Novel esterase and its use

Abstract

The present invention relates to novel esterases, more particularly to esterase variants having improved activity and / or improved thermostability compared to the esterase of SEQ ID N° 1 and their use for degrading polyester-containing materials (such as plastics). The esterases of the present invention are particularly suitable for degrading polyethylene terephthalate and materials containing polyethylene terephthalate.

Description

Technical Field

[0001] The present invention relates to novel esterases, more particularly to esterases having improved activity and / or improved thermostability compared to a parent esterase. The present invention also relates to the use of said novel esterases for degrading polyester-containing materials (such as plastics). The esterases of the present invention are particularly suitable for degrading polyethylene terephthalate and materials containing polyethylene terephthalate. Background Art

[0002] Esterase can catalyze the hydrolysis of various polymers (comprising polyester). In this article, esterase demonstrates promising effects in many industrial applications, comprises being applied to dishwashing and washing clothes as detergent, is used for processing biomass and food as degradation enzyme, is used for the biocatalyst of environmental pollutant detoxification or is used for the processing of polyester fabric in textile industry.Esterase is particularly useful as degradation enzyme for the purposes of hydrolyzing polyethylene terephthalate (PET).In fact, PET is used for many technical fields, such as being used for making clothes, carpet, or being used for making packaging or automotive plastics etc. in the form of thermosetting resin, makes the accumulation of PET in landfill become increasing ecological problem.

[0003] Enzymatic degradation of polyesters, in particular PET, is considered an interesting solution to reduce the accumulation of plastic waste. Indeed, enzymes can accelerate the hydrolysis of polyester-containing materials, more particularly plastic products, even down to the monomer level. Furthermore, the hydrolysis products (i.e. monomers and oligomers) can be recycled as materials for the synthesis of new polymers.

[0004] In this article, several esterases have been identified as candidate degrading enzymes for polyesters, and some variants of these esterases have been developed. Among esterases, cutinase, also known as cutin hydrolase (EC 3.1.1.74), is of particular concern. Cutinase 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, metagenomics approaches have led to the identification of other esterases.

[0005] However, there is still a need for esterases having improved activity and / or improved thermostability compared to known esterases, in order to provide a more efficient and thus more competitive polyester degradation process.

[0006] Brief description of the invention

[0007] The present invention provides novel esterases showing increased activity and / or increased thermostability compared to a parent or wild-type esterase having an amino acid sequence as shown in SEQ ID N° 1. The wild-type esterase corresponds to the esterase referenced by UPI0007C1BBA7 in the UniParc database and has polyester degradation activity as described. The esterases of the present invention are particularly useful in processes for degrading plastic products, more particularly plastic products containing PET.

[0008] In this context, the object of the present invention is to provide an esterase which (i) has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the full-length amino acid sequence as shown in SEQ ID N° 1, and (ii) has at least one amino acid substitution at a position corresponding to a residue selected from the group consisting of T12, E13, S14, S15, I16, E17, A18, V19, R20, R32, A35, F38, G62, F63, T64, A65, G66, Q67, E68, S69, I70, A71, W72, P75, R76, D88, T89, R92, L93, D94, Q95, P96, D97, H130, M132, G133, G134, G135, G136, G137, G138, G139, G140, G141, G142, G143, G144, G145 133, S139, W156, T158, R159, V172, Q175, T178, 1179, A180, P181, S184, F189, L204, R205, G206, A207, S208, V211, S212, N213, T214, P215, D216, T217, T218, T219, A220, K221, Y222, Q239, F240, L241, P243, A244, P245, D246, D247, F248, A249, 1250, S251, G136, T169, wherein the positions are as referenced by SEQ ID NO: 1 The esterase is numbered according to the amino acid sequence shown in SEQ ID N°1, and (iii) exhibits increased polyester degradation activity and / or increased thermal stability compared to the esterase of SEQ ID N°1.

[0009] Another object of the present invention is to provide an esterase which (i) has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the full-length amino acid sequence as shown in SEQ ID N°1, and (ii) has at least one amino acid substitution at position L210, wherein the position is numbered by reference to the amino acid sequence as shown in SEQ ID N°1, and wherein the substitution is different from L210F.

[0010] Another object of the present invention is to provide an esterase which (i) has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the full-length amino acid sequence as shown in SEQ ID N°1, and (ii) contains at least one amino acid substitution selected from the group consisting of L210A / C / D / E / G / H / I / K / M / N / P / Q / R / S / T / V / W / Y, R205C, S251C, Q95G / P, G136A, T169Q, V172I, S184E or N213M / D, wherein the positions are numbered by reference to the amino acid sequence as shown in SEQ ID N°1.

[0011] Preferably, the esterase comprises at least one substitution or combination of substitutions selected from L210T / A / R / W / H or R205C+S251C, preferably L210T. In a specific embodiment, the esterase comprises at least the combination of substitutions L210T+V172I.

[0012] Preferably, the esterase further comprises at least one amino acid residue selected from the group consisting of S131, D177 and H209, preferably the combination S131+D177+H209.

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

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

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

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

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

[0018] Another object of the present invention is to provide a method for degrading polyester, which comprises:

[0019] (a) contacting the polyester with an esterase according to the invention or a host cell according to the invention or a composition according to the invention; and, optionally

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

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

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

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

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

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

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

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

[0029] definition

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

[0031] As used herein, the terms "peptide", "polypeptide", "protein", "enzyme" refer to a chain of amino acids linked by peptide bonds, regardless of the number of amino acids forming the chain. Amino acids are represented herein 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; N: asparagine (Asn); P: proline (Pro); Q: glutamine (Gln); R: arginine (Arg); S: serine (Ser); T: threonine (Thr); V: valine (Val); W: tryptophan (Trp) and Y: tyrosine (Tyr).

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

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

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

[0035] "Substitution" refers to the replacement of an amino acid residue by another amino acid residue. Preferably, the term "substitution" refers to the replacement of an amino acid residue with another selected from the standard 20 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, alloisoleucine, N-methylisoleucine, N-methylvaline, pyroglutamine, aminobutyric acid, ornithine, norleucine, norvaline) and non-naturally occurring amino acid residues (e.g., cyclohexylalanine) prepared synthetically. Preferably, the term "substitution" refers to the replacement of an amino acid residue with another selected from the standard 20 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. In this document, the following terms are used to represent substitutions: L82A means that the amino acid residue (leucine, L) at position 82 of the parent sequence is replaced by alanine (A). A121V / I / M means that the amino acid residue (alanine, A) at position 121 of the parent sequence is replaced by one of the following amino acids: valine (V), isoleucine (I) or methionine (M). Substitutions can be conservative or non-conservative substitutions. Examples of conservative substitutions include the following groups: basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine, asparagine and threonine), hydrophobic amino acids (methionine, leucine, isoleucine, cysteine ​​and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine) and small amino acids (glycine, alanine and serine).

[0036] Unless otherwise indicated, positions disclosed in this application are numbered with reference to the amino acid sequence as shown in SEQ ID N°1.

[0037] As used herein, the term "sequence identity" or "identity" refers to the number (or fraction expressed as a percentage %) of matches (same 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. In particular, depending on the length of the two sequences, any of a variety of mathematical global or local alignment algorithms can be used to determine sequence identity. It is preferred to use a global alignment algorithm (e.g., Needleman and Wunsch algorithm; Needleman and Wunsch, 1970) to align sequences of similar length, which optimally align sequences over the entire length, while sequences of substantially different lengths preferably use a local alignment algorithm (e.g., Smith and Waterman algorithm (Smith and Waterman, 1981) or Altschul algorithm (Altschul et al., 1997; Altschul et al., 2005)). Comparison for determining the amino acid sequence identity percentage purpose can be achieved in various ways known to those skilled in the art, for example, using publicly available computer software that can be obtained on an Internet website (such as http: / / blast.ncbi.nlm.nih.gov / or http: / / www.ebi.ac.uk / Tools / emboss / ). Those skilled in the art can determine the suitable parameters for measuring the comparison, including any algorithm required for maximum comparison on the full length of the compared sequence. For purposes of this paper, % amino acid sequence identity values ​​refer to the values ​​produced using the paired sequence alignment program EMBOSS Needle, which uses the Needleman-Wunsch algorithm to produce the best global comparison of two sequences, wherein all search parameters are set to default values, i.e., scoring matrix=BLOSUM62, gap beginning=10, gap extension=0.5, terminal gap penalty=false, terminal gapgap=10 and terminal gap extension=0.5.

[0038] "Polymer" refers to a chemical compound or mixture of compounds whose structure consists of multiple monomers (repeating units) connected by covalent chemical bonds. In the context of the present invention, the term polymer includes natural or synthetic polymers consisting of a single type of repeating unit (i.e., homopolymer) or a mixture of different repeating units (i.e., copolymer or heteropolymer). According to the present invention, "oligomer" refers to a molecule containing 2 to about 20 monomers.

[0039] In the context of the present invention, "polyester-containing material" or "polyester-containing article" refers to an article, such as a plastic article, comprising at least one polyester in crystalline, semi-crystalline or completely amorphous form. In a specific embodiment, polyester-containing material refers to any article made of at least one plastic material, such as plastic sheets, tubes, rods, profiles, moldings, films, bulk, etc., which contains at least one polyester and possible other substances or additives, such as plasticizers, minerals or organic fillers. In another specific embodiment, polyester-containing material refers to a molten or solid plastic compound or plastic preparation, which is suitable for the preparation of plastic articles. In another specific embodiment, polyester-containing material refers to a textile, fabric or fiber comprising at least one polyester. In another specific embodiment, polyester-containing material refers to plastic waste or fiber waste comprising at least one polyester.

[0040] In the present specification, 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), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furandicarboxylate (PEF), polycaprolactone (PCL), polyethylene adipate (PEA), polyethylene naphthalate (PEN) and blends / mixtures of these polymers.

[0041] Novel esterase

[0042] The present invention provides novel esterases having improved activity and / or improved thermostability compared to the parent esterase. More particularly, the inventors have designed novel enzymes that are particularly suitable for industrial processes. The esterases of the present invention are particularly suitable for degrading polyesters, more particularly PET, including materials containing PET and in particular plastic products containing PET. In a specific embodiment, the esterases show increased activity and increased thermostability.

[0043] Therefore, an object of the present invention is to provide an esterase showing improved activity compared to the esterase having the amino acid sequence shown in SEQ ID N°1.

[0044] In particular, the inventors have identified specific amino acid residues in SEQ ID N°1 that are intended to contact the polymer substrate in the X-ray crystal structure (i.e., the folded 3D structure) of an esterase that can be advantageously modified to facilitate contact of the substrate with the esterase and result in increased adsorption of the polymer and / or increased activity of the esterase on the polymer.

[0045] In the context of the present invention, the term "increased activity" or "increased degradation activity" means an increased ability of the esterase to degrade polyester at a given temperature and / or an increased adsorption capacity on polyester compared to the degradation and / or adsorption capacity of the esterase of SEQ ID N°1 on the same polyester at the same temperature. In particular, the esterase of the present invention has an increased PET degradation activity. This increase may be at least 10%, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130% or more higher than the PET degradation activity of the esterase of SEQ ID N°1. In particular, the degradation activity is a depolymerization activity to obtain monomers and / or oligomers of the polyester, which can further be recovered and optionally reused.

[0046] The "degradation activity" of esterase can be evaluated by those skilled in the art according to methods known per se in the art. For example, the degradation activity can be evaluated by measuring the depolymerization activity rate of a specific polymer, measuring the rate of degradation of a solid polymer compound dispersed in an agar plate, or measuring the depolymerization activity rate of a polymer in a reactor. In particular, the degradation activity can be evaluated by measuring the "specific degradation activity" of the esterase. The "specific degradation activity" of the esterase to PET corresponds to the μmol / min of PET hydrolyzed per minute during the initial period of the reaction (i.e., the first 24 hours) or the mg / hour and per mg enzyme of the equivalent TA produced, and is determined by the linear part of the hydrolysis curve of the reaction, which is established by several samplings performed at different times during the first 24 hours. As another example, the "degradation activity" can be evaluated by measuring the rate and / or yield of oligomers and / or monomers released under suitable temperature, pH and buffer conditions when a polymer or a plastic product containing a polymer is contacted with a degrading enzyme after a defined period of time.

[0047] Those skilled in the art can evaluate the adsorption capacity of the enzyme on the substrate according to methods known in the art per se.For example, the adsorption capacity of the enzyme on the substrate can be measured from a solution containing the enzyme, and wherein the enzyme has been incubated with the substrate in advance under suitable conditions.

[0048] The inventors have also identified target amino acid residues in SEQ ID N° 1 which may advantageously be modified to improve the stability of the corresponding esterase at high temperatures, and advantageously at temperatures above 60° C., preferably above 70° C. (ie improved thermostability).

[0049] Therefore, an object of the present invention is to provide a novel esterase showing improved thermostability compared with the thermostability of the esterase having the amino acid sequence shown in SEQ ID N°1.

[0050] In the context of the present invention, the term "increased thermostability" means that the esterase has an increased ability to resist changes in its chemical and / or physical structure at elevated temperatures, particularly at temperatures between 40°C and 80°C, compared to the esterase of SEQ ID N°1.

[0051] In particular, thermal stability can be assessed by the melting temperature (Tm) of the esterase. In the context of the present invention, "melting temperature" refers to the temperature at which half of the enzyme population under consideration is unfolded or misfolded. Typically, the esterase of the present invention shows an increase of about 1°C, 2°C, 3°C, 4°C, 5°C, 10°C or more Tm compared to the Tm of the esterase of SEQ ID N°1. In particular, the esterase of the present invention may have an increased half-life at a temperature of 40°C-80°C compared to the esterase of SEQ ID N°1.

[0052] The melting temperature (Tm) of esterase can be measured by those skilled in the art according to methods known in the art per se. For example, DSF can be used to quantitatively measure the change in the thermal denaturation temperature of esterase, thereby determining its Tm. Alternatively, Tm can be assessed by analyzing protein folding using circular dichroism. Preferably, Tm is measured using DSF or circular dichroism as disclosed in the experimental section. In the context of the present invention, Tm is compared with Tm measured under the same conditions (e.g., pH, the nature and amount of polyester, etc.).

[0053] Alternatively, thermal stability can be assessed by measuring the esterase activity and / or polyester depolymerization activity of the esterase after incubation at different temperatures and comparing it to the esterase activity and / or polyester depolymerization activity of the parent esterase. The ability to perform multiple rounds of polyester depolymerization assays at different temperatures can also be assessed. A quick and useful test can be to assess the ability of the esterase to degrade solid polyester compounds dispersed in agar plates after incubation at different temperatures by halo diameter measurement.

[0054] Therefore, one object of the present invention is to provide an esterase which (i) has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the full-length amino acid sequence as shown in SEQ ID N° 1, (ii) has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the full-length amino acid sequence as shown in SEQ ID N° 1, N ° 1, compared to the position corresponding to the residue selected from the group consisting of: T12, E13, S14, S15, I16, E17, A18, V19, R20, R32, A35, F38, G62, F63, T64, A65, G66, Q67, E68, S69, I70, A71, W72, P75, R76, D88, T89, R92, L93, D94, Q95, P96, D97, H130, M132, G133, S139, W156, T158, R159, V17 2, Q175, T178, 1179, A180, P181, S184, F189, L204, R205, G206, A207, S208, V211, S212, N213, T214, P215, D216, T217, T218, T219, A220, K221, Y222, Q239, F240, L241, P243, A244, P245, D246, D247, F248, A249, 1250, S251, G136, T169, and (iii) exhibits increased polyester degradation activity and / or increased thermal stability compared to the esterase of SEQ ID N° 1.

[0055] In a specific embodiment, the esterase contains at least one amino acid substitution at a position corresponding to a residue selected from the group consisting of: T12, E13, S14, S15, I16, E17, A18, V19, R20, R32, A35, F38, G62, F63, T64, A65, G66, Q67, E68, S69, I70, A71, W72, P75, R76, D88, T89, R92, L93, D94, Q95, P96, D97, H130, M132, G133, S139, W156 , T158, R159, V172, Q175, T178, 1179, A180, P181, S184, F189, L204, R205, G206, A207, S208, V211, S212, N213, T214, P215, D216, T217, T218, T219, A220, K221, Y222, Q239, F240, L241, P243, A244, P245, D246, D247, F248, A249, 1250, or S251. In one embodiment, the esterase contains at least one amino acid substitution at a position corresponding to a residue selected from the group consisting of: G136 or T169.

[0056] Unless otherwise indicated, positions disclosed in this application are numbered with reference to the amino acid sequence as shown in SEQ ID N°1.

[0057] According to the present invention, the target amino acid can be replaced by any one of 19 other amino acids.

[0058] In a specific embodiment, the esterase has an amino acid sequence as shown in SEQ ID N° 1, which has a single amino acid substitution at a position corresponding to a residue selected from the group consisting of: T12, E13, S14, S15, I16, E17, A18, V19, R20, R32, A35, F38, G62, F63, T64, A65, G66, Q67, E68, S69, I70, A71, W72, P75, R76, D88, T89, R92, L93, D94, Q95, P96, D97, H130, M132, G133, S139, W156, T158, , P245, D246, D247, F248, A249, 1250, S251, G136 or T169. In particular, the esterase has the amino acid sequence of SEQ ID NO: 1. N ° 1, which has a single amino acid substitution at a position corresponding to a residue selected from the group consisting of: T12, E13, S14, S15, I16, E17, A18, V19, R20, R32, A35, F38, G62, F63, T64, A65, G66, Q67, E68, S69, I70, A71, W72, P75, R76, D88, T89, R92, L93, D94, Q95, P96, D97, H130, M132, G133, S139, W156, , T241, Y242, Q243, F244, F245, L246, D247, F248, A249, I250, or S251. In particular, the esterase comprises a single substitution at a position corresponding to a residue selected from: Q95, V172, S184, R205, N213 and S251, preferably the single substitution is selected from Q95G / P, V172I, S184E, R205C, N213M / D and S251C.More preferably, the esterase comprises a single substitution at a position corresponding to a residue selected from Q95, V172, S184 and N213, preferably, the single substitution is selected from Q95G / P, V172I, S184E or N213M / D. Alternatively, the esterase has an amino acid sequence as shown in SEQ ID N°1, having a single amino acid substitution at a position corresponding to a residue selected from G136 or T169, preferably, the single substitution is selected from G136A and T169Q.

[0059] In an embodiment of the invention, the esterase comprises at least one substitution at a position corresponding to a residue selected from the group consisting of: T12, E13, S14, S15, I16, E17, A18, V19, R20, G62, F63, T64, A65, G66, Q67, E68, S69, I70, A71, W72, P75, R76, D88, T89, R92, L93, D94, Q95, P96, D97, H130, M132, G133, S139, W15 6, T158, R159, V172, T178, 1179, A180, P181, S184, F189, L204, G206, A207, S208, V211, S212, N213, T214, P215, D216, T217, T218, T219, A220, K221, Y222, Q239, F240, L241, P243, A244, P245, D246, D247, F248, A249 or 1250. Preferably, the esterase comprises a substitution at a position corresponding to a residue selected from the group consisting of Q95, V172, S184 and N213. Preferably, the at least one substitution is selected from Q95G / P, V172I, S184E and N213M / D.

[0060] In an embodiment of the invention, the esterase comprises at least one substitution at a position corresponding to a residue selected from R32, A35 or F38.

[0061] In an embodiment of the invention, the esterase comprises at least one substitution at a position corresponding to a residue selected from the group consisting of Q175, I179, R205, G206 or S251. In a specific embodiment, the esterase comprises at least one substitution at position Q175 and exhibits an increased activity compared to the esterase of SEQ ID N°1. In another specific embodiment, the esterase comprises at least one substitution at a position selected from I179 or G206 and exhibits an increased thermostability compared to the esterase of SEQ ID N°1. In one embodiment, the esterase comprises at least a combination of substitutions at positions R205+S251, preferably at least a combination of substitutions R205C+S251C. In a preferred embodiment, the esterase comprises a combination of substitutions R205C+S251C and exhibits an increased thermostability compared to the esterase of SEQ ID N°1.

[0062] In a specific embodiment, the esterase comprises at least one substitution at a position corresponding to a residue selected from the group consisting of: G136 or T169. In particular, the esterase comprises at least one substitution selected from G136A or T169Q.

[0063] In one embodiment, the esterase comprises at least one substitution at a position selected from the group consisting of T64, E68, Q95, V172, N213, P215, G136, S184 or T169. In particular, the esterase comprises at least one substitution at a position selected from the group consisting of T64M, E68T, Q95G / P, V172I, N213D / M, P215D, G136A, S184E or T169Q. Preferably, the esterase comprises at least one substitution at a position selected from the group consisting of T64, E68, Q95, V172, N213 or P215, in particular selected from T64M, E68T, Q95G / P, V172I or N213D / M. More preferably, the esterase comprises at least one substitution at position V172, preferably V172I.

[0064] In a preferred embodiment, the esterase further comprises at least one substitution at position L210, more preferably selected from L210T / A / R / W / H / F, even more preferably L210T. In one embodiment, the esterase further comprises the substitution L210F. In particular, the esterase of the present invention comprises at least one combination of substitutions at positions selected from L210+R205+S251, preferably selected from L210T / A / R / W / H / F+R205C+S251C, more preferably L210T+R205C+S251C. In one embodiment, the esterase comprises an amino acid sequence consisting of the amino acid sequence shown in SEQ ID N°1, having a combination of substitutions L210T+R205C+S251C.

[0065] In one embodiment, the esterase comprises at least one substitution at position V172 and at least one substitution at a position selected from the group consisting of: Q95, N213, G136, T169, S184 or L210. Preferably, the esterase comprises at least V172I substitution and at least one substitution selected from the group consisting of: Q95G / P, N213D / M, G136A, T169Q, S184E or L210T / A / R / W / H / F.

[0066] In particular, the esterase comprises at least one combination of substitutions at positions L210+V172, preferably at least one combination of substitutions selected from L210T / A / R / W / H / F+V172I, more preferably L210T+V172I.

[0067] In a specific embodiment, the esterase comprises at least a combination of substitutions selected from L210T / A / R / W / H / F+V172I and at least one substitution at T64, E68, Q95, N213, P215, G136, T169 or S184, preferably selected from T64M, E68T, Q95G / P, N213D / M, G136A, T169Q, S184E.

[0068] In one embodiment, the esterase comprises at least one combination of substitutions selected from L210+V172+Q95, L210+V172+Q95+S184, L210+V172+N213 or L210+V172+Q95+G136+T169+S184. In particular, the esterase comprises at least one combination of substitutions selected from L210T+V172I+Q95G, L210T+V172I+Q95G+S184E, L210T+V172I+N213M or L210T+V172I+Q95G+G136A+T169Q+S184E. In a specific embodiment, the esterase comprises a combination of substitutions selected from L210T+V172I+Q95G, L210T+V172I+Q95G+S184E or L210T+V172I+N213M, and exhibits increased degradation activity and increased thermal stability compared to the esterase of SEQ ID N°1.

[0069] In another specific embodiment, the esterase further comprises at least one substitution at a position selected from A2, D233 or S255. In particular, the esterase further comprises at least one substitution selected from A2E, D233N or S255A.

[0070] In one embodiment, the esterase further comprises a combination of substitutions A2E+L210F+D233N+S255A. Preferably, the esterase comprises a combination of substitutions R205C+S251C+A2E+L210F+D233N+S255A. In one embodiment, the esterase comprises a combination of substitutions R205C+S251C+A2E+L210T / A / R / W / H+D233N+S255A. In one embodiment, the esterase comprises an amino acid sequence consisting of the amino acid sequence as shown in SEQ ID N°1, having a combination of substitutions R205C+S251C+A2E+L210T / A / R / W / H+D233N+S255A.

[0071] In one embodiment, the esterase comprises at least one combination of substitutions selected from L210+V172+Q95+A2+D233+S255, L210+V172+Q95+S184+A2+D233+S255, L210+V172+N213+A2+D233+S255, or L210+V172+Q95+G136+T169+S184+A2+D233+S255. In particular, the esterase comprises at least one combination of substitutions selected from L210T+V172I+Q95G+A2E+D233N+S255A, L210T+V172I+Q95G+S184E+A2E+D233N+S255A, L210T+V172I+N213M+A2E+D233N+S255A or L210T+V172I+Q95G+G136A+T169Q+S184E+A2E+D233N+S255A.

[0072] In a preferred embodiment, esterase of the present invention comprises at least one amino acid residue selected from S131, D177 and H209 as in the parent esterase, i.e. esterase of the present invention is not modified at one, two or all of these positions. Preferably, the esterase comprises the combination S131+D177+H209 as in the parent esterase. Alternatively, or in addition, the esterase comprises at least one amino acid residue selected from C242 or C257 as in the parent esterase. Preferably, the esterase comprises the combination C242+C257 as in the parent esterase. More preferably, the esterase comprises the combination S131+D177+H209+C242+C257 as in the parent esterase. In one embodiment, the esterase further comprises the amino acid residue P215 as in the parent esterase.

[0073] Another object of the present invention is to provide an esterase which (i) has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the full-length amino acid sequence as shown in SEQ ID N°1, (ii) contains at least one substitution at position L210, wherein the positions are numbered by reference to the amino acid sequence as shown in SEQ ID N°1, and wherein the substitution is different from L210F. In one embodiment, the esterase comprises at least one substitution in L210A / C / D / E / G / H / I / K / M / N / P / Q / R / S / T / V / W / Y. In one embodiment, the esterase comprises at least one substitution in L210A / C / D / E / G / H / K / M / N / P / Q / R / S / T / V / W / Y. In a preferred embodiment, the esterase comprises at least one substitution in L210T / A / R / W / H. Preferably, the esterase comprises at least the substitution L210T.

[0074] In particular, the esterase comprises a substitution in L210T / A / R and exhibits an increased degradation activity compared to the esterase of SEQ ID N° 1. In another specific embodiment, the esterase comprises a substitution in L210T / W / H and exhibits an increased thermostability compared to the esterase of SEQ ID N° 1. Preferably, the esterase comprises the substitution L210T and exhibits an increased degradation activity and an increased thermostability compared to the esterase of SEQ ID N° 1.

[0075] In a preferred embodiment, esterase of the present invention comprises at least one amino acid residue selected from S131, D177 and H209 as in the parent esterase, i.e. esterase of the present invention is not modified at one, two or all of these positions. Preferably, the esterase comprises the combination S131+D177+H209 as in the parent esterase. Alternatively, or additionally, the esterase comprises at least one amino acid residue selected from C242 or C257 as in the parent esterase. Preferably, the esterase comprises the combination C242+C257 as in the parent esterase. More preferably, the esterase comprises the combination S131+D177+H209+C242+C257 as in the parent esterase. In one embodiment, the esterase further comprises the amino acid residue P215 as in the parent esterase.

[0076] In a specific embodiment, the esterase of the invention further comprises at least one amino acid substitution at a position selected from the group consisting of: T12, E13, S14, S15, I16, E17, A18, V19, R20, R32, A35, F38, G62, F63, T64, A65, G66, Q67, E68, S69, I70, A71, W72, P75, R76, D88, T89, R92, L93, D94, Q95, P96, D97, H130, M132, G133, S139, W156, T158 , R159, V172, Q175, T178, I179, A180, P181, S184, F189, L204, R205, G206, A207, S208, V211, S212, N213, T214, P215, D216, T217, T218, T219, A220, K221, Y222, Q239, F240, L241, P243, A244, P245, D246, D247, F248, A249, I250, S251, G136, or T169. In particular, the esterase of the present invention further comprises at least one amino acid substitution at a position selected from the group consisting of: T12, E13, S14, S15, I16, E17, A18, V19, R20, R32, A35, F38, G62, F63, T64, A65, G66, Q67, E68, S69, I70, A71, W72, P75, R76, D88, T89, R92, L93, D94, Q95, P96, D97, H130, M132, G133, S139, W156, T157, 58, R159, V172, Q175, T178, I179, A180, P181, S184, F189, L204, R205, G206, A207, S208, V211, S212, N213, T214, P215, D216, T217, T218, T219, A220, K221, Y222, Q239, F240, L241, P243, A244, P245, D246, D247, F248, A249, I250, or S251.

[0077] In particular, the esterase further comprises at least one substitution at a position corresponding to a residue selected from R32, A35 or F38. Alternatively, or in addition, the esterase further comprises at least one substitution at a position corresponding to a residue selected from Q175, I179, R205, G206 or S251. In particular, the esterase comprises at least one substitution at position Q175 and shows an increased degradation activity compared to the esterase of SEQ ID N°1, and / or the esterase comprises at least one substitution at a position selected from I179 or G206 and shows an increased thermostability compared to the esterase of SEQ ID N°1. Preferably, the esterase further comprises a combination of substitutions at positions R205+S251, preferably R205C+S251C, and shows an increased thermostability compared to the esterase of SEQ ID N°1.

[0078] In particular, the esterase of the present invention further comprises at least one amino acid substitution at a position selected from G136 or T169. Preferably, the esterase further comprises at least one amino acid substitution selected from G136A or T169Q.

[0079] In one embodiment, the esterase further comprises at least one substitution at a position selected from the group consisting of: T64, E68, Q95, V172, N213, P215, G136, S184 or T169. Preferably, the esterase comprises at least one substitution selected from the group consisting of: T64M, E68T, Q95G / P, V172I, N213D / M, P215D, G136A, S184E or T169Q. In particular, the esterase further comprises at least one substitution at a position selected from the group consisting of: T64, E68, Q95, V172, N213 or P215, preferably selected from the group consisting of: T64M, E68T, Q95G / P, V172I or N213D / M.

[0080] In one embodiment, the esterase comprises at least one substitution selected from L210T / A / R / W / H and at least one substitution at position V172, preferably V172I. Preferably, the esterase comprises at least one substitution combination selected from L210T / A / R / W / H+V172I, more preferably L210T+V172I.

[0081] In a specific embodiment, the esterase comprises at least one combination of substitutions selected from L210T / A / R / W / H+V172I and at least one substitution at a position selected from the following positions: Q95, N213, G136, T169 or S184, preferably selected from Q95G / P, N213D / M, G136A, T169Q or S184E.

[0082] In one embodiment, the esterase comprises at least one substitution selected from L210T / A / R / W / H and a combination of at least one substitution at positions V172+Q95, V172+Q95+S184, V172+N213 or V172+Q95+G136+T169+S184. In particular, the esterase comprises at least one substitution selected from the group consisting of L210T+V172I+Q95G, L210T+V172I+Q95G+S184E, L210T+V172I+N213M or L210T+V172I+Q95G+G136A+T169Q+S184E.

[0083] In one embodiment, the esterase further comprises at least one substitution at a position selected from A2, D233 or S255. Preferably, the esterase comprises at least one substitution selected from A2E, D233N or S255A, preferably a combination of substitutions A2E+D233N+S255A.

[0084] In particular, the esterase comprises at least one combination of substitutions selected from the group consisting of L210T+V172I+Q95G+A2E+D233N+S255A, L210T+V172I+Q95G+S184E+A2E+D233N+S255A, L210T+V172I+N213M+A2E+D233N+S255A or L210T+V172I+Q95G+G136A+T169Q+S184E+A2E+D233N+S255A.

[0085] An object of the present invention is to provide an esterase which (i) has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the full-length amino acid sequence as shown in SEQ ID N°1, (ii) contains at least one amino acid substitution selected from the group consisting of L210A / C / D / E / G / H / I / K / M / N / P / Q / R / S / T / V / W / Y, R205C, S251C, Q95G / P, G136A, T169Q, V172I, S184E or N213M / D, wherein the positions are numbered by reference to the amino acid sequence as shown in SEQ ID N°1, and (iii) has increased degradation activity and / or increased thermal stability compared to the esterase of SEQ ID N°1.

[0086] In one embodiment, the esterase comprises at least one amino acid substitution selected from L210A / C / D / E / G / H / I / K / M / N / P / Q / R / S / T / V / W / Y, R205C or S251C, preferably selected from L210A / C / D / E / G / H / K / M / N / P / Q / R / S / T / V / W / Y, R205C or S251C. In a preferred embodiment, the esterase comprises at least one amino acid substitution selected from L210T / A / R / W / H, R205C or S251C.

[0087] In one embodiment the esterase comprises at least one amino acid substitution selected from Q95G / P, G136A, T169Q, V172I, S184E or N213M / D, preferably selected from Q95G, V172I, S184E or N213M.

[0088] In one embodiment, the esterase comprises at least one amino acid substitution selected from the group consisting of L210T / A / R / W / H, R205C, S251C, Q95G / P, G136A, T169Q, V172I, S184E or N213M / D. Preferably, the esterase of the invention comprises at least one substitution or combination of substitutions selected from the group consisting of L210T / A / R / W / H, R205C+S251C, Q95G, G136A, T169Q, V172I, S184E or N213M, preferably selected from the group consisting of L210T / A / R / W / H and R205C+S251C, more preferably selected from the group consisting of L210T and R205C+S251C. In particular, the esterase of the invention comprises at least the combination of substitutions L210T / A / R / W / H+R205C+S251C. Preferably, the esterase of the invention comprises the combination L210T+R205C+S251C.

[0089] In a preferred embodiment, the esterase of the present invention comprises at least one amino acid residue selected from S131, D177 and H209 as in the parent esterase, i.e. the esterase of the present invention is not modified at one, two or all of these positions. Preferably, the esterase comprises the combination S131+D177+H209 as in the parent esterase. Alternatively, or in addition, the esterase comprises at least one amino acid residue selected from C242 or C257 as in the parent esterase. Preferably, the esterase comprises the combination C242+C257 as in the parent esterase. More preferably, the esterase comprises the combination S131+D177+H209+C242+C257 as in the parent esterase.

[0090] In one embodiment, the esterase further comprises amino acid residue P215 as in the parent esterase.

[0091] In a specific embodiment, the esterase of the invention further comprises at least one amino acid substitution at a position selected from the group consisting of: T12, E13, S14, S15, I16, E17, A18, V19, R20, R32, A35, F38, G62, F63, T64, A65, G66, Q67, E68, S69, I70, A71, W72, P75, R76, D88, T89, R92, L93, D94, P96, D97, H130, M132, G133, S139 , W156, T158, R159, Q175, T178, I179, A180, P181, F189, L204, G206, A207, S208, V211, S212, T214, P215, D216, T217, T218, T219, A220, K221, Y222, Q239, F240, L241, P243, A244, P245, D246, D247, F248, A249, I250, G136, or T169. In particular, the esterase of the present invention further comprises at least one amino acid substitution at a position selected from the group consisting of: T12, E13, S14, S15, I16, E17, A18, V19, R20, R32, A35, F38, G62, F63, T64, A65, G66, Q67, E68, S69, I70, A71, W72, P75, R76, D88, T89, R92, L93, D94, Q95, P96, D97, H130, M132, G133, S139, W140, 56, T158, R159, V172, Q175, T178, I179, A180, P181, S184, F189, L204, G206, A207, S208, V211, S212, N213, T214, P215, D216, T217, T218, T219, A220, K221, Y222, Q239, F240, L241, P243, A244, P245, D246, D247, F248, A249, or I250.

[0092] In particular, the esterase comprises at least one substitution at a position corresponding to a residue selected from R32, A35 or F38. Alternatively or additionally, the esterase comprises at least one substitution at a position corresponding to a residue selected from Q175, I179 or G206. In particular, the esterase comprises at least one substitution at position Q175 and exhibits increased activity compared to the esterase of SEQ ID N°1, and / or the esterase comprises at least one substitution at a position selected from I179 or G206 and exhibits increased thermostability compared to the esterase of SEQ ID N°1.

[0093] In one embodiment, the esterase further comprises at least one substitution at a position selected from the group consisting of T64, E68, Q95, V172, N213, P215, G136, S184 or T169, preferably selected from the group consisting of T64, E68, Q95, V172, N213 or P215. In particular, the esterase comprises at least one substitution selected from the group consisting of T64M, E68T, Q95G / P, V172I, N213D / M, P215D, G136A, S184E or T169Q, preferably selected from the group consisting of T64M, E68T, Q95G / P, V172I or N213D / M, more preferably selected from the group consisting of T64M, E68T, Q95G, V172I or N213M.

[0094] In one embodiment, the esterase comprises at least two substitutions selected from the group consisting of L210A / C / D / E / G / H / I / K / M / N / P / Q / R / S / T / V / W / Y, R205C, S251C, Q95G / P, G136A, T169Q, V172I, S184E or N213M / D, preferably selected from the group consisting of L210T / A / R / W / H, R205C, S251C, Q95G, G136A, T169Q, V172I, S184E or N213M. Preferably, the esterase comprises at least two substitutions or a combination of substitutions selected from the group consisting of L210T / A / R / W / H, R205C+S251C, Q95G, G136A, T169Q, V172I, S184E or N213M.

[0095] In one embodiment, the esterase comprises at least one substitution selected from L210T / A / R / W / H and at least one substitution at position V172, preferably V172I. Preferably, the esterase comprises at least one combination of substitutions selected from L210T / A / R / W / H+V172I, more preferably L210T+V172I.

[0096] In a specific embodiment, the esterase comprises at least one combination of substitutions selected from L210T / A / R / W / H+V172I and at least one substitution at a position selected from the following positions: Q95, N213, G136, T169 or S184, preferably selected from Q95G / P, N213D / M, G136A, T169Q or S184E.

[0097] In one embodiment, the esterase comprises a combination of at least one substitution selected from L210T / A / R / W / H and at least one substitution at positions V172+Q95, V172+Q95+S184, V172+N213 or V172+Q95+G136+T169+S184.

[0098] In one embodiment, the esterase comprises at least one combination of substitutions selected from the group consisting of R205C+S251C, L210T+V172I+Q95G, L210T+V172I+Q95G+S184E, L210T+V172I+N213M, or L210T+V172I+Q95G+G136A+T169Q+S184E. In one embodiment, the esterase comprises a combination of substitutions selected from the group consisting of L210T+V172I+Q95G, L210T+V172I+Q95G+S184E, or L210T+V172I+N213M, and exhibits increased degradation activity and increased thermal stability compared to the esterase of SEQ ID N°1.

[0099] In one embodiment, the esterase further comprises at least one substitution at a position selected from A2, D233 or S255. Preferably, the esterase comprises at least one substitution selected from A2E, D233N or S255A, preferably a combination of substitutions A2E+D233N+S255A.

[0100] In particular, the esterase comprises at least one substitution combination selected from the following: L210T+V172I+Q95G+A2E+D233N+S255A, L210T+V172I+Q95G+S184E+A2E+D233N+S255A, L210T+V172I+N213M+A2E+D233N+S255A or L210T+V172I+Q95G+G136A+T169Q+S184E+A2E+D233N+S255A.

[0101] Polyester degradation activity of variants

[0102] It is an object of the present invention to provide novel enzymes having esterase activity.In a particular embodiment, the enzyme of the present invention exhibits cutinase activity.

[0103] In a specific embodiment, the esterase of the present invention has polyester degradation activity, preferably polyethylene terephthalate (PET) degradation activity, and / or polybutylene adipate terephthalate (PBAT) degradation activity, and / or polycaprolactone (PCL) degradation activity, and / or polybutylene succinate (PBS) activity, more preferably polyethylene terephthalate (PET) degradation activity, and / or polybutylene adipate terephthalate (PBAT) degradation activity. Even more preferably, the esterase of the present invention has polyethylene terephthalate (PET) degradation activity.

[0104] Advantageously, the esterase of the present invention exhibits polyester degradation activity at least in the temperature range of 20°C-90°C, preferably 40°C-80°C, more preferably 60°C-70°C. In a specific embodiment, the esterase exhibits polyester degradation activity at 60°C. In a specific embodiment, the esterase exhibits polyester degradation activity at 70°C. In a specific embodiment, the polyester degradation activity is still measurable at a temperature of 70°C-80°C.

[0105] In a specific embodiment, the esterase of the invention has an increased polyester degradation activity at a given temperature, more particularly at a temperature of 40°C-80°C, more preferably at a temperature of 50°C-70°C, compared to the esterase of SEQ ID N°1.

[0106] In a specific embodiment, the polyester degradation activity of the esterase at 65°C is at least 5%, preferably at least 10%, 20%, 50%, 100% or more higher than the polyester degradation activity of the esterase of SEQ ID N°1.

[0107] In a specific embodiment, the esterase of the present invention shows measurable esterase activity at least in the range of pH 5-11, preferably in the range of pH 6-9, more preferably in the range of pH 6.5-9, even more preferably in the range of pH 6.5-8.

[0108] Nucleic acid, expression cassette, vector, host cell

[0109] It is a further object of the present invention to provide a nucleic acid encoding an esterase as defined above.

[0110] As used herein, the terms "nucleic acid", "nucleic acid sequence", "polynucleotide", "oligonucleotide" and "nucleotide sequence" refer to sequences of deoxyribonucleic acid and / or ribonucleic acid. The nucleic acid may be DNA (cDNA or gDNA), RNA or a mixture thereof. It may be in single-stranded form or in duplex form or a mixture thereof. It may be of recombinant, artificial and / or synthetic origin, and it may contain modified nucleotides, which contain, for example, modified bonds, modified purine or pyrimidine bases or modified sugars. The nucleic acid of the present invention may be in an isolated or purified form and prepared, isolated and / or manipulated by techniques known per se in the art, such as cloning and expression of cDNA libraries, amplification, enzymatic synthesis or recombinant techniques. The nucleic acid may also be synthesized in vitro by well-known chemical synthesis techniques, as described, for example, in Belousov (1997) Nucleic Acids Res. 25: 3440-3444.

[0111] The present invention also encompasses nucleic acids that hybridize to nucleic acids encoding esterases as defined above under stringent conditions. Preferably, such stringent conditions include incubating the hybridization filter in 2XSSC / 0.1%SDS at about 42°C for about 2.5 hours, followed by washing the filter four times in 1XSSC / 0.1%SDS at 65°C for 15 minutes each. The protocols used are described in references such as 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)).

[0112] The present invention also encompasses nucleic acids encoding the esterases of the present invention, wherein the sequence of said nucleic acid, or at least a portion of said sequence, has been engineered with optimized codon usage.

[0113] Alternatively, nucleic acid according to the present invention can be derived from the sequence of an esterase according to the present invention, and the usage of codons can be adjusted according to the host cell in which the nucleic acid is transcribed. These steps can be carried out according to methods well known to those skilled in the art, some of which are described in the reference manual Sambrook etc. (Sambrook etc., 2001).

[0114] The nucleic acid of the present invention may also contain other nucleotide sequences, such as regulatory regions, ie, promoters, enhancers, silencers, terminators, signal peptides, etc., which can be used to cause or regulate the expression of the polypeptide in a selected host cell or system.

[0115] The present invention also relates to an expression cassette comprising a nucleic acid according to the present invention operably linked to one or more control sequences directing the expression of said nucleic acid in a suitable host cell.

[0116] The term "expression" as used herein refers to any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0117] The term "expression cassette" refers to a nucleic acid construct comprising an operably linked coding region (ie, a nucleic acid of the invention) and regulatory regions (ie, comprising one or more control sequences).

[0118] Typically, the expression cassette comprises or consists of a nucleic acid according to the present invention that is operably connected to a control sequence (such as a transcription promoter and / or a transcription terminator). The control sequence may include a promoter recognized by a host cell or an in vitro expression system for the expression of a nucleic acid encoding an esterase according to the present invention. The promoter contains a transcription control sequence that mediates enzyme expression. The promoter may be any polynucleotide that shows transcriptional activity in a host cell, including mutant, truncated and hybrid promoters, and may be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to the host cell. The control sequence may also be a transcription terminator that is recognized by the host cell to terminate transcription. The terminator is operably connected to the 3'-end of the nucleic acid encoding the esterase. Any terminator that is functional in a host cell may be used for the present invention. Typically, the expression cassette comprises or consists of a nucleic acid according to the present invention that is operably connected to a transcription promoter and a transcription terminator.

[0119] The invention also relates to a vector comprising the nucleic acid or the expression cassette as defined above.

[0120] The term "vector" or "expression vector" as used herein refers to a DNA or RNA molecule comprising an expression cassette of the present invention, which is used as a vector for transferring recombinant genetic material into a host cell. The main types of vectors are plasmids, bacteriophages, viruses, cosmids and artificial chromosomes. The vector itself is usually a DNA sequence consisting of an insert (heterologous nucleic acid sequence, transgene) and a larger sequence that serves as the "skeleton" of the vector. The purpose of the vector for transferring genetic information to the host is typically to separate, propagate or express the insert in the target cell. The vector referred to as an expression vector (expression construct) is particularly suitable for expressing heterologous sequences in target cells and usually has a promoter sequence that drives the expression of heterologous sequences encoding polypeptides. Generally, the regulatory elements present in the expression vector include a transcriptional promoter, a ribosome binding site, a terminator and an optional operator. Preferably, the expression vector also contains a replication origin for autonomous replication in the host cell, a selection marker, a limited number of useful restriction enzyme sites and the potential for high copy number. Examples of expression vectors are cloning vectors, modified cloning vectors, specially designed plasmids and viruses. Expression vectors that provide suitable levels of polypeptide expression in different hosts are well known in the art. The choice of vector typically depends on the compatibility of the vector with the host cell into which the vector is to be introduced.Preferably, the expression vector is a linear or circular double-stranded DNA molecule.

[0121] Another object of the present invention is to provide a host cell comprising the above-mentioned nucleic acid, expression cassette or vector. Therefore, the present invention relates to the use of nucleic acid, expression cassette or vector according to the present invention for transforming, transfecting or transducing a host cell. The selection of the vector typically depends on the compatibility of the vector with the host cell to be introduced.

[0122] According to the present invention, host cells can be transformed, transfected or transduced in a transient or stable manner. The expression cassette or vector of the present invention is introduced into the host cell so that the expression cassette or vector is maintained as a chromosomal integrant or as a self-replicating extrachromosomal vector. The term "host cell" also encompasses any offspring of a parent host cell that is different from the parent host cell due to mutations during replication. The host cell can be any cell that can be used to produce variants of the present invention, such as a prokaryote or a eukaryote. The prokaryotic host cell can be any gram-positive bacteria or gram-negative bacteria. The host cell can also be a eukaryotic cell, such as a yeast, fungus, mammal, insect or plant cell. In a specific embodiment, the host cell is selected from the group consisting of Escherichia coli, Bacillus, Streptomyces, Trichoderma, Aspergillus, Saccharomyces, Pichia, Vibrio or Yarrowia.

[0123] The nucleic acid, expression cassette or expression vector according to the invention can be introduced into the host cell by any method known to those skilled in the art, such as electroporation, conjugation, transduction, transformation of competent cells, protoplast transformation, protoplast fusion, biolistic "gene gun" transformation, PEG-mediated transformation, lipid-assisted transformation or transfection, chemical-mediated transfection, lithium acetate-mediated transformation, liposome-mediated transformation.

[0124] Optionally, more than one copy of a nucleic acid, cassette or vector of the invention can be inserted into the host cell to increase production of the variant.

[0125] In a specific embodiment, the host cell is a recombinant microorganism. The present invention does allow engineering of microorganisms with improved ability to degrade polyester-containing materials. For example, the sequences of the present invention can be used to supplement wild-type strains of fungi or bacteria known to be able to degrade polyester to improve and / or increase strain capabilities.

[0126] Esterase production

[0127] Another object of the present invention is to provide a method for producing the esterase of the present invention, comprising expressing a nucleic acid encoding the esterase and optionally recovering the esterase.

[0128] In particular, the present invention relates to an in vitro method for producing an esterase of the present invention, comprising (a) contacting a nucleic acid, a cassette or a vector of the present invention with an in vitro expression system; and (b) recovering the produced esterase. In vitro expression systems are well known to those skilled in the art and are commercially available.

[0129] Preferably, the production method comprises:

[0130] (a) culturing a host cell comprising a nucleic acid encoding an esterase of the invention under conditions suitable for expression of said nucleic acid; and optionally

[0131] (b) recovering the esterase from the cell culture.

[0132] Advantageously, the host cell is a recombinant Bacillus, a recombinant Escherichia coli, a recombinant Aspergillus, a recombinant Trichoderma, a recombinant Streptomyces, a recombinant Saccharomyces, a recombinant Pichia pastoris, a recombinant Vibrio, or a recombinant Yarrowia.

[0133] Host cells are cultured in a nutrient medium suitable for producing polypeptides using methods known in the art. For example, cells may be cultured by shaking flasks, or by small-scale or large-scale fermentations (including continuous, batch, fed-batch or solid-state fermentations) in suitable medium and under conditions that allow enzyme expression and / or separation in a laboratory or industrial fermentor. Cultivation is carried out in a suitable nutrient medium, which is from a commercial supplier or prepared according to a disclosed composition (e.g., in the catalog of the American Type Culture Collection).

[0134] If the esterase is secreted into the nutrient medium, the esterase can be directly recovered from the culture supernatant. Conversely, the esterase can be recovered from cell lysates or after osmosis. The esterase can be recovered using any method known in the art. For example, the esterase can be recovered from the nutrient medium by conventional methods, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation or precipitation. Optionally, the esterase can be partially or completely purified by a variety of methods known in the art, including but not limited to chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing and size exclusion), electrophoretic methods (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE or extraction to obtain substantially pure polypeptides.

[0135] Esterase can be used as is in purified form, alone or in combination with other enzymes, to catalyze the enzymatic reactions involved in the degradation and / or recovery of polyester and / or polyester-containing materials (such as polyester-containing plastics). The esterase can be in soluble form, or on a solid phase. In particular, it can be bound to a cell membrane or lipid vesicle, or to a synthetic support (such as glass, plastic, polymer, filter, membrane), for example in the form of microbeads, columns, plates, etc.

[0136] Composition

[0137] Another object of the present invention is to provide a composition comprising the esterase or host cell of the present invention or an extract thereof.In the context of the present invention, the term "composition" encompasses any kind of composition comprising the esterase or host cell of the present invention.

[0138] Based on the total weight of the composition, the composition of the present invention may comprise 0.1%-99.9%, preferably 0.1%-50%, more preferably 0.1%-30%, even more preferably 0.1%-5% by weight of esterase. Alternatively, the composition may comprise 5-10% by weight of the esterase of the present invention.

[0139] The composition may be liquid or dry, such as in powder form. In some embodiments, the composition is a lyophilisate.

[0140] The composition may further comprise excipients and / or reagents, etc. Suitable excipients include buffers commonly used in biochemistry, reagents for adjusting pH, preservatives (such as sodium benzoate, sodium sorbate or sodium ascorbate), retainers, protective agents or stabilizers (such as starch), dextrin, gum arabic, salts, sugars (such as sorbitol, trehalose or lactose), glycerol, polyethylene glycol, polypropylene glycol, propylene glycol, chelating agents (such as EDTA), reducing agents, amino acids, carriers (such as solvents or aqueous solutions), etc. The composition of the present invention can be obtained by mixing the esterase with one or more excipients.

[0141] In a specific embodiment, the composition comprises 0.1%-99.9%, preferably 50%-99.9%, more preferably 70%-99.9%, even more preferably 95%-99.9% by weight of excipients based on the total weight of the composition. Alternatively, the composition may comprise 90%-95% by weight of excipients.

[0142] In a specific embodiment, the composition may further comprise other polypeptides showing enzymatic activity.The amount of the esterase of the invention will be easily adjusted by a person skilled in the art according to, for example, the nature of the polyester to be degraded and / or other enzymes / polypeptides comprised in the composition.

[0143] In a specific embodiment, the esterase of the present invention is dissolved in an aqueous medium together with one or more excipients, especially excipients that can stabilize or protect the polypeptide from degradation. For example, the esterase of the present invention can be dissolved in water, and other components such as glycerol, sorbitol, dextrin, starch, glycols (such as propylene glycol), salts, etc. are finally added. Then, the resulting mixture can be dried to obtain a powder. The method of drying this mixture is well known to those skilled in the art, including but not limited to freeze drying, freeze drying, spray drying, supercritical drying, down-draft evaporation, thin layer evaporation, centrifugal evaporation, conveyor drying, fluidized bed drying, drum drying or any combination thereof.

[0144] In a particular embodiment, the composition is in powder form and comprises an esterase and a stabilizing / solubilizing amount of glycerol, sorbitol or a dextrin (such as maltodextrin and / or cyclodextrin), starch, a glycol (such as propylene glycol) and / or a salt.

[0145] In a specific embodiment, the composition of the invention comprises at least one recombinant cell expressing an esterase of the invention or an extract thereof. "Cell extract" refers to any fraction obtained from a cell, such as a cell supernatant, cell debris, cell wall, DNA extract, enzyme or enzyme preparation, or any preparation obtained from a cell by chemical, physical and / or enzymatic treatment, which is substantially free of living cells. Preferred extracts are enzyme active extracts. The composition of the invention may comprise one or more recombinant cells of the invention or an extract thereof, and optionally, one or more other cells.

[0146] In one embodiment, the composition consists of or comprises the culture medium of a recombinant microorganism expressing and secreting the esterase of the invention. In a specific embodiment, the composition comprises such a lyophilized culture medium.

[0147] Uses of esterase

[0148] Another object of the present invention is to provide a method for degrading and / or recovering polyester or polyester-containing materials under aerobic or anaerobic conditions using the esterase of the present invention. The esterase of the present invention is particularly suitable for degrading PET and PET-containing materials.

[0149] Therefore, one object of the present invention is to use the esterase of the present invention, or the corresponding recombinant cells or extracts, or compositions thereof, for the enzymatic degradation of polyesters.

[0150] In a specific embodiment, the polyester targeted by the esterase is selected from polyethylene terephthalate (PET), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furandicarboxylate (PEF), polycaprolactone (PCL), polyethylene adipate (PEA), polyethylene naphthalate (PEN) and blends / mixtures of these materials, preferably polyethylene terephthalate.

[0151] In a preferred embodiment, the polyester is PET, and at least monomers (e.g., monoethylene glycol or terephthalic acid) and / or oligomers (e.g., methyl-2-hydroxyethyl terephthalate (MHET), bis(2-hydroxyethyl terephthalate) (BHET), 1-(2-hydroxyethyl)-4-methyl terephthalate (HEMT) and dimethyl terephthalate (DMT)) are recovered.

[0152] Another object of the present invention is to use the esterase of the present invention, or the corresponding recombinant cell or extract thereof, or the composition thereof, for enzymatic degradation of at least one polyester in a polyester-containing material.

[0153] Another object of the present invention is to provide a method for degrading at least one polyester in a polyester-containing material, wherein the polyester-containing material is contacted with the esterase or host cell or extract or composition thereof of the present invention, thereby degrading at least one polyester in the polyester-containing material.

[0154] Advantageously, the polyester is depolymerized into monomers and / or oligomers.

[0155] In particular, the present invention provides a method for degrading PET of a PET-containing material, wherein the PET-containing material is contacted with an esterase or a host cell or a composition of the present invention, thereby degrading PET.

[0156] In one embodiment, at least one polyester is degraded into repolymerizable monomers and / or oligomers, which can be advantageously recovered for reuse. The monomers / oligomers obtained can be used for recycling (e.g., repolymerizing the polyester) or methanation. In a specific embodiment, at least one polyester is PET, and monoethylene glycol, terephthalic acid, methyl-2-hydroxyethyl terephthalate (MHET), bis(2-hydroxyethyl terephthalate) (BHET), 1-(2-hydroxyethyl)-4-methyl terephthalate (HEMT) and / or dimethyl terephthalate (DMT) are recovered.

[0157] In one embodiment, the polyester of the polyester-containing material is completely degraded.

[0158] The time required for degradation of the polyester-containing material may vary depending on the polyester-containing material itself (i.e., the nature and origin of the polyester-containing material, composition, shape, etc.), the type and amount of esterase used, and various process parameters (i.e., temperature, pH, other reagents, etc.). A person skilled in the art can easily adapt the process parameters to the polyester-containing material and the envisioned degradation time.

[0159] Advantageously, the degradation process is carried out at a temperature of 20°C-90°C, preferably 40°C-80°C, more preferably 60°C-70°C. In a specific embodiment, the degradation process is carried out at 60°C. In another specific embodiment, the degradation process is carried out at 65°C. In another specific embodiment, the degradation process is carried out at 70°C. More generally, the temperature is maintained below the inactivation temperature, which corresponds to the temperature at which the esterase is inactivated (i.e., loses more than 80% of its activity compared to its activity at its optimal temperature) and / or the recombinant microorganism no longer synthesizes the esterase. In particular, the temperature is kept below the glass transition temperature (Tg) of the target polyester.

[0160] Advantageously, the method is performed in a continuous flow process at a temperature at which the esterase can be used several times and / or recycled.

[0161] Advantageously, the degradation process is carried out at a pH of 5-11, preferably at a pH of 6-9, more preferably at a pH of 6.5-9, even more preferably at a pH of 6.5-8.

[0162] In specific embodiments, the polyester-containing material may be pretreated prior to contacting with the esterase in order to physically alter its structure, thereby increasing the contact surface between the polyester and the esterase.

[0163] Another object of the present invention is to provide a method for producing monomers and / or oligomers from a polyester-containing material, comprising exposing the polyester-containing material to an esterase of the present invention, or a corresponding recombinant cell or an extract or composition thereof, and optionally recovering the monomers and / or oligomers.

[0164] The monomers and / or oligomers produced by the 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.

[0165] The process of the present invention is particularly useful for producing monomers selected from monoethylene glycol and terephthalic acid, and / or oligomers selected from methyl-2-hydroxyethyl terephthalate (MHET), bis(2-hydroxyethyl)terephthalate (BHET), 1-(2-hydroxyethyl)-4-methyl terephthalate (HEMT) and dimethyl terephthalate (DMT) from PET and / or plastic articles comprising PET.

[0166] The recovered monomers and / or oligomers can be further purified and adjusted in a repolymerizable form using all suitable purification methods. Examples of purification methods include combined or uncombined stripping processes, aqueous separation, steam selective condensation, filtration and concentration of post-bioprocess media, separation, distillation, vacuum evaporation, extraction, electrodialysis, adsorption, ion exchange, precipitation, crystallization, concentration and acid dehydration and precipitation, nanofiltration, acid catalyst treatment, semi-continuous mode distillation or continuous mode distillation, solvent extraction, evaporative concentration, evaporative crystallization, liquid / liquid extraction, hydrogenation, azeotropic distillation process, adsorption, column chromatography, simple vacuum distillation and microfiltration.

[0167] The recovered repolymerizable monomers and / or oligomers can be reused, for example, in the synthesis of polyesters. Advantageously, polyesters of the same nature are repolymerized. However, the recovered monomers and / or oligomers can be mixed with other monomers and / or oligomers, for example, in order to synthesize new copolymers. Alternatively, the recovered monomers can be used as chemical intermediates to produce new target compounds.

[0168] The present invention also relates to a method for surface hydrolysis or surface functionalization of a polyester-containing material, comprising exposing the polyester-containing material to an esterase of the present invention, or a corresponding recombinant cell or extract thereof, or a composition. The method of the present invention is particularly suitable for increasing the hydrophilicity or water absorption of the polyester material. This increased hydrophilicity has particular significance in textile production, electronics and biomedical applications.

[0169] Another object of the present invention is to provide a polyester-containing material comprising an esterase of the present invention and / or a recombinant microorganism expressing and secreting the esterase. For example, a method for preparing such a polyester-containing material comprising an esterase of the present invention is disclosed in patent applications WO2013 / 093355, WO2016 / 198650, WO2016 / 198652, WO2019 / 043145 and WO2019 / 043134.

[0170] Therefore, an object of the present invention is to provide a polyester-containing material containing an esterase and / or a recombinant cell and / or a composition or an extract thereof of the present invention, and at least PET. According to one embodiment, the present invention provides a plastic article comprising PET and an esterase of the present invention having PET degradation activity.

[0171] Therefore, another object of the present invention is to provide a polyester-containing material containing an esterase and / or a recombinant cell and / or a composition thereof or an extract thereof of the present invention, and at least PBAT. According to one embodiment, the present invention provides a plastic product comprising PBAT and an esterase of the present invention having PBAT degradation activity.

[0172] Therefore, another object of the present invention is to provide a polyester-containing material containing the esterase and / or recombinant cell and / or composition thereof or extract thereof of the present invention and at least PBS. According to one embodiment, the present invention provides a plastic article comprising PBS and the esterase of the present invention having PBS degradation activity.

[0173] Therefore, another object of the present invention is to provide a polyester-containing material containing the esterase and / or recombinant cell and / or composition thereof or extract thereof of the present invention and at least PCL. According to one embodiment, the present invention provides a plastic article comprising PCL and an esterase of the present invention having PCL degradation activity.

[0174] Typically, esterases of the present invention can be used in detergents, foods, animal feeds, papermaking, textiles and pharmaceutical applications. More particularly, esterases of the present invention can be used as components of detergent compositions. Detergent compositions include, but are not limited to, hand-wash or machine-wash laundry detergent compositions (such as fabric softener compositions pretreated and laundry additive compositions and rinse-added fabrics suitable for staining), detergent compositions for general household hard surface cleaning operations, detergent compositions for hand-wash or machine-wash dishwashing operations. In a specific embodiment, esterases of the present invention can be used as detergent additives. Therefore, the invention provides detergent compositions comprising esterases of the present invention. In particular, esterases of the present invention can be used as detergent additives to reduce pilling and graying effects in textile cleaning processes.

[0175] The present invention also relates to methods of using the esterase of the present invention in animal feed, and feed compositions and feed additives comprising the esterase of the present invention. The terms "feed" and "feed composition" refer to any compound, preparation, mixture or composition suitable for or intended to be ingested by an animal. In another specific embodiment, the esterase of the present invention is used to hydrolyze proteins and produce a hydrolyzate comprising peptides. Such a hydrolyzate can be used as a feed composition or a feed additive.

[0176] Another object of the present invention is to provide a method of using the esterase of the present invention in the papermaking industry. More particularly, the esterase of the present invention can be used to remove stickies from the pulp and water lines of a paper machine. DETAILED DESCRIPTION

[0177] Example 1 - Construction, expression and purification of esterase

[0178] - Build

[0179] The esterase according to the invention has been produced using the plasmid construct pET26b-220 / 5-His. The plasmid contains the gene for cloning the esterase encoding SEQ ID N°1, which is optimized for E. coli expression between NdeI and XhoI restriction sites. Two site-directed mutagenesis kits were used to produce esterase variants according to the supplier's recommendations: the QuikChange II site-directed mutagenesis kit and the QuikChange Lightning MultiSite-Directed from Agilent (Santa Clara, California, USA).

[0180] - Expression and purification of esterase

[0181] Strain Stellar TM (Clontech, California, USA) and E.coli One BL21DE3 (Lifetechnologies, Carlsbad, California, USA) has been used continuously for cloning and recombinant expression in 50 mL LB-Miller medium or ZYM automatic induction medium (Studier et al., 2005-Prot. Exp. Pur. 41, 207-234). Induction in LB-Miller medium was performed at 16 ° C with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG, Euromedex, Souffelweyersheim, France). Cultivation was stopped by centrifugation (8000 rpm, 20 minutes at 10 ° C) in an Avanti J-26XP centrifuge (Beckman Coulter, Brea, USA). The cells were suspended in 20 mL Talon buffer (Tris-HCl 20 mM, NaCl 300 mM, pH 8). The cell suspension was then sonicated by an FB 705 sonicator (Fisherbrand, Illkirch, France) at 30% amplitude for 2 minutes (2 seconds on and 1 second off cycle). Then, a centrifugation step was achieved: centrifugation in an Eppendorf centrifuge at 11000 rpm, 10°C for 30 minutes. The soluble fraction was collected and subjected to affinity chromatography. Metal affinity resin (Clontech, CA, USA) was used to complete the purification step. The protein elution step was performed with Talon buffer 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.

[0182] Example 2 - Evaluation of the degradation activity of esterase

[0183] The degradation activity of the esterase was determined and compared with the activity of the esterase of SEQ ID N°1.

[0184] Several methods have been used to assess specific activity:

[0185] (1) Specific activity based on PET hydrolysis

[0186] (2) Degradation activity based on solid polyester

[0187] (3) Based on PET hydrolysis activity above 100 mL in the reactor

[0188] 2.1. Specific activity based on PET hydrolysis

[0189] 100 mg of amorphous PET in powder form (prepared according to WO2017 / 198786 to achieve a crystallinity of less than 20%) was weighed and placed in a 100 mL glass bottle. 1 mL of an esterase preparation containing SEQ ID NO1 esterase (as a reference control) prepared at 0.02 mg / mL in Talon buffer (Tris-HCl 20 mM, NaCl 0.3 M, pH 8) or an esterase of the present invention was placed in a glass bottle. Finally, 49 mL of 0.1 M potassium phosphate buffer pH 8 was added.

[0190] Disaggregation was initiated by incubating each glass bottle in a Max Q 4450 incubator (Thermo Fisher Scientific, Inc. Waltham, MA, USA) at 60°C, 65°C, or 70°C and 150 rpm.

[0191] The initial rate of the depolymerization reaction (in mg / hour of equivalent TA produced) was determined by sampling at different times during the first 24 hours and analyzed by ultra-high performance liquid chromatography (UHPLC). If necessary, the samples were diluted in 0.1 M potassium phosphate buffer (pH 8). Then, 150 μL of methanol and 6.5 μL of 6N HCl were added to 150 μL of sample or dilution. After mixing and filtering on a 0.45 μm syringe filter, the samples were loaded on the UHPLC to monitor the release of terephthalic acid (TA), MHET and BHET. The chromatography system used was the Ultimate 3000 UHPLC system (ThermoFisher Scientific. Inc. Waltham, MA, USA), including a pump module, an autosampler, a column oven thermostatted at 25°C and a UV detector at 240 nm. The column used was HS C18 HPLC column (150x4.6mm, 5μm, equipped with a precolumn, Supelco, Bellefonte, USA). TA, MHET and BHET were separated using a gradient of MeOH (30%-90%) in 1mM H2SO4 at 1mL / min. 20μL of sample was injected. TA, MHET and BHET were measured according to a standard curve made from commercially available TA and BHET and in-house synthesized MHET under the same conditions as the samples. The specific activity of PET hydrolysis (mg of equivalent TA / hour / mg of enzyme) was determined in the linear part of the hydrolysis curve of the reaction, which was established by sampling at different times during the first 24 hours. Equivalent TA corresponds to the sum of the measured TA and the TA contained in the measured MHET and BHET.

[0192] 2.2. Activity based on the degradation of polyester in solid form

[0193] 20 μL of enzyme preparation was deposited in the wells produced in the agar plates containing PET. The agar plates were prepared by dissolving 500 mg of PET in hexafluoro-2-propanol (HFIP) and pouring the medium into 250 mL of aqueous solution. After evaporation of HFIP at 52°C and 140 mbar, 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 plate and stored at 4°C.

[0194] After 2-24 hours at 60°C, 65°C or 70°C, the diameter or surface area of ​​the halos formed due to polyester degradation by the wild-type esterase and the variants were measured and compared.

[0195] 2.3. Activity based on PET hydrolysis in the reactor

[0196] In a 500 mL Minibio bioreactor (Applikon Biotechnology, Delft, The Netherlands), 0.69 μmol-2.07 μmol of purified esterase prepared in 80 mL 100 mM potassium phosphate buffer pH 8 was mixed with 20 g of amorphous PET (prepared according to WO2017 / 198786 to achieve a crystallinity of less than 20%). Temperature regulation of 60°C, 65°C or 70°C was performed by water bath immersion, and a single marine impeller was used at 250 rpm to maintain constant stirring. The pH of the PET depolymerization assay was adjusted to pH 8 by 6N NaOH and guaranteed by the my-Control biocontroller system (Applikon Biotechnology, Delft, The Netherlands). Alkali consumption was recorded during the assay and can be used to characterize the PET depolymerization assay.

[0197] The final yield of the PET depolymerization assay is determined by determining the residual PET weight or by determining the equivalent TA produced or by base consumption. At the end of the reaction, the weight determination of the residual PET is evaluated by filtering the reaction volume using a 12-15 μm grade 11 ash-free paper filter (Dutscher SAS, Brumath, France) and drying the residue before weighing. The determination of the equivalent TA produced is achieved using the UHPLC method described in 2.1, and the hydrolysis percentage is calculated based on the molar concentration (TA+MHET+BHET) at a given time compared to the total amount of TA contained in the initial sample. The acid monomers produced by PET depolymerization will be neutralized by base to be able to maintain the pH in the reactor. The determination of the equivalent TA produced is calculated using the corresponding molar base consumption, and the hydrolysis percentage is calculated based on the molar concentration of the equivalent TA at a given time compared to the total amount of TA contained in the initial sample.

[0198] The specific degradation activities of the esterases (variants) of the present invention are shown in the following Table 1. The specific degradation activity of the esterase of SEQ ID N° 1 was used as a reference and was considered as 100% specific degradation activity. The specific degradation activity was measured at 65°C as disclosed in Example 2.1.

[0199] Table 1: Specific degradation activity of variants of the invention

[0200] Variants Specific degradation activity V1:L210T 147％ V2:L210A 123％ V3:L210R 145％ V7: L210T+V172I+Q95G 137％ V8: L210T+V172I+Q95G+S184E 259％ V9: L210T+V172I+N213M 172％

[0201] Example 3 - Evaluation of the thermal stability of esterases of the present invention

[0202] The thermostability of the esterase of the present invention has been determined and compared with the thermostability of the esterase of SEQ ID N°1.

[0203] Different methodologies have been used to estimate thermal stability:

[0204] (1) Circular dichroism of proteins in solution;

[0205] (2) residual esterase activity after protein incubation under given temperature, time and buffer conditions;

[0206] (3) residual polyester depolymerization activity after protein incubation under given temperature, time and buffer conditions;

[0207] (4) The ability to degrade solid polyester compounds (such as PET or PBAT or the like) dispersed in an agar plate after protein incubation under given temperature, time, and buffer conditions;

[0208] (5) the ability to perform multiple rounds of polyester depolymerization assays under given conditions of temperature, buffer, protein concentration, and polyester concentration;

[0209] (6) Differential scanning fluorimetry (DSF);

[0210] The protocol details of these methods are given below.

[0211] 3.1 Circular dichroism

[0212] Circular dichroism (CD) spectroscopy was performed using a Jasco 815 apparatus (Easton, USA) to compare the melting temperatures (T m )(T m =79.8°C) and the T of the esterase of the present invention mTechnically, 400 μL of protein sample was prepared at 0.5 mg / mL in Talon buffer and used for CD. A first scan at 280-190 nm was performed to determine the two maximum intensities of CD corresponding to the correct folding of the protein. A second scan was then performed at 25°C-110°C at a long wave corresponding to this maximum intensity and provided a specific curve analyzed by Sigmaplot version 11.0 software (sigmoid 3 parameters y=a / (1+e^((x-x0) / b))) and Tm was determined when x=x0. The T obtained m Reflects the thermal stability of a given protein. m The higher it is, the more stable the variant is at high temperatures.

[0213] 3.2 Residual esterase activity

[0214] 1 mL of a solution of 40 mg / L (in Talon buffer) of the esterase of SEQ ID N°1 or the esterase of the invention was incubated at different temperatures (40°C, 50°C, 60°C, 65°C, 70°C, 75°C, 80°C and 90°C) for up to 10 days. Samples were taken regularly, diluted 1-500 times in 0.1 M potassium phosphate buffer pH 8.0, and subjected to p-nitrophenol-butyrate (pNP-B) assay. 20 μL of sample was mixed with 175 μL of 0.1 M potassium phosphate buffer pH 8.0 and 5 μL of a solution of pNP-B in 2-methyl-2-butanol (40 mM). The enzyme reaction was carried out at 30°C with stirring for 15 minutes, and the absorbance at 405 nm was obtained by a microplate spectrophotometer (Versamax, Molecular Devices, Sunnyvale, CA, USA). The activity of pNP-B hydrolysis (initial velocity expressed in μmol / min of pNPB) was determined using a standard curve of p-nitrophenol released in the linear part of the hydrolysis curve.

[0215] 3.3 Residual polyester depolymerization activity

[0216] 10 mL of 40 mg / L (in Talon buffer) esterase of SEQ ID N°1 and esterase of the present invention were incubated at different temperatures (40, 50, 60, 65, 70, 75, 80 and 90°C) for 1-30 days. 1 mL samples were taken regularly and transferred to a bottle containing 100 mg of amorphous PET micronized at 250-500 μm (prepared according to WO2017 / 198786 to achieve a crystallinity of less than 20%) and 49 mL of 0.1 M potassium phosphate buffer pH 8.0 and incubated at 60°C, 65°C or 0°C. 150 μL of buffer was sampled regularly. When necessary, the sample was diluted in 0.1 M potassium phosphate buffer pH 8. Then, 150 μL of methanol and 6.5 μL of 6N HCl were added to 150 μL of sample or dilution. After mixing and filtering on a 0.45 μm syringe filter, the samples were loaded on UHPLC to monitor the release of terephthalic acid (TA), MHET and BHET. The chromatography system used was an Ultimate 3000 UHPLC system (Thermofisher Scientific, Inc. Waltham, MA, USA), including a pump module, an autosampler, a column oven thermostated at 25°C and a UV detector at 240 nm. The column used was HS C18 HPLC column (150x4.6mm, 5μm, equipped with a precolumn, Supelco, Bellefonte, USA). TA, MHET and BHET were separated using a gradient of MeOH (30%-90%) in 1mM H2SO4 at 1mL / min. 20μL of sample was injected. TA, MHET and BHET were measured according to a standard curve prepared from commercially available TA and BHET and in-house synthesized MHET under the same conditions as the samples. The activity of PET hydrolysis (μmol / min of PET hydrolysis or mg / hour of equivalent TA produced) was determined in the linear part of the hydrolysis curve, which was established by sampling at different times during the first 24 hours. Equivalent TA corresponds to the sum of the measured TA and the TA contained in the measured MHET and BHET.

[0217] 3.4 Degradation of solid polyester

[0218] 1 mL of 40 mg / L (in Talon buffer) of the esterase of SEQ ID N°1 and the esterase of the present invention were incubated at different temperatures (65, 70, 75, 80 and 90°C) for 1-30 days. 20 μL of the enzyme preparation was deposited regularly in the wells produced in the agar plate containing PET. The preparation of the agar plate containing PET was achieved by dissolving 500 mg of PET in hexafluoro-2-propanol (HFIP) and pouring the culture medium into 250 mL of aqueous solution. After evaporation of HFIP at 52°C and 140 mbar, 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.

[0219] The diameter or surface area of ​​the halos formed due to polyester degradation by wild-type esterase and variants of the invention are measured and compared after 2-24 hours at 60° C., 65° C. or 70° C. The half-life of the enzyme at a given temperature corresponds to the time required for a 2-fold reduction in the diameter of the halos.

[0220] 3.5 Multi-round polyester depolymerization

[0221] The ability of esterases to perform continuous rounds of polyester depolymerization assays was evaluated in an enzyme reactor. A Minibio 500 bioreactor (Applikon Biotechnology BV, Delft, The Netherlands) was started with 3 g of amorphous PET (prepared according to WO2017 / 198786 to achieve a crystallinity of less than 20%) and 100 mL of 10 mM potassium phosphate buffer pH 8 containing 3 mg of LC-esterase. The stirring was set at 250 rpm using a marine impeller. The bioreactor was thermostated at 60 ° C, 65 ° C or 70 ° C by immersion in an external water bath. The pH was adjusted to 8 by adding 3 M of KOH. Different parameters (pH, temperature, stirring, addition of alkali) were monitored by BioXpert software V2.95. 1.8 g of amorphous PET (prepared according to WO2017 / 198786 to achieve a crystallinity of less than 20%) was added every 20 h. 500 μL of reaction medium was sampled regularly.

[0222] 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, USA) thermostated at 65°C. The eluent was 5 mM K2HPO4, 0.6 mL.min -1 The injection volume was 20 μL. The ethylene glycol was monitored using a refractometer.

[0223] The percentage of hydrolysis is calculated based on the ratio of the molar concentration at a given time (TA + MHET + BHET) to the total amount of TA contained in the initial sample, or based on the ratio of the molar concentration at a given time (EG + MHET + 2 × BHET) 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 per hour.

[0224] The half-life of the enzyme was assessed as the incubation time required to obtain a 50% loss in degradation rate.

[0225] 3.6 Differential Scanning Fluorometry (DSF)

[0226] Use DSF to evaluate the thermal stability of wild-type protein (SEQ ID N ° 1) and its variants by measuring its melting temperature (Tm) (temperature when half of the protein population is unfolded). Prepare protein samples with a concentration of 14 μM and store in a buffer A consisting of 20mMTris HCl pH 8.0, 300mM NaCl. First, the SYPRO orange dye 5000x stock solution in DMSO is diluted to 250x in water. Protein samples are loaded onto white transparent 96-well PCR plates (Bio-Rad cat#HSP9601), and each well contains a final volume of 25 μl. The final concentrations of protein and SYPRO orange dye in each well are 5 μM (0.14 mg / ml) and 10X respectively. The loading volume in each well is as follows: 15 μL buffer A, 9 μL 14 μM protein solution and 1 μL 250xSypro orange dilution solution. Then seal the PCR plate with an optical quality sealing tape and rotate at room temperature for 1 minute at 2000 rpm. Then, the DSF experiment was performed with a CFX96 real-time PCR system set to use 450 / 490 excitation and 560 / 580 emission filters. The samples were heated from 25°C to 100°C at a rate of 0.3°C / s. Fluorescence measurements were taken every 0.03 seconds. The melting temperature was determined from the peak of the first derivative of the melting curve using the Bio-Rad CFX Manager software.

[0227] Then, the esterase of SEQ ID N° 1 and the esterase of the present invention were compared based on their Tm values. Due to the high reproducibility between experiments on the same protein from different productions, a ΔT of 0.8°C m Considered significant for comparing variants. Tm values ​​correspond to the average of at least 3 measurements. The Tm of the esterase of SEQ ID N° 1 was evaluated at 79.8°C as disclosed in Example 3.6.

[0228] The thermostability of the esterase variants of the invention is shown in Table 2 below, expressed as Tm values ​​and evaluated according to Example 3.6. The increase in Tm compared to the esterase of SEQ ID N° 1 is shown in brackets.

[0229] Table 2: Tm of the esterase of the present invention compared with SEQ ID N°1

[0230] Variants Tm V1:L210T 81.2℃(+1.4℃) V4:L210W 83.8℃(+4.0℃) V5:L210H 81.8℃(+2.0℃) V6: R205C+S251C 80.8℃(+1.0℃) V7: L210T+V172I+Q95G 82.8℃(+3.0℃) V8: L210T+V172I+Q95G+S184E 85.2℃(+5.4℃) V9: L210T+V172I+N213M 82.0℃(+2.2℃) V10: L210T+V172I+Q95G+G136A+T169Q+S184E 82.0℃(+2.2℃)

Claims

1. An esterase which, based on the amino acid sequence shown in SEQ ID NO: 1, has only the substitution of L210T, or a combination of substitutions of L210T+V172I+Q95G, L210T+V172I+Q95G+S184E or L210T+V172I+N213M, and has improved polyester degradation activity and / or improved thermal stability compared to the esterase of SEQ ID NO:

1.

2. An esterase having only L210A or L210R substitution based on the amino acid sequence shown in SEQ ID NO: 1, and having improved polyester degradation activity compared to the esterase of SEQ ID NO:

1.

3. An esterase which has only the substitution of L210W or L210H, or a combination of substitutions of L210T+V172I+Q95G+G136A+T169Q+S184E, based on the amino acid sequence as shown in SEQ ID NO: 1, and has improved thermal stability compared to the esterase of SEQ ID NO:

1.

4. A nucleic acid encoding an esterase as defined in any one of claims 1 to 3.

5. An expression cassette or vector comprising the nucleic acid of claim 4.

6. A host cell comprising the nucleic acid of claim 4 or the expression cassette or vector of claim 5.

7. A composition comprising an esterase as defined in any one of claims 1 to 3, or a host cell or an extract thereof according to claim 6.

8. A method for degrading at least one polyester in a polyester-containing material, comprising: (a) contacting the polyester-containing material with an esterase according to any one of claims 1 to 3, or a host cell according to claim 6, or a composition according to claim 7.

9. The method of claim 8, further comprising step (b) after step (a), (b) recovering monomers and / or oligomers.

10. The method of claim 9, wherein the polyester is selected from the group consisting of polyethylene terephthalate, polytrimethylene terephthalate, polybutylene terephthalate, polyethylene isosorbide terephthalate, polylactic acid, polyhydroxyalkanoate, polybutylene succinate, polybutylene succinate adipate, polybutylene adipate terephthalate, polyethylene furandicarboxylate, polycaprolactone, polyethylene adipate, polyethylene naphthalate, and blends / mixtures thereof.

11. The method of claim 10, wherein the polyester is polyethylene terephthalate.

12. A polyester-containing material comprising the esterase according to any one of claims 1 to 3 or the host cell according to claim 6 or the composition according to claim 7.

13. A detergent composition comprising the esterase according to any one of claims 1 to 3 or the host cell according to claim 6 or the composition according to claim 7.

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