阅读说明：本技术 新酯酶及其用途 (Novel esterases and their use ) 是由 S·迪奎斯内 V·图尼尔 A·马蒂 于 2019-07-26 设计创作，主要内容包括：本发明涉及新酯酶,更特别地涉及与SEQ ID N°1的酯酶相比具有改进的活性和/或改进的热稳定性的酯酶变体及其在降解含聚酯材料如塑料制品中的用途。本发明的酯酶特别适合于降解聚对苯二甲酸乙二醇酯和含聚对苯二甲酸乙二醇酯的材料。(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 plastic articles. The esterases of the invention are particularly suitable for degrading polyethylene terephthalate and materials containing polyethylene terephthalate.)

1. An esterase (i) having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the full-length amino acid sequence set forth in SEQ ID N ° 1, (ii) comprising at least four substitutions at a position selected from F208, D203, S248, V170, V177, T176, T61, S65 or Y92 as compared to the amino acid sequence SEQ ID N ° 1, and (iii) exhibiting increased polyester degrading activity and/or increased thermostability as compared to the esterase of SEQ ID N ° 1.

2. The esterase according to claim 1, wherein said esterase comprises at least a combination of a substitution at position F208+ D203+ S248 and one substitution at a position selected from V170, V177, T176, T61, S65, N211, or Y92.

3. The esterase according to claim 1 or 2, wherein said esterase comprises at least a combination of a substitution selected from F208I + D203C + S248C or F208W + D203C + S248C and one substitution selected from V170I, V177I, T176N, T61M, S65T, N211D/M or Y92G/P/F.

4. The esterase according to claims 1-3, wherein said esterase comprises at least a substitution at position F208+ D203+ S248 and a combination of one or two substitutions selected from V170 or Y92.

5. The esterase according to any of the preceding claims, wherein said esterase comprises at least a combination of substitutions selected from the group consisting of: F208I + D203C + S248C + V170I, F208I + D203C + S248C + Y92G, F208I + D203C + S248C + V170I + Y92G, F208W + D203C + S248C + V170I, F208W + D203C + S248C + Y92G or F208W + D203C + S248C + V170I + Y92G.

6. The esterase according to any of the preceding claims, wherein the esterase further comprises at least one amino acid residue selected from S130, D175, or H207.

7. The esterase according to any of the preceding claims, wherein said esterase further comprises at least one substitution at a position corresponding to a residue selected from the group consisting of: t, R, a, W, R, a205, N214, a215, a216, I217, F238, V242, D244, P245, a246, L247, D, R138, D158, Q182, F187, P, L, D, N, S, Q, K159, a174, a125, S218, S, T, L202, N204, S212, V219, Y220, Q237, L239, N241, N243, a, L, D, P, M131, P210, a209, P179, R, G, R, S, a, R, H156, H183, a, T, S, F, L, G135, a140, N143, S145, a149, S164, V167, S206, N213, T252, E173, G, a121, T, N211, Y, D, or S.

8. The esterase according to any of the preceding claims, wherein said esterase further comprises at least one substitution selected from the group consisting of: A121S, N213P, S212F/T/I/L, A125G, N204D/I/L/Y/H/F, G135A, W69R, N214D/I/L/F/Y/H, N241P, N243P, R12F/Y/H, P179E, V242Y, V167Q or N211D/M.

9. The esterase according to any preceding claim, wherein the esterase comprises a combination of substitutions selected from the combinations of substitutions listed in any of Table 1, Table 2, and Table 3.

10. The esterase according to any of the preceding claims, wherein the esterase has an amino acid sequence shown as SEQ ID N ° 2 and has at least four substitutions at a position selected from the group consisting of F208, D203, S248, V170, V177, T61, T176, S65, N211, or F92, wherein said position is numbered by reference to the amino acid sequence shown as SEQ ID N ° 2, and the esterase shows increased polyester degrading activity and/or increased thermostability as compared to the esterase of SEQ ID N ° 1.

11. The esterase according to any of the preceding claims, wherein the esterase has an amino acid sequence shown in SEQ ID N ° 2 and has at least four substitutions at a position selected from the group consisting of F208, D203, S248, V170, V177, T61, N211, or F92, wherein said positions are numbered by reference to the amino acid sequence shown in SEQ ID N ° 2, and the esterase exhibits increased polyester degrading activity and/or increased thermostability as compared to the esterase of SEQ ID N ° 1.

12. The esterase according to claim 10 or 11, wherein the esterase comprises at least a combination of a substitution at position F208+ D203+ S248 and one substitution at a position selected from V170, V177, T176, T61, S65, N211 or F92.

13. The esterase according to any of claims 10 to 12, wherein said esterase comprises at least a combination of a substitution selected from F208I + D203C + S248C or F208W + D203C + S248C and one substitution selected from V170I, V177I, T176N, T61M, S65T, N211D/M or F92G/P.

14. The esterase according to any of the preceding claims, wherein the esterase further comprises at the N-terminus an amino acid sequence having at least 55%, 65%, 75%, 85% or 100% identity to the full-length amino acid sequence set forth in SEQ ID N ° 3.

15. The esterase according to claim 14, wherein said N-terminal amino acid sequence is selected from the group consisting of seq id no: the amino acid sequence shown as SEQ ID N degrees 4, SEQ ID N degrees 5, SEQ ID N degrees 6 or SEQ ID N degrees 7.

16. A nucleic acid encoding an esterase as defined in any of claims 1-15.

17. An expression cassette or vector comprising the nucleic acid of claim 16.

18. A host cell comprising the nucleic acid of claim 16 or the expression cassette or vector of claim 17.

19. A composition comprising an esterase as defined in any of claims 1 to 15, or a host cell according to claim 18 or an extract thereof.

20. A method of degrading a polyester, comprising:

(a) contacting the polyester with the esterase according to any of claims 1-15 or the host cell according to claim 18 or the composition according to claim 19; and optionally

(b) Recovering the monomer and/or oligomer.

21. A method of degrading at least one polyester of a polyester-containing material, comprising:

(a) contacting the polyester-containing material with the esterase according to any of claims 1-15 or the host cell according to claim 18 or the composition according to claim 19; and optionally

(b) Recovering the monomer and/or oligomer.

22. The method according to claim 20 or 21, wherein the polyester is selected from the group consisting of polyethylene terephthalate (PET), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polysorbate terephthalate (PEIT), polylactic acid (PLA), Polyhydroxyalkanoates (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), Polycaprolactone (PCL), poly (ethylene adipate) (PEA), polyethylene naphthalate (PEN) and blends/mixtures of these materials, preferably polyethylene terephthalate.

23. A polyester-containing material comprising the esterase according to any of claims 1-15 or the host cell according to claim 18 or the composition according to claim 19.

24. A detergent composition comprising the esterase according to any of claims 1-15 or the host cell according to claim 18 or the composition according to claim 19.

Background

Esterases are capable of catalyzing the hydrolysis of various polymers, including polyesters. In this context, esterases have shown promising results in many industrial applications, including as detergents for dishwashing and laundry applications, as degrading enzymes for treating biomass and food, as biocatalysts in the detoxification of environmental pollutants or for treating polyester fabrics in the textile industry. The use of esterases as degrading enzymes to hydrolyze polyethylene terephthalate (PET) is of particular interest. Indeed, PET is used in numerous technical fields, for example for the manufacture of clothing, carpets or in the form of thermosetting resins for the manufacture of packaging materials or automotive plastic articles, etc., so that the accumulation of PET in landfills becomes an increasing ecological problem.

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

In this context, several esterases have been identified as candidate degrading enzymes for polyesters, and several variants of this esterase have been developed. Among esterases, cutinases, also known as keratohydrolases (EC 3.1.1.74), are of particular interest. Cutinases have been identified from various fungi (P.E.Kolattukudy in "Lipases", Ed.B.Borg-str Loam and H.L.Brockman, Elsevier 1984, 471-504), bacterial and plant pollen. More recently, other esterases have been identified by the metagenomic approach.

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

Summary of The Invention

The present invention provides novel esterases that exhibit increased activity and/or increased thermostability as compared to a parent or wild-type esterase having an amino acid sequence as set forth in SEQ ID N ° 1. This wild-type esterase corresponds to amino acids 36-293 of the amino acid sequence of the metagenome-derived cutinase described in Sulaiman et al, Appl Environ microbiol.2012mar and is designated G9BY57 in SwissProt. The esterases of the invention are particularly useful in a process for degrading plastics, especially plastics containing PET.

In this respect, it is an object of the present invention to provide an esterase which (i) has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the full-length amino acid sequence shown as SEQ ID N ° 1, (ii) comprises at least four substitutions at a position selected from the group consisting of F208, D203, S248, V170, V177, T176, T61, S65 or Y92 compared to the amino acid sequence SEQ ID N ° 1, and (iii) shows an increased polyester degrading activity and/or an increased thermostability compared to the esterase of SEQ ID N ° 1.

It is another object of the invention to provide an esterase which (i) has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the full-length amino acid sequence shown in SEQ ID N ° 1, (ii) comprises at least four substitutions at a position selected from F208, D203, S248, V170, V177, T176, T61, S65, N211 or Y92 compared to the amino acid sequence SEQ ID N ° 1, and (iii) shows increased polyester degrading activity and/or increased thermostability compared to the esterase of SEQ ID N ° 1.

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

It is a further object of the invention to provide nucleic acids encoding the esterases of the invention. The invention also relates to expression cassettes or expression vectors comprising said nucleic acids, and to host cells comprising said nucleic acids, expression cassettes or vectors.

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

It is a further object of the present invention to provide a process for producing an esterase of the invention, which comprises:

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

(b) Recovering the esterase from the cell culture.

It is a further object of the present invention to provide a method of degrading a polyester comprising:

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

(b) Recovering the monomer and/or oligomer.

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

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

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

(b) Recovering monomers and/or oligomers of the at least one polyester.

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

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

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

Detailed description of the inventionThe above-mentioned

Definition of

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

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. Herein, amino acids are represented by their one-letter or three-letter codes according to the following nomenclature: a: alanine (Ala); c: cysteine (Cys); d: aspartic acid (Asp); e: glutamic acid (Glu); f: phenylalanine (Phe); g: glycine (Gly); h: histidine (His); i: isoleucine (Ile); k: lysine (Lys); l: leucine (Leu); m: methionine (Met); n: asparagine (Asn); p: proline (Pro); q: glutamine (Gln); r: arginine (Arg); s: serine (Ser); t: threonine (Thr); v: valine (Val); w: tryptophan (Trp) and Y: tyrosine (Tyr).

The term "esterase" refers to an enzyme belonging to a class of hydrolases classified under EC 3.1.1 according to enzyme nomenclature that catalyzes the hydrolysis of an ester to an acid and an alcohol. The term "cutinase" or "keratolytic enzyme" refers to an esterase classified according to enzyme nomenclature as EC 3.1.1.74, which is capable of catalyzing a chemical reaction that produces keratinous monomers from keratin and water.

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

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

Thus, the terms "modified" and "altered" as used herein in relation to a particular position means that the amino acid at that particular position has been modified as compared to the amino acid at that particular position in the wild-type protein.

"substitution" refers to the substitution of one amino acid residue with another amino acid residue. Preferably, the term "substitution" refers to the replacement of one amino acid residue by another amino acid residue selected from the group consisting of naturally occurring 20 standard amino acid residues, naturally occurring rare amino acid residues (e.g., hydroxyproline, hydroxylysine, allohydroxylysine, 6-N-methyllysine, N-ethylglycine, N-methylglycine, N-ethylasparagine, alloisoleucine, N-methylisoleucine, N-methylvaline, pyroglutamide, aminobutyric acid, ornithine, norleucine, norvaline), and non-naturally occurring amino acid residues that are typically synthetic (e.g., cyclohexyl-alanine). Preferably, the term "substitution" refers to the replacement of one amino acid residue by another selected from the naturally occurring 20 standard amino acid residues (G, P, A, V, L, I, M, C, F, Y, W, H, K, R, Q, N, E, D, S and T). The symbol "+" indicates a combination of substitutions. In this document, the following terms are used to denote substitutions: L82A shows the substitution of amino acid residue 82 (leucine, L) of the parent sequence with alanine (A). A121V/I/M indicates that the amino acid residue at position 121 (alanine, A) of the parent sequence is substituted with one of the following amino acids: valine (V), isoleucine (I) or methionine (M). Substitutions may be conservative or non-conservative substitutions. Examples of conservative substitutions are within the 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).

Unless otherwise indicated, the positions disclosed herein are referenced to the amino acid sequence numbering shown in SEQ ID N ° 1.

The term "sequence identity" or "identity" as used herein refers to the number (or in the hundreds) of matches (identical amino acid residues) between two polypeptide sequencesFraction expressed in% percent). Sequence identity is determined by comparing sequences when aligned so as to maximize overlap and identity while minimizing sequence gaps. In particular, sequence identity can be determined using any of a number of mathematical global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar length are preferably aligned using a global alignment algorithm (e.g., Needleman and Wunsch algorithms; Needleman and Wunsch, 1970) that optimally aligns the sequences over their full length, while sequences of substantially different length are preferably aligned using a local alignment algorithm (e.g., Smith and Waterman algorithms (Smith and Waterman, 1981) or Altschul algorithms (Altschul et al, 1997; Altschul et al, 2005)). Alignment for determining percent amino acid sequence identity can be achieved in a variety of ways within the skill in the art, for example, using an Internet website such ashttp://blast.ncbi.nlm.nih.gov/Orhttp:// www.ebi.ac.uk/Tools/emboss/Publicly available computer software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the full length of the sequences being compared. For purposes herein,% amino acid sequence identity values refer to values generated using the paired sequence alignment program EMBOSS Needle that generates the best global alignment of two sequences using the Needleman-Wunsch algorithm, with all search parameters set to default values, i.e., scoring matrix BLOSUM62, gap open 10, gap extension 0.5, end gap penalty error, end gap open 10, and end gap extension 0.5.

"Polymer" refers to a compound or mixture of compounds whose structure is composed of multiple monomers (repeating units) joined 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. a homopolymer) or a mixture of different repeating units (i.e. a copolymer or heteropolymer). According to the present invention, "oligomer" refers to a molecule containing from 2 to about 20 monomers.

In the context of the present invention, "polyester-containing material" or "polyester-containing product" refers to a product, such as a plastic article, comprising at least one polyester in a crystalline, semi-crystalline or completely amorphous form. In a particular embodiment, polyester-containing material refers to any article made of at least one plastic material, such as plastic sheets, tubes, bars, profiles (profiles), patterns (flaps), films, chunks, etc., comprising at least one polyester, and possibly other substances or additives, such as plasticizers, mineral or organic fillers. In another embodiment, polyester-containing material refers to a plastic compound or plastic formulation in a molten or solid state suitable for use in the manufacture of plastic articles. In another embodiment, polyester-containing material refers to a textile, fabric or fiber comprising at least one polyester. In another embodiment, polyester-containing material refers to plastic waste or fiber waste that includes at least one polyester.

In the present description, the term "polyester" encompasses, but is not limited to, polyethylene terephthalate (PET), polypropylene terephthalate (PTT), polybutylene terephthalate (PBT), polysorbate terephthalate (PEIT), polylactic acid (PLA), Polyhydroxyalkanoates (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), Polycaprolactone (PCL), poly (ethylene adipate) (PEA), polyethylene naphthalate (PEN), and blends/mixtures of these polymers.

Novel esterases

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

It is therefore an object of the present invention to provide esterases which exhibit an increased activity compared to esterases having the amino acid sequence shown in SEQ ID No 1.

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

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

The "degradation activity" of the esterase can be assessed by the person skilled in the art according to methods known per se in the art. For example, the degradation activity can be assessed by measuring the rate of depolymerization activity of a particular polymer, measuring the rate of degradation of solid polymer compounds dispersed in an agar plate, or measuring the rate of depolymerization activity of a polymer in a reactor. In particular, the degradation activity can be assessed by measuring the "specific degradation activity" of the esterase. The "specific degradation activity" of esterase on PET corresponds to μmol/min or mg equivalent TA/hour of hydrolyzed PET in the early phase of the reaction (i.e. the first 24 hours) and per mg of esterase and is determined from the linear part of the reaction hydrolysis curve, which is established by several samples taken at different times during the first 24 hours. As another example, "degradation activity" can be assessed by measuring the rate of oligomer and/or monomer release upon contacting the polymer or polymer-containing plastic article with a degrading enzyme under suitable temperature, pH and buffer conditions after a defined period of time.

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

The inventors have also identified the target amino acid residue in SEQ ID N ° 1, which can advantageously be modified to improve the stability of the corresponding esterase at high temperature (i.e. improved thermostability), and advantageously at a temperature above 50 ℃, preferably above 70 ℃.

It was therefore an object of the present invention to provide novel esterases which show an increased thermostability in comparison with the thermostability of an esterase having an amino acid sequence as shown in SEQ ID No 1.

In the context of the present invention, the term "increased thermostability" means an increased ability of the esterase to resist changes in its chemical and/or physical structure at elevated temperatures, in particular at temperatures of from 50 ℃ to 90 ℃, compared to the esterase of SEQ ID N ° 1.

In particular, the thermal stability can be assessed by assessing 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 invention shows an increase in Tm of about 1 ℃, 2 ℃, 3 ℃, 4 ℃, 5 ℃, 10 ℃, 12 ℃ or more compared to the Tm of the esterase of SEQ ID N ° 1. In particular, the esterase of the invention may have an increased half-life at a temperature of 50 ℃ to 90 ℃ compared to the esterase of SEQ ID N ° 1.

The melting temperature (Tm) of the esterase can be measured by a person skilled in the art according to methods known per se in the art. For example, DSF can be used to quantify the change in the thermal denaturation temperature of an 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 described in the experimental section. In the context of the present invention, a comparison of Tm is performed with Tm measured under the same conditions (e.g. pH, nature and amount of polyester, etc.).

Alternatively, thermostability can be assessed by measuring esterase activity and/or polyester depolymerization activity of the esterase after incubation at different temperatures and comparing 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 evaluated. A rapid and valuable test may include assessing the ability of the enzyme to degrade solid polyester compounds dispersed in agar plates after incubation at different temperatures by halo diameter measurements.

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

According to the invention, the targeted amino acid can be substituted with any of 19 other amino acids.

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

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

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

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

According to a particular embodiment, the esterase comprises at least a substituted combination selected from the group consisting of: f208+ D203+ S248+ V170, F208+ D203+ S248+ V177, F208+ D203+ S248+ Y92, F208+ D203+ S248+ V170+ Y92, F208+ D203+ S248+ T61, F208+ D203+ S248+ T176, F208+ D203+ S248+ S65 and F208+ D203+ S65.

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

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

In a specific embodiment, the variant of the invention further comprises at least one substitution at a position selected from: t, R, a, W, R, a205, N214, a215, a216, I217, F238, V242, D244, P245, a246, L247, D, R138, D158, Q182, F187, P, L, D, N, S, Q, K159, a174, a125, S218, S, T, L202, N204, S212, V219, Y220, Q237, L239, N241, N243, a, L, D, P, M131, P210, a209, P179, R, G, R, S, a, R, H156, H183, a, T, S, F, L, G135, a140, N143, S145, a149, S164, V167, S206, N213, T252, E173, G, a121, T, N211, Y, D, or S.

In a specific embodiment, the variant of the invention further comprises at least one substitution at a position selected from: n213, N211, a121, N204, S212, a125, G135, W69, N214, N241, N243, R12, P179, V242 or V167.

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

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

In one embodiment, the esterase of the invention further comprises the substitution a 121S. In particular, the variant comprises at least a combination of substitutions selected from: F208I + D203C + S248C + V170I + Y92G + A121S, F208I + D203C + S248C + V170I + A121S or F208I + D203C + S248C + Y92G + A121S.

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

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

In one embodiment, the esterase of the invention comprises the substitution a 125G. In particular, the variant comprises at least a combination of substitutions selected from: F208I + D203C + S248C + V170I + Y92G + A125G, F208I + D203C + S248C + V170I + A125G or F208I + D203C + S248C + Y92G + A125G.

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

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

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

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

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

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

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

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

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

In one embodiment, the esterase of the invention comprises the substitution a 140S.

In one embodiment, the esterase of the invention further comprises at least one substitution at a position selected from the group consisting of: t11, R12, a14, W69, R73, a205, N214, a215, a216, F238, V242, D244, P245, a246, L247, Q182, F187, or S218. More preferably, the substitution is selected from T11M/E/I/S/N/D/Q, R Q/D/N/P/F/V/E/L/Y, R12Y, R, A14Y, R/M/E/Y, R/G/M/D/E/S/C/Q/F/N/Y, R205Y, R, N214Y, R/E/Y, R214Y, R/L/F/Y/Y, R215Y, R, A216Y, R, F238Y, R, V242Y, R/Y, R244Y, R/Y, R245Y, R/Y/Y, R/D/H/Y, R, Q182Y, R/36187Y, R/I or S218 36247. In one embodiment, the esterase comprises at least one substitution selected from the group consisting of: T11M/I/S/N/D, R12N/G/P/V/L, A14E, W69M, R73I/G/D/S/C/Q/F/N/V, A205D, N214E/C, A215N, P245Y or A246D/H. In another specific embodiment, the variant of the invention further comprises at least two substitutions at positions selected from: t, R, a, W, R, a205, N214, a215, a216, I217, F238, V242, D244, P245, a246, L247, D, R138, D158, Q182, F187, P, L, D, N, S, Q, K159, a174, a125, S218, S, T, L202, N204, S212, V219, Y220, Q237, L239, N241, N243, a, L, D, P, M131, P210, a209, P179, R, G, R, S, a, R, H156, H183, a, T, S, F, L, G135, a140, N143, S145, a149, S164, V167, S206, N213, T252, E173, G, a121, T, N211, Y, D, or S.

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

In one embodiment, the esterase of the invention comprises at least two substitutions selected from N213P, G135A, a140S, V167Q, N241P or R12H. In particular, the variant comprises at least a combination of substitutions selected from: F208I + D203C + S248C + V170I + Y92G + N213P + G135A, F208I + D203C + S248C + V170I + Y92G + R12H + N241P, F208I + D203C + S248C + V170I + Y92G + R12H + V167Q, F208I + D203C + S248C + V170I + Y92G + A140S + V167Q or F208I + D203C + S248C + V170I + Y92G + N241P + V167Q.

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

In one embodiment, the esterase of the invention comprises at least one substitution, preferably at least two substitutions, selected from a17T, T27S, S48T, F90L, L82I, G135A, a140S, N143I, S145T, a149G, S164P, V167Q, S206T, N213P or T252S. In particular, the variant comprises at least a combination of substitutions selected from: f208+ D203+ S248+ Y92 + A17 + T27 + S48 + L82 + G135 + A140 + N143 + S145 + A149 + S164 + V167 + S206 + N213 + T252, F208+ D203+ S248+ Y92 + A17 + T27 + S48 + L82 + F90 + G135 + A140 + N143 + S145 + S149 + S164 + V167 + S206 + N213 + T252 or F208+ D203+ S248+ Y92 + T27 + S48 + L82 + F90 + G135 + A140 + N143 + S145 + A149 + S164 + V167 + S206 + N213 + T252.

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

In another specific embodiment, the esterase of the invention further comprises at least one amino acid residue selected from the group consisting of: g59, Y60, T61, D63, S65, S66, N85, T86, R89, F90, H129, W155, T157, T176, V177, a178 and N211, i.e. the esterase of the invention is not modified in one of these positions compared to SEQ ID N ° 1. Preferably, the esterase comprises the amino acid residue F90 as in the parent esterase.

It is a further object of the present invention to provide an esterase (i) having the amino acid sequence shown in SEQ ID N ° 2, (ii) having at least four substitutions at a position selected from the group consisting of F208, D203, S248, V170, V177, T176, S65, T61 or F92, wherein said position is numbered by reference to the amino acid sequence shown in SEQ ID N ° 2, and (iii) showing an increased polyester degrading activity and/or an increased thermostability compared to the esterase of SEQ ID N ° 1.

It is a further object of the present invention to provide an esterase (i) having the amino acid sequence shown in SEQ ID N ° 2, (ii) having at least four substitutions at a position selected from the group consisting of F208, D203, S248, V170, V177, T176, S65, T61, N211 or F92, wherein said positions are numbered by reference to the amino acid sequence shown in SEQ ID N ° 2, and (iii) showing an increased polyester degrading activity and/or an increased thermostability as compared to the esterase of SEQ ID N ° 1.

The amino acid sequence shown in SEQ ID N ° 2 corresponds to a variant of the amino acid sequence of SEQ ID N ° 1 having the combination of the substitutions a17T + T27S + S48T + L82I + F90L + Y92F + G135A + a140S + N143I + S145T + a149G + S164P + V167Q + S206T + N213P + T252S compared to SEQ ID N ° 1.

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

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

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

In a specific embodiment, the esterase comprises at least a combination of a substitution at position F208+ D203+ S248 and one substitution at a position selected from V170, V177, T176, T61 or F92 compared to the esterase of SEQ ID N ° 2. In a specific embodiment, the esterase comprises at least a combination of a substitution at position F208+ D203+ S248 and one substitution at a position selected from V170, V177, T176, T61, S65, N211 or F92 compared to the esterase of SEQ ID N ° 2. In a particular embodiment, the esterase comprises a combination of at least one substitution at a position selected from the group consisting of: f208+ D203+ S248+ V170, F208+ D203+ S248+ V177, F208+ D203+ S248+ T61, F208+ D203+ S248+ F92, F208+ D203+ S248+ T176, F208+ D203+ S248+ S65, F208+ D203+ S248+ N211 or F208+ D203+ S248+ V170+ F92. In particular, the esterase comprises at least a combination of a substitution selected from F208I + D203C + S248C or F208W + D203C + S248C and one substitution selected from T61M, V170I, V177I, T176N, S65T, N211D/M or F92G/P.

According to a particular embodiment, the esterase comprises at least a combination of substitutions selected from the group consisting of: f208+ D203+ S248+ V170, F208+ D203+ S248+ V177, F208+ D203+ S248+ F92, F208+ D203+ S248+ V170+ F92, F208+ D203+ S248+ T61, F208+ D203+ S248+ T176, F208+ D203+ S248+ S65 and F208+ D203+ S248+ S65.

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

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

In a specific embodiment, the variant of the invention further comprises at least one substitution at a position selected from: t11, R12, a14, W69, R73, a205, N214, a215, a216, I217, F238, V242, D244, P245, a246, L247, D94, R138, D158, Q182, F187, P10, L15, D18, N87, S88, S95, Q99, K159, a174, a125, S218, S99, T99, L202, N204, S212, V219, Y220, Q237, L239, N241, N243, a 99, L99, D99, P99, M131, P210, a209, P179, R99, G99, R99, S99, a 36156, R36156, H183, E173, G99, a121, T69, N157, Y99, or N99, wherein the amino acid sequence is numbered by the amino acid ID degrees shown in SEQ 99.

In a specific embodiment, the variant of the invention further comprises at least one substitution at a position selected from: n211, a121, N204, S212, a125, W69, N214, N241, N243, R12, P179, or V242.

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

In one embodiment, the esterase of the invention comprises the substitution a121S compared to the esterase of SEQ ID N ° 2. In particular, the variant comprises at least a combination of substitutions selected from: F208I + D203C + S248C + V170I + F92G + A121S, F208I + D203C + S248C + V170I + A121S or F208I + D203C + S248C + F92G + A121S.

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

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

In one embodiment, the esterase of the invention comprises the substitution a125G compared to the esterase of SEQ ID N ° 2. In particular, the variant comprises at least a combination of substitutions selected from: F208I + D203C + S248C + V170I + F92G + A125G, F208I + D203C + S248C + V170I + A125G or F208I + D203C + S248C + F92G + A125G.

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

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

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

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

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

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

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

In one embodiment, the esterase of the invention further comprises at least one substitution at a position selected from the group consisting of: t11, R12, a14, W69, R73, a205, N214, a215, a216, F238, V242, D244, P245, a246, L247, Q182, F187, or S218. The substitution is more preferably selected from T11M/E/I/S/N/D/Q, R Q/D/N/P/F/V/E/L/Y, R12Y, R, A14Y, R/M/E/Y, R/G/M/D/E/S/C/Q/F/N/Y, R205Y, R, N214Y, R/E/Y, R214Y, R/L/F/Y/Y, R215Y, R, A216Y, R, F238Y, R, V242Y, R/Y, R244Y, R/Y, R245Y, R/Y/Y, R/D/H/Y, R, Q182Y, R/36187Y, R/I or S218 36247. In one embodiment, the esterase comprises at least one substitution selected from the group consisting of: T11M/I/S/N/D, R12N/G/P/V/L, A14E, W69M, R73I/G/D/S/C/Q/F/N/V, A205D, N214E/C, A215N, P245Y or A246D/H.

In one embodiment, the variant of the invention further comprises at least two substitutions at positions selected from: t11, R12, a14, W69, R73, a205, N214, a215, a216, I217, F238, V242, D244, P245, a246, L247, D94, R138, D158, Q182, F187, P10, L15, D18, N87, S88, S95, Q99, K159, a174, a125, S218, S99, T99, L202, N204, S212, V219, Y220, Q237, L239, N241, N243, a 99, L99, D99, P99, M131, P210, a209, P179, R99, G99, R99, S99, a 36156, R36156, H183, E173, G99, a121, T69, N157, Y99, or N99, wherein the amino acid sequence is numbered by the amino acid ID degrees shown in SEQ 99.

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

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

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

In another embodiment, the esterase of the invention further comprises at least one amino acid residue selected from the group consisting of G59, Y60, T61, D63, S65, S66, N85, T86, R89, H129, W155, T157, T176, V177, a178 and N211 as in SEQ ID N ° 2, i.e. the esterase of the invention is not modified at one of these positions compared to SEQ ID N ° 2.

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

In a particular embodiment, the esterase variant comprises a combination of substitutions selected from the group consisting of: F208I + D203C + S248C + V170I, F208I + D203C + S248C + Y92G, F208I + D203C + S248C + V170I + Y92G, F208I + D203C + S248C + V177I, F208W + D203C + S248C + Y92G, F208W + D203C + S248C + V177I, F208I + D203C + S248C + N211M, F208W + D203C + S248C + N211M. These esterases show increased polyester degrading activity and increased thermostability compared to esterases of SEQ ID N ° 1 or SEQ ID N ° 2.

In a particular embodiment, the esterase variant comprises a combination of substitutions selected from the group consisting of: f208+ D203+ S248+ V170, F208+ D203+ S248+ Y92, F208+ D203+ S248+ V170+ Y92, F208+ D203+ S248+ V177, F208+ D203+ S248+ N211, F208+ D203+ S248+ V170, F208+ D203+ S248+ T176, F208+ D203+ S248+ T121, F208+ D203+ S248+ Y92 + A121, F208+ D203+ S248+ S65, F208+ D203+ S248, F208+ D248 + S248+ V170+ Y92 + N204, F208+ D203+ S248+ V203 + V + S248+ V203 + V2 + S248, F208+ D203+ V203 + S248+ V203 + S248, F203 + D + V203 + S248, F203 + V203 + N248, F203 + V203 + N248 + S248, F203 + D + S248, F203 + D203+ V203 + S248, F203 + D + V203 + S248, F203 + N203 + S248, F203 + S248+ V203 + S248, F203 + V203 + S248, F203 + D203+ S248, F203 + V, F208I + D203C + S248C + V170I + Y92G + N213P + G135A + V167Q, F208I + D203C + S248C + V170C + Y92C + N213C + G135C + N241C, F208C + D203C + S248C + V170C + Y92C + N213C + G135C + R12C, F208C + D203C + S248C + V170C + Y92C + N C, F208C + D203C + Y C + V167C + V C or F208C + D C + S203 + S C + V170+ V C. These esterases show increased polyester degrading activity and increased thermostability compared to esterases of SEQ ID N ° 1 or SEQ ID N ° 2.

Preferably, the esterase variant comprises a combination of substitutions selected from the group consisting of: F208W + D203C + S248C + V177C, F208C + D203C + S248C + T176C, F208C + D203C + S248C + S65C, F208C + D203C + S248C + V170+ C + Y92C + N213C + G135C + N241C, F208C + D203C + S248C + V170+ Y92C + N213C + G135C + R12C, F208C + D203C + S248C + Y92C, F208C + D C + S C + V170+ Y C + Y92C, F208C + D203C + S248+ S C + Y92 + N-N5 + F203 + N203 + S C + N203 + N72 + V170+ N203 + N72 + N203 + N72.

Polyester degrading Activity of variants

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

In a particular embodiment, the esterase of the invention has polyester degrading activity, preferably polyethylene terephthalate (PET) degrading activity and/or polybutylene adipate terephthalate (PBAT) degrading activity and/or polybutylene succinate (PBS) degrading activity and/or Polycaprolactone (PCL) degrading activity, more preferably polyethylene terephthalate (PET) degrading activity and/or polybutylene adipate terephthalate (PBAT) degrading activity and/or Polycaprolactone (PCL) degrading activity. Even more preferably. The esterase of the invention has the degradation activity of polyethylene terephthalate (PET).

Advantageously, the esterase of the invention exhibits polyester degrading activity at least in the temperature range of 20 ℃ to 90 ℃, preferably 40 ℃ to 80 ℃, more preferably 50 ℃ to 70 ℃, even more preferably 60 ℃ to 70 ℃. In a specific embodiment, the esterase exhibits polyester degrading activity at 65 ℃. In a specific embodiment, the esterase exhibits polyester degrading activity at 70 ℃. In one embodiment, the polyester degrading activity may still be measured at a temperature of 60 ℃ to 90 ℃.

In a specific embodiment, the esterase of the invention has an increased polyester degrading activity at a given temperature, more particularly at a temperature of 40 ℃ to 80 ℃, more preferably at a temperature of 50 ℃ to 70 ℃, even more preferably at a temperature of 60 ℃ to 70 ℃, even more preferably at a temperature of 65 ℃ compared to the esterase of SEQ ID N ° 1.

In a specific embodiment, the polyester degrading activity of the esterase at 65 ℃ is at least 5% higher, preferably at least 10%, 20%, 50%, 100%, 130% or more higher than the polyester degrading activity of the esterase of SEQ ID N ° 1.

In a particular embodiment, the esterase of the invention exhibits measurable esterase activity at least in the pH range of 5 to 11, preferably in the pH range of 6 to 9, more preferably in the pH range of from pH to 6.5 to 9, even more preferably in the pH range of 6.5 to 8.

Nucleic acids, expression cassettes, vectors, host cells

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

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

The invention also encompasses nucleic acids which hybridize under stringent conditions to nucleic acids encoding an esterase as defined above. Preferably, such stringent conditions include incubating the hybridization filter in 2 XSSC/0.1% SDS at about 42 ℃ for about 2.5 hours, followed by washing the filter in 1 XSSC/0.1% SDS at 65 ℃ 4 times for 15 minutes each. The protocol used is described in the reference of Sambrook et al. (Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor N.Y. (1988)) and Ausubel (Current Protocols in Molecular Biology (1989)).

The invention also encompasses a nucleic acid encoding an esterase of the invention, wherein the sequence of said nucleic acid, or at least a part of said sequence, has been engineered using optimized codon usage.

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

The nucleic acids of the invention may further comprise additional nucleotide sequences, such as regulatory regions, i.e., promoters, enhancers, silencers, terminators, signal peptides, and the like, that may be used to cause or regulate expression of the polypeptide in a selected host cell or system.

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

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

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

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

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

As used herein, the term "vector" or "expression vector" refers to a DNA or RNA molecule comprising an expression cassette of the invention, which serves as a medium for transferring recombinant genetic material into a host cell. The main types of vectors are plasmids, phages, 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 as the "backbone" of the vector. The purpose of the vector to transfer the genetic information to the host is typically to isolate, propagate or express the insert in the target cell. Vectors referred to as expression vectors (expression constructs) are particularly suitable for expressing heterologous sequences in target cells, and typically have a promoter sequence that drives expression of the heterologous sequence encoding the polypeptide. Typically, the regulatory elements present in an expression vector include a transcription promoter, a ribosome binding site, a terminator and optionally an operator. Preferably, the expression vector also comprises an origin of replication for autonomous replication in the host cell, a selectable marker, a limited number of useful restriction enzyme sites and the possibility of a high copy number. Examples of expression vectors are cloning vectors, modified cloning vectors, specifically designed plasmids and viruses. Expression vectors that provide suitable levels of expression of a polypeptide in various hosts are well known in the art. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. Preferably, the expression vector is a linear or circular double stranded DNA molecule.

It is a further object of the present invention to provide a host cell comprising a nucleic acid, expression cassette or vector as described above. The present invention therefore relates to the use of a nucleic acid, expression cassette or vector according to the invention for transforming, transfecting or transducing a host cell. The choice of vector will typically depend on the compatibility of the vector with the host cell into which it must be introduced.

According to the present invention, the host cell may be transformed, transfected or transduced in a transient or stable manner. The expression cassette or vector of the invention is introduced into a host cell such that the cassette or vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector. The term "host cell" also encompasses any progeny of a parent host cell that differs from the parent host cell due to mutations that occur during replication. The host cell may be any cell used to produce a variant of the invention, e.g., a prokaryotic cell or a eukaryotic cell. The prokaryotic host cell may be any gram-positive or gram-negative bacterium. The host cell may also be a eukaryotic cell, such as a yeast, fungal, mammalian, insect or plant cell. In a specific embodiment, the host cell is selected from the group consisting of: coli (Escherichia coli), Bacillus (Bacillus), Streptomyces (Streptomyces), Trichoderma (Trichoderma), Aspergillus (Aspergillus), Saccharomyces (Saccharomyces), Pichia (Pichia), Vibrio (Vibrio) or Yarrowia (Yarrowia).

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

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

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

Production of esterase

It is a further object of the invention to provide a process for producing an esterase of the invention, which comprises expressing a nucleic acid encoding the esterase and optionally recovering the esterase.

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

Preferably, the production method comprises

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

(b) Recovering the esterase from the cell culture.

Advantageously, the host cell is a recombinant Bacillus, a recombinant Escherichia coli, a recombinant Aspergillus, a recombinant Trichoderma, a recombinant Streptomyces, a recombinant Saccharomyces, a recombinant Pichia, a recombinant Vibrio or a recombinant yarrowia.

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

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

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

Composition comprising a metal oxide and a metal oxide

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

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

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

The composition may further comprise excipients and/or agents and the like. Suitable excipients encompass buffers commonly used in biochemistry; an agent for adjusting pH; preservatives, for example sodium benzoate, sodium sorbate or sodium ascorbate; conservatives, protectants or stabilizers, such as starch, dextrin, gum arabic, salt, sugar (e.g., sorbitol, trehalose or lactose), glycerol, polyethylene glycol, polypropylene glycol, propylene glycol; chelating agents (such as EDTA); a reducing agent; an amino acid; a carrier (e.g., a solvent or aqueous solution), and the like. The composition of the invention may be obtained by mixing the esterase with one or more excipients.

In a particular embodiment, the composition comprises from 0.1% to 99.9%, preferably from 50% to 99.9%, more preferably from 70% to 99.9%, even more preferably from 95% to 99.9% by weight of excipients, based on the total weight of the composition. Alternatively, the composition may comprise from 90% to 95% by weight excipient.

In a specific embodiment, the composition may further comprise other polypeptides exhibiting enzymatic activity. The esterase content of the invention can be readily adjusted by the skilled person, for example depending on the nature of the polyester to be degraded and/or the other enzymes/polypeptides contained in the composition.

In a particular embodiment, the esterase of the invention is dissolved in an aqueous medium together with one or more excipients, in particular excipients capable of stabilizing or protecting the polypeptide from degradation. For example, the esterase of the invention may be finally dissolved in water together with other components, such as glycerol, sorbitol, dextrin, starch, glycols (e.g., propylene glycol), salts, and the like. The resulting mixture may then be dried to obtain a powder. Methods of drying such mixtures are well known to those skilled in the art and include, but are not limited to, lyophilization, freeze drying, spray drying, supercritical drying, downdraft evaporation, thin layer evaporation, centrifugal evaporation, conveyor drying, fluidized bed drying, drum drying, or any combination thereof.

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

In a particular embodiment, the composition of the invention comprises at least one recombinant cell expressing an esterase of the invention or an extract thereof. By "cell extract" is meant any fraction obtained from a cell, such as cell supernatant, cell debris, cell wall, DNA extract, enzyme or enzyme preparation or any preparation derived from a cell by chemical, physical and/or enzymatic treatment, which is substantially free of living cells. Preferred extracts are enzymatically 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 several additional cells.

In one embodiment, the composition comprises or consists of a culture medium of a recombinant microorganism expressing and secreting an esterase of the invention. In a particular embodiment, the composition comprises such a medium that is lyophilized.

Use of esterases

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

Accordingly, an object of the present invention is the use of an esterase of the invention, or a corresponding recombinant cell or an extract thereof, or a composition for the enzymatic degradation of a polyester.

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

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

Another object of the present invention is the use of the esterase of the invention, or of its corresponding recombinant cells or extracts, or of the composition, for the enzymatic degradation of at least one polyester of a polyester-containing material.

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

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

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

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

In one embodiment, the polyester containing polyester material is completely degraded.

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

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

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

Advantageously, the degradation process is carried out at a pH of from 5 to 11, preferably at a pH of from 6 to 9, more preferably at a pH of from 6.5 to 9, even more preferably at a pH of from 6.5 to 8.

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

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

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

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

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

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 may be mixed with other monomers and/or oligomers, for example, to synthesize new copolymers. Alternatively, the recovered monomer can be used as a chemical intermediate to produce a new target compound.

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

It is a further object of the present invention to provide polyester-containing materials comprising the esterase of the invention and/or a recombinant microorganism expressing and secreting said esterase. As an example, methods for preparing such polyester-containing materials comprising the esterase of the invention are disclosed in patent applications WO2013/093355, WO 2016/198650, WO 2016/198652, WO 2019/043145 and WO 2019/043134.

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

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

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

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

Typically, the esterases of the invention may be used in detergent, food, animal feed and pharmaceutical applications. More particularly, the esterases of the invention may be used as a component of detergent compositions. Detergent compositions include, but are not limited to, hand or machine laundry detergent compositions, such as laundry additive compositions suitable for pre-treating dyed fabrics and rinse-added fabric softener compositions, detergent compositions for general household hard surface cleaning operations, detergent compositions for hand or machine dishwashing operations. In one embodiment, the esterase of the invention can be used as a detergent additive. Accordingly, the present invention provides detergent compositions comprising the esterase of the invention. In particular, the esterases of the invention can be used as detergent additives to reduce pilling and greying effects during textile cleaning.

The invention also relates to methods of using the esterases of the invention in animal feed, as well as to feed compositions and feed additives comprising the esterases of the invention. The terms "feed" and "feed composition" refer to any compound, formulation, mixture, or composition suitable for or intended for ingestion by an animal. In another embodiment, the esterase of the invention is used to hydrolyze a protein and produce a hydrolysate comprising peptides. The hydrolysate can be used as feed composition or feed additive.

Detailed Description

Example 1 construction, expression and purification of esterase

Construction of

Construction of pET26b-LCC-His using a plasmid produced an esterase according to the invention. The plasmid consists in cloning the gene encoding the esterase of SEQ ID N ° 1 between NdeI and XhoI restriction sites, optimized for escherichia coli expression. Two site-directed mutagenesis kits were used according to the supplier's recommendations to generate esterase variants: QuikChange II site-directed mutagenesis kit and QuikChange Lightning multiple site-directed mutagenesis kit from Agilent (Santa Clara, California, USA).

Expression and purification of the esterase

The strain Stellar was used continuously in 50mL LB-Miller medium or ZYM self-induction medium (Studier et al, 2005-prot. exp. Pur.41, 207-234)^TM(Clontech, California, USA) and E.coli OneBL21 DE3(Life technologies, Carlsbad, California, USA) was cloned and expressed recombinantly. LB-Miller culture with 0.5mM isopropyl beta-D-1-thiogalactopyranoside (IPTG, Euromedex, Souffelweyerheim, France) at 16 deg.CInduction in nutrient. The culture was terminated by centrifugation (8000rpm, 20 minutes at 10 ℃) in an Avanti J-26XP centrifuge (Beckman Coulter, Brea, USA). The cells were suspended in 20mL of Talon buffer (Tris-HCl 20mM, NaCl 300mM, pH 8). The cell suspension was then sonicated by an FB 705 sonicator (Fisherbrand, ilkirch, France) with an amplitude of 30% (2 second ON and 1 second OFF cycles) over 2 minutes. Then, a centrifugation step is carried out: in an Eppendorf centrifuge at 11000rpm, 10 ℃ for 30 minutes. The soluble fractions were collected and subjected to affinity chromatography. For the purification stepMetal Affinity Resin (Clontech, CA, USA) was done. Protein elution was performed with a Talon buffer step supplemented with imidazole. The purified protein was dialyzed against Talon buffer, then quantitated using the Bio-Rad protein assay according to the manufacturer's instructions (Life science Bio-Rad, France) and stored at +4 ℃.

Example 2 evaluation of the degrading Activity of esterases

The degradation activity of the esterase was determined and compared with that of the esterase of SEQ ID N.cndot.1.

Various methods have been used to assess specific activity:

(1) specific activity based on PET hydrolysis;

(2) activity based on polyester degradation in the solid state;

(3) based on the activity of PET hydrolysis in greater than 100mL reactor.

2.1 specific Activity based on hydrolysis of PET

100mg of amorphous PET (in powder form and prepared according to WO 2017/198786 so that its crystallinity is below 20%) are weighed and introduced into a 100mL glass bottle. 1mL of esterase preparation comprising SEQ ID N.cndot.1 (as reference control) or esterase of the invention prepared in Talon buffer (Tris-HCl 20mM, NaCl 0.3M, pH8) at 0.02 or 0.03mg/mL was introduced into a glass vial. Finally, 49mL of 0.1M potassium phosphate buffer (pH8) was added.

Depolymerization was initiated by incubating each glass vial in a Max Q4450 incubator (Thermo Fisher Scientific, inc. waltham, MA, USA) at 60 ℃, 65 ℃ or 70 ℃ and 150 rpm.

The initial rate of depolymerization reaction was determined by sampling at various times during the first 24 hours and by Ultra High Performance Liquid Chromatography (UHPLC) analysis, in mg of equivalent TA/hour produced. If necessary, the sample was diluted in 0.1M potassium phosphate buffer, pH8. Then, 150. mu.L of methanol and 6.5. mu.L of HCl 6N were added to 150. mu.L of the sample or dilution. After mixing and filtration on a 0.45 μm syringe filter, samples were loaded onto UHPLC to monitor the release of Terephthalic Acid (TA), MHET and BHET. The chromatographic system used was an Ultimate 3000UHPLC system (Thermo Fisher Scientific, inc. waltham, MA, USA) comprising a pump module, an autosampler, a thermostatted column oven at 25 ℃ and a UV detector at 240 nm. The column used isHSC18HPLC column (150X 4.6mm, 5 μm, equipped with a pre-column, Supelco, Bellefonte, USA). TA, MHET and BHET were separated at 1mL/min using a gradient of MeOH in 1mM H2SO4 (30% -90%). The injection volume was 20. mu.L of sample. TA, MHET and BHET were measured under the same conditions as the samples according to standard curves prepared from commercial TA and BHET and internally synthesized MHET. The specific activity of PET hydrolysis (mg equivalent TA/hr/mg enzyme) was determined in the linear portion of the hydrolysis curve of the reaction, which was established by sampling at different times over the first 24 hours. The equivalent TA corresponds to the sum of the measured TA and the TA contained in the measured MHET and BHET.

2.2 Activity based on degradation of polyesters in solid form

The induced cells, semi-purified protein extract or purified protein may be used as a composition comprising the esterase of the invention to assess the activity of such esterase.

The induced cells correspond to cell culture samples obtained after cultivation in ZYM auto-induction medium or after induction by IPTG in LB-Miller medium (as described in example 1).

Under the following protocol, a semi-purified protein extract (as described in example 1) was obtained after culture from ZYM auto-induction medium or after induction by IPTG in LB-Miller medium. The culture was stopped by centrifugation (8000rpm, 20 minutes at 10 ℃) in an Avanti J-26XP centrifuge (Beckman Coulter, Brea, USA). The cell pellet was suspended in lysis buffer (20mM Tris-HCl, pH8, 300mM NaCl). Cells were disrupted in 2h freeze/thaw cycles at-80 ℃ and then 1 μ L of lysonase bioprocessing reagent (Merck Millipore, Darmstadt, Germany) was added and incubated at 28 ℃ for 1h, including vortexing for homogenization every 15 min. Lysates were clarified by centrifugation (2250 Xg, 15min, 4 ℃). To generate a semi-purified fraction, the lysate was treated at 70 ℃ for 1h and clarified by centrifugation (2250 Xg, 15min, 4 ℃). The protein concentration of this fraction was quantified using the Bio-Rad protein assay according to the manufacturer's instructions (Life science Bio-Rad, France).

Purified protein was obtained as described in example 1.

Samples of the composition were placed on a surface or in wells created in an agar multi-purpose tray (agar omni) containing PET or another solid polyester compound (e.g., PBAT or the like) prepared as follows. Agar plates containing PET were prepared by dissolving 500mg of PET in hexafluoro-2-propanol (HFIP) and the medium was poured into 250mL of aqueous solution. After evaporation of the HFIP at 52 ℃ and 140mbar, the solution was mixed in v/v with 0.2M potassium phosphate buffer (pH8) containing 3% agar. Each multipurpose dish was prepared using about 30mL of the mixture and stored at 4 ℃.

After 2 to 24 hours, the surface area or diameter of the halo formed by degradation of the polyester by the wild-type esterase and the variant of the invention was measured and compared at 60 ℃, 65 ℃ or 70 ℃.

2.3 Activity based on hydrolysis of PET in the reactor

0.69. mu. mol to 2.07. mu. mol of The purified esterase prepared in 80mL of 100mM potassium phosphate buffer (pH8) was mixed with 20g of amorphous PET (prepared according to WO 2017/198786 so that its crystallinity is below 20%) in a 500mL Minibio bioreactor (Applikon Biotechnology, Delft, The Netherlands). The temperature was adjusted by water bath immersion at 60 ℃ and maintained with constant stirring at 250rpm using a single marine impeller. The pH of The PET depolymerization assay was adjusted to 8 by 6N NaOH and confirmed by my-Control Biocontroller System (Applikon Biotechnology, Delft, The Netherlands). Base consumption is recorded during the assay and can be used for characterization of the PET depolymerization assay.

The final yield of the PET depolymerization assay was determined by measuring the weight of PET remaining or by measuring the equivalents TA and EG produced or by base consumption. At the end of the reaction, the weight of the residual PET was assessed by filtering the reaction volume through a 12-15 μm grade 11 ashless paper filter (Dutscher SAS, Brumath, France) and drying such retentate before weighing. The equivalents TA and EG produced were determined using the UHPLC method described in 2.1 and the percent hydrolysis was calculated based on the ratio of the molar concentration ratio at a given time (TA + MHET + BHET) to the total amount of TA contained in the initial sample. Depolymerization of PET produces acidic monomers that will be neutralized with a base to be able to maintain the pH in the reactor. The equivalent TA produced was calculated using the corresponding molar amount of base consumed and the percent hydrolysis was calculated based on the ratio of the molar concentration of equivalent TA at a given time to the total amount of TA contained in the initial sample.

The specific degradation activity of the esterases (variants) of the invention is shown in Table 1 below. The specific degradation activity of the esterase of SEQ ID N ° 1 was used as reference and was considered as 100% specific degradation activity. The specific degradation activity was measured as described in example 2.1.

Table 1: specific degradation Activity of variants of the invention

The variants V1-V83 have the exact amino acid sequence shown as SEQ ID N ° 1, except for the combinations of substitutions listed in table 1, respectively.

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

Table 2: the variants of the invention have a PET depolymerization rate after 24 hours and a time to reach a PET depolymerization rate of 90%

Example 3 evaluation of the thermal stability of the esterase of the invention

The thermal stability of the esterase of the invention was determined and compared with that of the esterase of SEQ ID No 1.

Different methods were used to estimate the thermal stability:

(1) circular dichroism of proteins in solution;

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

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

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

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

(6) differential Scanning Fluorescence (DSF);

details of the protocol of these methods are given below.

3.1 circular dichroism

Circular Dichroism (CD) was performed with a Jasco 815 apparatus (Easton, USA) to compare the melting of the esterase of SEQ ID N ° 1Transition temperature (T)_m) (Tm. RTM.84.7 ℃) and the Tm of the esterase of the invention.

Technically, 400. mu.L of protein sample was prepared at 0.5mg/mL in Talon buffer and used for CD. A first scan from 280nm to 190nm was effected to determine the two maximum intensities of CD corresponding to the correct folding of the protein. Then, a second scan from 25 ℃ to 110 ℃ is performed at a wavelength corresponding to this maximum intensity and provides a specific curve (sigmoid 3 parameter y ═ a/(1+ e ^ ((x-x0)/b))) which is analyzed by Sigmaplot version 11.0 software, and T is determined when x ═ x0_m. Obtained T_mReflecting the thermostability of a given protein. T is_mThe higher the variant, the more stable it is at high temperatures.

3.2 residual esterase Activity

1mL of a 40mg/L (in Talon buffer) solution of the esterase of SEQ ID N.cndot.1 or of the esterase of the invention were incubated for 10 days at different temperatures (65, 70, 75, 80 and 90 ℃). Samples were taken periodically and diluted 1 to 500-fold in 0.1M potassium phosphate buffer, pH8.0, for p-nitrophenol-butyrate (pNP-B) assay. mu.L of the sample was mixed with 175. mu. L0.1M potassium phosphate buffer pH8.0 and 5. mu.L of pNP-B in 2-methyl-2-butanol (40 mM). The enzymatic reaction was carried out at 30 ℃ for 15 minutes with stirring, and the absorbance at 405nm was obtained by a microplate spectrophotometer (Versamax, Molecular Devices, Sunnyvale, Calif., USA). The activity of pNP-B hydrolysis was determined using a standard curve of p-nitrophenol released in the linear part of the hydrolysis curve (initial rate expressed as. mu. mol pNPB/min).

3.3 depolymerization Activity of residual polyester

10mL of a solution of the esterase of SEQ ID No 1 and of the esterase of the invention at 40mg/L (in Talon buffer) were incubated for 1 to 30 days at different temperatures (65, 70, 75, 80 and 90 ℃ C.), respectively. Periodically, 1mL of a sample was taken and transferred to a vial containing 100mg of amorphous PET micronized to 250-500 μ M (prepared according to WO 2017/198786 so that its crystallinity is less than 20%) and 49mL of 0.1M potassium phosphate buffer pH8.0, and incubated at 65 ℃. Periodically 150. mu.L of buffer was sampled. If necessary, the sample was diluted in 0.1M potassium phosphate buffer, pH8. Then, 150. mu.L of methanol and 6.5. mu.L of HCl 6N were added to 150. mu.L of the sample orIn a diluent. After mixing and filtration on a 0.45 μm syringe filter, samples were loaded onto UHPLC to monitor the release of Terephthalic Acid (TA), MHET and BHET. The chromatographic system used was an Ultimate 3000UHPLC system (Thermo Fisher Scientific, inc. waltham, MA, USA) comprising a pump module, an autosampler, a thermostatted column oven at 25 ℃ and a UV detector at 240 nm. The column used isHSC18HPLC columns (150X 4.6mm, 5 μm, equipped with a pre-column, Supelco, Bellefonte, USA). 1mM H was used₂SO₄Gradient MeOH (30% -90%) in 1mL/min separated TA, MHET, and BHET. The injection volume was 20. mu.L of sample. TA, MHET and BHET were measured according to commercial TA and BHET and internally synthesized MHET under the same conditions as the samples. The activity of PET hydrolysis (μmol hydrolyzed PET/min or equivalent TA/hour of mg produced) was determined in the linear portion of the hydrolysis curve, which was established by sampling at different times over the first 24 hours. The equivalent TA corresponds to the sum of the measured TA and the TA contained in the measured MHET and BHET. 3.4 degradation of the polyester in solid form

1mL of a 40mg/L (in Talon buffer) solution of the esterase of SEQ ID No 1 and the esterase of the invention, respectively, were incubated at different temperatures (65, 70, 75, 80 and 90 ℃) for 1 to 30 days. Periodically, 20. mu.L of enzyme preparation was placed in wells created on agar plates containing PET. Agar plates containing PET were prepared by dissolving 500mg of PET in hexafluoro-2-propanol (HFIP) and the medium was poured into 250mL of aqueous solution. After evaporation of the HFIP at 52 ℃ and 140mbar, the solution was mixed in v/v with 0.2M potassium phosphate buffer (pH8) containing 3% agar. Each multipurpose dish was prepared using about 30mL of the mixture and stored at 4 ℃.

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

3.5 depolymerization of the polyesters in multiple rounds

Esterases were evaluated in enzyme reactors for their ability to perform several successive rounds of polyester depolymerization assays. The Minibio500 bioreactor (Applikon Biotechnology B.V., Delft, The Netherlands) was started with 3g amorphous PET (prepared according to WO 2017/198786 to give a crystallinity of less than 20%) and 100mL of 10mM potassium phosphate buffer (pH8) containing 3mg LC-esterase. The agitation was set to 250rpm using a marine impeller. The bioreactor was thermostatted at 60 ℃, 65 ℃ or 70 ℃ by immersion in an external water bath. The pH was adjusted to 8 by addition of 3M KOH. The different parameters (pH, temperature, stirring, addition of base) were monitored by the BioXpert software V2.95. 1.8g of amorphous PET was added every 20 hours. Periodically, 500. mu.L of the reaction medium were sampled.

The contents of TA, MHET and BHET were determined by HPLC as described in example 2.3. EG content was determined using an Aminex HPX-87K column (Bio-Rad Laboratories, Inc, Hercules, California, United States) thermostated at 65 ℃. The eluent is 0.6mL.min^-1K of₂HPO₄5 mM. The injection volume was 20. mu.L. Ethylene glycol was monitored using a refractometer.

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

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

3.6 Differential Scanning Fluorescence (DSF)

DSF was used to assess the thermostability of the wild-type protein (SEQ ID N ° 1) and variants thereof by determining its melting temperature (Tm, i.e. the temperature at which half of the protein population was expanded). Protein samples were prepared at a concentration of 14. mu.M (0.4mg/mL) and stored in buffer A consisting of 20mM Tris HCl (pH 8.0), 300mM NaCl. A stock solution of SYPRO orange dye 5000x in DMSO was first diluted to 250x in water. Protein samples were loaded onto white clear 96-well PCR plates (Bio-Rad cat # HSP9601) where each well contained a final volume of 25. mu.l. The final concentration of protein and SYPRO Orange dye in each well was 5. mu.M (0.14mg/ml) and 10X, respectively. The volumes loaded per well were as follows: mu.L of buffer A, 9. mu.L of 0.4mg/mL protein solution and 1. mu.L of 250 XSypro Orange dilution. The PCR plate was then sealed with optical quality sealing tape and spun at 2000rpm for 1 minute at room temperature. Then, DSF experiments were performed using a CFX96 real-time PCR system configured to use 450/490 excitation and 560/580 emission filters. The sample was heated from 25 ℃ to 100 ℃ at a rate of 0.3 ℃/sec. A single fluorescence measurement was taken every 0.03 seconds. The melting temperature was determined from the peak of the first derivative of the melting curve using Bio-Rad CFX Manager software.

Then, the esterase of SEQ ID N.cndot.1 and the esterase of the invention were compared based on their Tm values. Due to the high reproducibility between experiments from different produced identical proteins, the Δ Tm at 0.8 ℃ was considered significant for the comparative variants. The Tm values correspond to the average of at least 3 measurements. The Tm of the esterase of SEQ ID N.cndot.1 was evaluated at 84.7 ℃.

The thermostability of the esterase variants of the invention is shown in table 3 below, expressed as Tm values and evaluated according to example 2.6. The increase in Tm compared to the esterase of SEQ ID N.cndot.1 is shown in parentheses.

Table 3: tm of the esterase of the invention compared with SEQ ID No 1

The variants V1-V83 have the exact amino acid sequence shown as SEQ ID N ° 1, except for the combinations of substitutions listed in table 3, respectively.