated, and heterozygous promoters, and may be obtained from a gene encoding an extracellular or intracellular polypeptide homologous or heterologous to that of the host cell. The control sequence may also be a transcription terminator, which is recognized by the host cell to terminate transcription. The terminator is operably linked to the 3' end of the nucleic acid encoding the esterase. Any terminator functional in a host cell may be used in the present invention. Typically, the expression cassette comprises, or consists of, a nucleic acid according to the invention operably linked to, a transcription promoter and a transcription terminator.

[0123] The present invention also relates to vectors comprising nucleic acids or expression cassettes as defined above.

[0124] The term "vector" refers to a DNA molecule used as a medium to transfer recombinant genetic material into a host cell. The main types of vectors are plasmids, bacteriophages, viruses, granules, and artificial chromosomes. A vector itself is typically a DNA sequence consisting of an insert (a heterologous nucleic acid sequence, a transgene) and a larger sequence serving as the vector's "backbone." The purpose of vectors that transfer genetic information to a host is usually to isolate, propagate, or express the insert in target cells. Vectors called expression vectors (expression constructs) are particularly well-suited for expressing heterologous sequences in target cells and typically have a promoter sequence that drives the expression of the heterologous sequence encoding a polypeptide. Generally, regulatory elements present in expression vectors include transcription promoters, ribosome binding sites, terminators, and optionally, operons. Preferably, expression vectors also contain origins of replication for autonomous replication in the host cell, selection markers, a limited number of useful restriction enzyme sites, and the potential for high copy numbers. Examples of expression vectors are cloning vectors, modified cloning vectors, specifically designed plasmids, and viruses. Expression vectors that provide appropriate polypeptide expression levels in different hosts are well known in the art. The choice of vector generally depends on the compatibility of the vector with the host cell to which it is to be introduced.

[0125] Another object of the present invention is to provide host cells comprising the nucleic acids, expression cassettes, or vectors described above. Therefore, the present invention relates to the use of the nucleic acids, expression cassettes, or vectors according to the present invention for transforming, transfecting, or transducing host cells. The choice of vector generally depends on the compatibility of the vector with the host cells to which it must be introduced.

[0126] According to the present invention, host cells can be transformed, transfected, or transduced in a transient or stable manner. The expression cassette or vector of the present invention is introduced into a host cell such that the cassette or vector remains a chromosomal integrontoon or a self-replicating extrachromosomal vector. The term "host cell" also includes any progeny of a parent host cell that differs from the parent host cell due to mutations occurring during replication. The host cell can be any cell used to generate variants of the present invention, such as prokaryotic or eukaryotic cells. Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. Host cells can also be eukaryotic cells, such as yeast, fungi, mammalian, insect, or plant cells. In one specific embodiment, the host cell is selected from *Escherichia coli*, *Bacillus*, *Streptomyces*, *Trichoderma*, *Aspergillus*, *Saccharomyces*, *Pichia*, or *Yarrowia*.

[0127] The nucleic acid, expression cassette, or expression vector according to the invention can be introduced into host cells by any method known to those skilled in the art, such as electroporation, conjugation, transduction, competent cell transformation, protoplast transformation, protoplast fusion, biological projectile "gene gun" transformation, PEG-mediated transformation, lipid-assisted transformation or transfection, chemically mediated transfection, lithium acetate-mediated transformation, and liposome-mediated transformation.

[0128] Optionally, more than one copy of the nucleic acid, cassette, or vector of the present invention may be inserted into a host cell to increase the generation of variants.

[0129] In one specific embodiment, the host cell is a recombinant microorganism. This invention does indeed allow for the engineering of microorganisms with improved capabilities to degrade polyester-containing materials. For example, the sequences of this invention can be used to supplement wild-type strains of fungi or bacteria known to be capable of degrading polyesters to improve and / or increase the strain's capabilities.

[0130] Production of esterase variants

[0131] Another object of the present invention is to provide a method for producing the esterase variant of the present invention, comprising expressing a nucleic acid encoding the esterase and optionally recovering the esterase.

[0132] In particular, the present invention relates to an in vitro method for generating the esterase of the present invention, comprising (a) contacting the nucleic acid, cassette, or vector of the present invention with an in vitro expression system; and (b) recovering the generated esterase. The in vitro expression system is well known to those skilled in the art and is commercially available.

[0133] Preferably, the production method includes

[0134] (a) Culture host cells containing nucleic acids encoding the esterase of the present invention under conditions suitable for nucleic acid expression; and optionally...

[0135] (b) The esterase is recovered from the cell culture.

[0136] Advantageously, the host cell is a recombinant Bacillus, recombinant Escherichia coli, recombinant Aspergillus, recombinant Trichoderma, recombinant Streptomyces, recombinant Saccharomyces cerevisiae, recombinant Pichia pastoris, or recombinant Yarrowia lipolytica.

[0137] Host cells are cultured in a nutrient medium suitable for peptide production using methods known in the art. For example, cells can be cultured by shake flask culture or by small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid-state fermentation) in a laboratory or industrial fermenter under suitable medium and conditions that allow for the expression and / or isolation of enzymes. Culture is carried out in a suitable nutrient medium prepared from a commercial supplier or according to a published composition (e.g., in the catalogue of the U.S. Center for Typical Culture Collections).

[0138] If the esterase is secreted into the nutrient medium, it can be recovered directly from the culture supernatant. Conversely, it can be recovered from cell lysates or after permeabilization. Esterases can be recovered using any method known in the art. For example, esterases can be recovered from the nutrient medium by conventional methods, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. Optionally, esterases can be partially or completely purified by a variety of methods known in the art, including but not limited to chromatography (e.g., ion exchange, affinity, hydrophobicity, chromatographic focusing, and size exclusion), electrophoresis procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction, to obtain substantially pure polypeptides.

[0139] Esterases can be used alone or in combination with other enzymes in purified form to catalyze enzymatic reactions involved in the degradation and / or recycling of polyester-containing materials (e.g., polyester-containing plastic articles). Esterases can be in soluble or solid-phase form. In particular, they can bind to cell membranes or lipid vesicles, or to synthetic supports such as glass, plastics, polymers, filters, membranes, in the form of beads, columns, plates, etc.

[0140] Composition

[0141] Another object of the present invention is to provide compositions comprising the esterase or host cell of the present invention. In the context of the present invention, the term "composition" includes any kind of composition comprising the esterase of the present invention. In one specific embodiment, the esterase is in an isolated or at least partially purified form.

[0142] The composition may be liquid or dry, such as in powder form. In some embodiments, the composition is lyophilized. For example, the composition may contain an esterase and / or recombinant cells encoding the esterase of the present invention or extracts thereof, as well as optional excipients and / or reagents, etc. Suitable excipients include buffers commonly used in biochemistry; reagents for pH adjustment; preservatives, such as sodium benzoate, sodium sorbate, or sodium ascorbate; conserving agents, protectants, or stabilizers, such as starch, dextrin, gum arabic, salts, sugars such as sorbitol, trehalose, or lactose, glycerol, polyethylene glycol, polyethylene glycol, polypropylene glycol, propylene glycol; chelating agents such as EDTA; reducing agents; amino acids; carriers such as solvents or aqueous solutions, etc. The compositions of the present invention can be obtained by mixing the esterase with one or more excipients.

[0143] The compositions of the present invention may contain 0.1%-99.9%, preferably 0.1%-50%, more preferably 0.1%-30%, and even more preferably 0.1%-5% by weight of the esterase of the present invention and 0.1%-99.9%, preferably 50%-99.9%, more preferably 70%-99.9%, and even more preferably 95%-99.9% by weight of excipients. Preferred compositions contain 0.1-5% by weight of the esterase of the present invention.

[0144] In one specific embodiment, the composition may further comprise other polypeptides exhibiting enzymatic activity. For example, depending on the nature of the polyester-containing material to be degraded and / or other enzymes / polypeptides contained in the composition, those skilled in the art can readily adjust the esterase content of the present invention.

[0145] In one specific embodiment, the esterase of the present invention is dissolved in an aqueous medium together with one or more excipients, said excipients being particularly capable of stabilizing or protecting the polypeptide from degradation. For example, the esterase of the present invention may ultimately be dissolved in water together with other components, such as glycerol, sorbitol, dextrin, starch, glycols such as propylene glycol, salts, etc. The resulting mixture can then be dried to obtain a powder. Methods for drying such mixtures are well known to those skilled in the art and include, but are not limited to, lyophilization, freeze-drying, spray drying, supercritical drying, downward draft evaporation, thin-layer evaporation, centrifugal evaporation, conveying drying, fluidized bed drying, drum drying, or any combination thereof.

[0146] In another specific embodiment, the compositions of the present invention comprise at least one recombinant cell expressing the esterase of the present invention, or an extract thereof. “Cell extract” means any portion obtained from cells, such as cell supernatant, cell debris, cell wall, DNA extract, enzyme or enzyme preparation, or any preparation derived from cells by chemical, physical, and / or enzymatic treatment, which substantially does not contain living cells. Preferred extracts are enzyme-active extracts. The compositions of the present invention may comprise one or more recombinant cells of the present invention, or extracts thereof, and optionally one or more additional cells.

[0147] In one embodiment, the composition comprises or is composed of a lyophilized culture medium of recombinant microorganisms expressing and secreting the esterase of the present invention. In one embodiment, the powder comprises the esterase of the present invention and a stable / solubilizing amount of glycerol, sorbitol or dextrin such as maltodextrin and / or cyclodextrin, starch, glycol such as propylene glycol and / or salt.

[0148] Uses of the esterase of this invention

[0149] Another object of the present invention is to provide a method for degrading and / or recycling polyester-containing materials (e.g., plastic articles made of or containing polyester) under aerobic or anaerobic conditions using the esterase of the present invention. The variant esterase of the present invention is particularly suitable for degrading plastic articles containing PET.

[0150] Therefore, one object of the present invention is the use of the esterase of the present invention, or the corresponding recombinant cell or its extract, or composition thereof, in the enzymatic degradation of polyester-containing materials, such as PET-containing materials.

[0151] Another object of the present invention is to provide a method for degrading plastic articles comprising at least one polyester, wherein the plastic article is contacted with an esterase or host cell or composition of the present invention, thereby degrading the plastic article. Advantageously, the polyester containing the polyester material is depolymerized into monomers and / or oligomers.

[0152] In one embodiment of the degradation method, at least one polyester is degraded to produce repolymerizable monomers and / or oligomers, which are advantageously recycled for reuse.

[0153] In one implementation, the polyester containing the polyester material is completely degraded.

[0154] In one specific embodiment, the plastic article comprises at least one polyester selected from the following: polyethylene terephthalate (PET), polypropylene terephthalate (PTT), polybutylene terephthalate (PBT), polyisosorbate terephthalate (PEIT), polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene adipate succinate (PBSA), polybutylene terephthalate adipate (PBAT), polyfuran acetate (PEF), polycaprolactone (PCL), polyethylene adipate (PEA), polyethylene naphthalate (PEN), and blends / mixtures of these materials, preferably polyethylene terephthalate. In a preferred embodiment, the polyester material comprises PET, and at least monomers such as monoethylene glycol or terephthalic acid, and / or oligomers such as methyl 2-hydroxyethyl terephthalate (MHET), bis(2-hydroxyethyl) terephthalate (BHET), 2-hydroxyethyl benzoate (HEB), and dimethyl terephthalate (DMT) are recycled, for example, for recycling or methanation.

[0155] The present invention also relates to a method for preparing monomers and / or oligomers from polyester-containing materials, comprising exposing the polyester-containing material to the esterase of the present invention, or a corresponding recombinant cell or its extract, or a composition, and optionally recovering the monomers and / or oligomers. The method of the present invention is particularly suitable for preparing monomers selected from monoethylene glycol and terephthalic acid, and / or oligomers selected from methyl 2-hydroxyethyl terephthalate (MHET), bis(2-hydroxyethyl) terephthalate (BHET), 2-hydroxyethyl benzoate (HEB), and dimethyl terephthalate (DMT).

[0156] The time required to degrade polyester-containing materials can vary depending on the polyester-containing material itself (i.e., the nature and source of the plastic product, its composition, shape, etc.), the type and content of the esterase used, and various process parameters (i.e., temperature, pH, other reagents, etc.). Those skilled in the art can easily adapt the process parameters to polyester-containing materials.

[0157] Advantageously, the degradation method is carried out at a temperature of 20°C-90°C, preferably 40°C-80°C, more preferably 50°C-70°C, even more preferably 60°C-70°C, and even more preferably 65°C. In another specific embodiment, the degradation method is carried out at 70°C. More generally, the temperature is maintained below the inactivation temperature, which corresponds to the temperature at which the esterase is inactivated and / or the recombinant microorganisms no longer synthesize the esterase. In particular, the temperature is maintained below the glass transition temperature (Tg) of the polyester in the polyester-containing material. More specifically, the method is carried out in a continuous manner and at a temperature at which the esterase can be used several times and / or recycled.

[0158] Advantageously, the degradation method is carried out at a pH of 5-11, preferably a pH of 6-9, more preferably a pH of 6.5-9, and even more preferably a pH of 6.5-8.

[0159] In one specific embodiment, the polyester-containing material may be pretreated before contact with the esterase to physically alter its structure, thereby increasing the contact surface between the polyester and the variant of the invention.

[0160] Optionally, the monomers and / or oligomers generated by depolymerization can be recovered sequentially or continuously. Depending on the starting polyester-containing material, a single type of monomer and / or oligomer or several different types of monomers and / or oligomers can be recovered.

[0161] The recovered monomers and / or oligomers can be further purified and formulated into repolymerizable forms using all suitable purification methods. Examples of purification methods include stripping, separation by aqueous solution, selective steam condensation, post-bioprocess media filtration and concentration, separation, distillation, vacuum evaporation, extraction, electrodialysis, adsorption, ion exchange, precipitation, crystallization, concentration and acid addition dehydration and precipitation, nanofiltration, acid catalyst treatment, semi-continuous or continuous distillation, solvent extraction, evaporative concentration, evaporative crystallization, liquid / liquid extraction, hydrogenation, azeotropic distillation, adsorption, column chromatography, simple vacuum distillation and microfiltration, in combination or without combination.

[0162] The repolymerizable monomers and / or oligomers can then be reused, for example, to synthesize polyesters. Advantageously, polyesters with the same properties are repolymerized. However, the recycled monomers and / or oligomers can be mixed with other monomers and / or oligomers to, for example, synthesize new copolymers. Alternatively, the recycled monomers can be used as chemical intermediates to produce new target compounds.

[0163] This invention also relates to methods for surface hydrolysis or surface functionalization of polyester-containing materials, including exposing the polyester-containing material to the esterase of the present invention, or corresponding recombinant cells or extracts thereof, or compositions thereof. The methods of the present invention are particularly suitable for increasing the hydrophilicity or water absorption of polyester materials. This increased hydrophilicity may be of particular interest in textile production, electronic, and biomedical applications.

[0164] Another object of the present invention is to provide a polyester-containing material comprising the esterase of the present invention and / or recombinant microorganisms expressing and secreting said esterase. In one specific embodiment, such polyester-containing material may be a plastic compound. Therefore, an object of the present invention is to provide a plastic compound comprising the esterase of the present invention and / or recombinant cells and / or compositions or extracts thereof; and at least one polyester. In a preferred embodiment, the polyester is PET. Detailed Implementation

[0165] Example 1 - Construction, expression and purification of esterase

[0166] -Build

[0167] The pET26b-LCC-His plasmid was constructed to generate the esterase variant. This plasmid is used to clone the gene encoding the esterase SEQ ID N°1 between the NdeI and XhoI restriction sites, which was optimized for expression in *E. coli*. Two site-directed mutagenesis kits were used to generate the esterase variant as recommended by the vendor: the QuikChange II site-directed mutagenesis kit from Agilent and the QuikChange Lightning multi-site mutagenesis kit (Santa Clara, California, USA).

[0168] - Expression and purification of esterases

[0169] The strain Stellar was continuously used in 50 mL LB-Miller medium or ZYM self-induction medium (Studier et al., 2005-Prot.Exp.Pur.41, 207-234). TM (Clontech, California, USA) and E. coli One BL21 DE3 cells (Life Technologies, Carlsbad, California, USA) were cloned and recombinantly expressed. Induction was performed in LB-Miller medium at 16°C using 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG, Euromedex, Souffelweyersheim, France). Culture was terminated by centrifugation (8000 rpm, 10°C for 20 min) in an Avanti J-26XP centrifuge (Beckman Coulter, Brea, USA). Cells were resuspended in 20 mL of Talon buffer (Tris-HCl 20 mM, NaCl 300 mM, pH 8). The cell suspension was then sonicated for 2 minutes using an FB 705 sonicator (Fisherbrand, Illkirch, France) with an amplitude of 30% (2 sec ON and 1 sec OFF cycles). Then, a centrifugation step was performed: 11,000 rpm, 10°C for 30 minutes in an Eppendorf centrifuge. The soluble fraction was collected and subjected to affinity chromatography. This purification step used... Metal Affinity Resin (Clontech, CA, USA) was used. Protein elution was performed using a Talon buffer gradient supplemented with imidazole. The purified protein was dialyzed against Talon buffer and then quantified using the Bio-Rad protein assay according to the manufacturer's instructions (Lifescience Bio-Rad, France), and stored at +4°C.

[0170] Example 2 - Evaluation of the thermal stability of the esterase of the present invention

[0171] The thermal stability of the esterase variant was determined and compared with that of the esterase in SEQ ID N°1.

[0172] Using different methods to estimate thermal stability:

[0173] (1) Circular dichroism of proteins in solution;

[0174] (2) The activity of residual esterases after incubation of proteins under given temperature, time and buffer conditions;

[0175] (3) Depolymerization activity of residual polyester after incubation of protein under given temperature, time and buffer conditions;

[0176] (4) The ability to degrade solid polyester compounds (such as PET or PBAT or similar compounds) dispersed in agar plates after incubation of proteins under given temperature, time and buffer conditions.

[0177] (5) The ability to perform multiple rounds of polyester depolymerization determination under given temperature, buffer, protein concentration and polyester concentration conditions;

[0178] (6) Differential scanning fluorescence (DSF);

[0179] The details of these methods are given below.

[0180] 2.1 Circular Dichroism

[0181] Circular dichroism (CD) was performed using a Jasco 815 device (Easton, USA) to compare the fusion temperature (T) of the esterase of SEQ ID N°1 and the esterase variant of the present invention. m ). T m The temperature corresponding to 50% protein denaturation.

[0182] Technically, 400 μL of protein sample was prepared in Talon buffer at 0.5 mg / mL for CD. A first scan from 280 nm to 190 nm was performed to determine the two maximum intensities corresponding to the correct folding of the protein in CD. A second scan from 25 °C to 110 °C was then performed at a longer wavelength corresponding to these maximum intensities, providing a specific curve (sigmoid 3 parameter y = a / (1 + e^((x - x0) / b))), which was analyzed using Sigmaplot version 11.0 software, determining T when x = x0. m The obtained T m This reflects the thermal stability of a given protein. m The higher the value, the more stable the variant is at high temperatures.

[0183] 2.2 Activity of residual esterases

[0184] A 40 mg / L solution (in Talon buffer) of 1 mL of the esterase or esterase variant of SEQ ID N°1 was incubated for 10 days at different temperatures (65, 70, 75, 80, and 90 °C). Samples were periodically taken and diluted 1 to 500 times in 0.1 M potassium phosphate buffer (pH 8.0) for p-nitrophenol-butyrate (pNP-B) determination. 20 μL of sample was mixed with 175 μL of 0.1 M potassium phosphate buffer (pH 8.0) and 5 μL of pNP-B in 40 mM 2-methyl-2-butanol. The enzymatic reaction was carried out at 30 °C with stirring for 15 min, and the absorbance was obtained at 405 nm using a microplate spectrophotometer (Versamax, Molecular Devices, Sunnyvale, CA, USA). The hydrolytic activity of pNP-B (initial rate expressed as μmol pNPB / min) was determined using a standard curve of p-nitrophenol released in the linear portion of the hydrolysis curve. The half-life of an enzyme at a given temperature corresponds to the time required for 50% of its initial activity to be lost.

[0185] 2.3 Depolymerization activity of residual polyester

[0186] Crushed Cristal preforms were immersed in liquid nitrogen and micronized to a fine powder of <500 μm using an Ultra Centrifugal Mill ZM 200 system. The resulting powder was then sieved. Only the fraction with a size of 250 μm–500 μm was used for depolymerization testing. The crystallinity of this fraction was measured to be 11.5% using a Mettler Toledo DSC 3 at a heating rate of 10 °C / min.

[0187] 10 mL solutions of the esterase and its variants of SEQ ID N°1 at 40 mg / L (in Talon buffer) were incubated at different temperatures (65, 70, 75, 80, and 90 °C) for 10 to 30 days. Periodically, 1 mL samples were taken and transferred to 49 mL of 0.1 M potassium phosphate buffer containing 100 mg of amorphous PET micronized to 250–500 μm and pH 8.0, and incubated at 65 °C. Periodically, 150 μL of buffer was taken. If necessary, the sample was diluted in 0.1 M potassium phosphate buffer at pH 8. Then, 150 μL of methanol and 6.5 μL of HCl 6N were added to 150 μL of sample or diluent. After mixing and filtering through a 0.45 μm syringe filter, the sample was loaded onto a UHPLC system to monitor the release of terephthalic acid (TA), MHET, and BHET. The chromatographic system used was an Ultimate 3000U HPLC system (Thermo Fisher Scientific, Inc., Waltham, MA, USA), including a pump module, autosampler, a 25°C constant-temperature column oven, and a 240nm UV detector. The column used was... HSC18 HPLC column (150 × 4.6 mm, 5 μm, equipped with pre-column, Supelco, Bellefonte, USA). TA, MHET, and BHET were separated at 1 mL / min using a MeOH gradient (30%–90%) in 1 mM H₂SO₄. 20 μL of sample was injected. TA, MHET, and BHET were measured under the same conditions as the samples, according to commercial TA and BHET and internally synthesized MHET. The hydrolytic activity of PET was determined in the linear portion of the hydrolysis curve (μmol / min of hydrolyzed PET or mg / h of the equivalent TA produced). The equivalent TA corresponds to the sum of the measured TA and the TA contained in the measured MHET and BHET. The half-life of the enzyme at a given temperature corresponds to the time required for 50% loss of initial activity.

[0188] 2.4 Degradation of solid polyester

[0189] 20 μL of enzyme preparation was placed in the wells of an agar plate containing PET. The agar plate was prepared by dissolving 500 mg of PET in HFIP and then adding this medium to 250 mL of aqueous solution. After evaporating the HFIP at 52 °C, the solution was mixed v / v with 0.2 M potassium phosphate buffer (pH 8) containing 3% agar. Approximately 30 mL of the mixture was used to prepare each omnitray and stored at 4 °C.

[0190] After 24 hours, the diameter of the halo formed due to polyester degradation was measured at 60 or 65 °C. The half-life of the enzyme at a given temperature corresponds to the time required for the halo diameter to decrease by a factor of 2.

[0191] 2.5 Multi-round depolymerization of polyester

[0192] The ability of esterase to perform consecutive rounds of polyester depolymerization assays was evaluated in an enzyme reactor. A Minibio 500 bioreactor (Applikon Biotechnology BV, Delft, The Netherlands) was started with 3 g of amorphous PET and 100 mL of 10 mM potassium phosphate buffer (pH 8) containing 3 mg of LC-esterase. The agitation was set to 250 rpm using a marine impeller. The bioreactor was kept at 65 °C by immersion in an external water bath. The pH was adjusted to 8 by adding 3 M KOH. Different parameters (pH, temperature, agitation, alkali addition) were monitored using BioXpert software V2.95. 1.8 g of amorphous PET was added every 20 hours. 500 μL of the reaction medium was sampled periodically.

[0193] As described in Example 2.3, the contents of TA, MHET, and BHET were determined by HPLC. The content of EG was determined using an Aminex HPX-87K column (Bio-Rad Laboratories, Inc., Hercules, California, United States) at a constant temperature of 65°C. The eluent was K₂HPO₄ 5mM, and the concentration was determined at 0.6 mL / min. -1 The injection volume was 20 μL. Ethylene glycol was monitored using a refractometer.

[0194] The percentage of hydrolysis is 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+2x BHET) at a given time to the total amount of EG contained in the initial sample. The degradation rate is calculated as mg of total TA released per hour or mg of total EG released per hour.

[0195] The half-life of the enzyme was assessed as the incubation time required to achieve a 50% loss in degradation rate.

[0196] 2.6 Differential Scanning Fluorescence (DSF)

[0197] DMSO was used to assess the thermal stability of wild-type protein (SEQ ID N°1) and its variants by determining their melting temperature (Tm, i.e., the temperature at which half of the protein population unfolds). Protein samples were prepared at a concentration of 14 μM (0.4 mg / mL) and stored in buffer A consisting of 20 mM Tris HCl (pH 8.0) and 300 mM NaCl. The 5000x stock solution of SYPRO Orange dye in DMSO was first diluted to 250x in water. Protein samples were loaded onto white, clear 96-well PCR plates (Bio-Rad cat#HSP9601), with each well containing a final volume of 25 μL. The final concentrations of protein and SYPRO Orange dye in each well were 5 μM (0.14 mg / mL) and 10X, respectively. The loading volumes per well were as follows: 15 μL buffer A, 9 μL 0.4 mg / mL protein solution, and 1 μL 250x Sypro Orange dilution. The PCR plate was then sealed with optical quality sealing tape and rotated at 2000 rpm for 1 minute at room temperature. DSF experiments were then performed using a CFX96 real-time PCR system configured with 450 / 490 excitation and 560 / 580 emission filters. The sample was heated from 25°C to 100°C at a rate of 1.1°C / min. Single fluorescence measurements were performed every 0.3°C. The melting temperature was determined by curve fitting to the Boltzmann equation.

[0198] Wild-type proteins and variants were then compared based on their Tm values. Due to the high reproducibility between experiments on the same protein from different productions, a ΔTm of 0.8 °C was considered significant for comparing variants. The Tm value corresponded to the average of at least two measurements.

[0199] The comparative thermostability of the esterase variants of the present invention is shown in Table 2 below, expressed as Tm values ​​and evaluated according to Example 2.6. The increase in Tm compared to the esterase of SEQ ID NO1 is shown in parentheses.

[0200] Table 2: Tm of the esterase of the present invention

[0201]

[0202]

[0203] Example 3 - Evaluation of the thermostability and activity of the esterase variant of the present invention

[0204] The specific degradation activity of the esterase variant of the present invention was evaluated on PET and compared with the specific degradation activity of the esterase of SEQ ID N°1.

[0205] Weigh 100 mg of amorphous PET and transfer it to a 100 mL glass vial. Prepare 1 mL of esterase preparation (as a control) or variant preparation at a concentration of 0.02 or 0.03 mg / mL in Talon buffer (Tris-HCl 20 mM, NaCl 0.3 M, pH 8) and transfer it to the glass vial. Finally, add 49 mL of 0.1 M potassium phosphate buffer (pH 8).

[0206] Depolymerization was initiated by incubating each vial at 65°C and 150 rpm in a Max Q 4450 incubator (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

[0207] The initial rate of depolymerization was determined by ultra-high performance liquid chromatography (UHPLC) analysis of samples taken at different times during the initial 24-hour period, expressed in mg of equivalent TA produced per hour. If necessary, samples were diluted in 0.1 M potassium phosphate buffer at pH 8. Then, 150 μL of methanol and 6.5 μL of HCl 6N were added to 150 μL of sample or diluent. After mixing and filtering on a 0.45 μm syringe filter, the sample was 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, autosampler, 25 °C constant column oven, and a 240 nm UV detector. The column used was... HSC 18 HPLC column (150 × 4.6 mm, 5 μm, equipped with pre-column, Supelco, Bellefonte, USA). TA, MHET, and BHET were separated at 1 mL / min using a MeOH gradient (30%–90%) in 1 mM H₂SO₄. 20 μL of sample was injected. TA, MHET, and BHET were measured under the same conditions as the sample, based on standard curves prepared according to commercial TA and BHET and internally synthesized MHET. The specific degradation activity of PET (mg equivalent TA / h / mg enzyme) was determined in the linear portion of the hydrolysis curve.

[0208] Table 3 shows the degradation specific activity and thermal stability results of the esterase variants of the present invention.

[0209] The specific degradation activity of the esterase of SEQ ID N°1 was used as a reference and considered as 100% degradation activity. Degradation activity (mg equivalent TA / h / mg enzyme) was measured as described in Example 3. Equivalent TA corresponds to the sum of the measured TA and the TA contained in the measured MHET and BHET. Thermostability is expressed as a Tm value (measured according to Example 2.6), and the increase in Tm compared to the Tm of the esterase of SEQ ID N°1 is shown in parentheses.

[0210] Table 3: Specific activity and Tm of the esterase of the present invention

[0211]

Claims

1. An esterase variant wherein, compared with the amino acid sequence shown in SEQ ID NO:1, the amino acid substitutions or combinations of substitutions are as follows: Y92G, Y92P, or Y92P + F208W, wherein the esterase variant has polyester degradation activity and exhibits increased thermal stability compared with the esterase of SEQ ID NO:

1.

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

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

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

5. A method for producing esterase, comprising: (a) Culturing the host cells according to claim 4 under suitable conditions to express nucleic acids encoding esterases; and optional (b) The esterase is recovered from the cell culture.

6. A composition comprising the esterase according to claim 1 and / or the nucleic acid according to claim 2, and / or the expression cassette or vector according to claim 3, and / or the host cell according to claim 4, and optionally one or more excipients or additives.

7. A method for degrading plastic articles containing at least one polyester, comprising: (a) Contacting the plastic article with the esterase according to claim 1, the host cell according to claim 4, or the composition according to claim 6, thereby degrading the plastic article; and optional (b) Recover monomers and / or oligomers.

8. The method of claim 7, wherein the plastic article comprises at least one polyester selected from the group consisting of polyethylene terephthalate (PET), polypropylene terephthalate (PTT), polybutylene terephthalate (PBT), polyisosorbate terephthalate (PEIT), polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene adipate succinate (PBSA), polybutylene adipate terephthalate (PBAT), polyfuran acetate (PEF), polycaprolactone (PCL), polyethylene adipate (PEA), polyethylene naphthalate (PEN), and blends / mixtures of these materials.

9. The method of claim 8, wherein the plastic article comprises at least polyethylene terephthalate.

10. The method according to any one of claims 7-9, wherein step (a) is performed at a temperature of 50°C-90°C.

11. The method according to any one of claims 7-9, wherein step (a) is performed at a temperature of 60°C-70°C.

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