Enzyme variants and uses thereof

Abstract

The present disclosure relates generally to polypeptides having mono-(2-hydroxyethyl)terephthalate hydrolase (MHETase) activity, and more specifically, to polypeptides comprising an amino acid sequence that (i) has at least 70% sequence identity to SEQ ID NO:1, and (ii) differs from SEQ ID NO:1 by amino acid substitutions at one or more positions selected from the group consisting of positions corresponding to amino acids 156-396, 398-410, and 425-603 of SEQ ID NO:1.

Description

[Technical field]

[0001] The present invention relates to a novel enzyme, more particularly to a recombinant enzyme that hydrolyzes the ester bond of mono-(2-hydroxyethyl)terephthalic acid, and uses thereof. [Background technology]

[0002] All references, including any patents or patent applications cited in this specification are hereby incorporated by reference in order to enable a full understanding of the present invention, however such references should not be construed as amounting to an acknowledgement that any of these documents form part of the common general knowledge in the art, in Australia or in any other country.

[0003] Global industrialization has had a major impact on the environment, most notably the increased production of and reliance on plastics and plastic products. Although there have been increasing efforts to find suitable and environmentally sustainable alternatives to plastics, including their production and disposal, such products remain a major problem and contribute to the majority of environmental pollution. One of the major contributors to this problem is polyethylene terephthalate (PET), which is produced in millions of tons each year around the world, and its waste. The environmental significance of this problem is due, at least in part, to the chemical properties of plastics, particularly PET-based products, as they do not decompose easily in nature.

[0004] Approaches to addressing the problem of plastic waste have typically included incineration, landfilling, and mechanical decomposition. However, these approaches also have significant environmental impacts. For example, incineration of plastic produces potentially harmful by-products that are released into the atmosphere; the rate at which plastic decomposes in landfills is very slow, risking the leaching of toxic substances into groundwater; and mechanical decomposition is relatively expensive, often limiting the use of the by-products.

[0005] More recently, biological (enzymatic) degradation of plastics has been considered an alternative approach to reduce waste plastic accumulation. This approach involves the use of PETase, an enzyme of the esterase class that catalyzes the hydrolysis of PET to the monomer mono-2-hydroxyethyl terephthalate (MHET). MHETase can also be used to hydrolyze MHET to terephthalate and ethylene glycol. These hydrolysates can then be suitably recycled as materials for the production of new products, including plastics. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Palm et al. (2019, Nat. Comms. 10:1717) [Non-Patent Document 2] Sagong et al. (2020, ACS Catal. 10:4805) [Non-Patent Document 3] Yoshida et al. (2020, Science, 352(6278):1196) [Non-Patent Document 4] http: / / blast.ncbi.nlm.nih.gov / [Non-Patent Document 5] http: / / www.ebi.ac.uk / Tools / emboss / [Non-Patent Document 6] Belousov (1997) Nucleic Acids Res. 25:3440~3444 [Non-Patent Document 7] Raducanu et al. (2020, Journal of Biological Chemistry 295(34):12214~12223) Summary of the Invention [Problem to be solved by the invention]

[0007] Although the enzymatic degradation of plastics is an attractive alternative for reducing the environmental impact of plastic waste and their disposal, it has not yet seen widespread adoption due to reasons including its relative inefficiency, slow enzymatic degradation rates, and low enzyme expression levels in common industrial host strains. Thus, there remains a pressing need for improved methods and reagents for the enzymatic degradation of plastics. [Means for solving the problem]

[0008] In one aspect disclosed herein, there is provided a polypeptide having mono-(2-hydroxyethyl)terephthalate hydrolase (MHETase) activity, the polypeptide comprising an amino acid sequence that (i) has at least 70% sequence identity to SEQ ID NO:1 and (ii) differs from SEQ ID NO:1 by amino acid substitutions at one or more positions selected from the group consisting of positions corresponding to amino acids 156-396, 398-410, and 425-603 of SEQ ID NO:1.

[0009] In another aspect, (i) a position corresponding to amino acid 156 of SEQ ID NO:1; (ii) a position corresponding to amino acid 159 of SEQ ID NO:1; (iii) a position corresponding to amino acid 192 of SEQ ID NO:1; and (iv) a position corresponding to amino acid 503 of SEQ ID NO:1 The present invention provides a polypeptide comprising an amino acid sequence that differs from SEQ ID NO:1 by an amino acid substitution at one or more positions selected from the group consisting of:

[0010] In one embodiment, the polypeptide differs from SEQ ID NO: 1 by amino acid substitutions at positions corresponding to amino acids 159, 252, and 503 of SEQ ID NO: 1. In another embodiment, the amino acid substitutions are T159V, Y252F, and Y503W, or conservative amino acid substitutions for any of the foregoing.

[0011] In another embodiment, the polypeptide differs from SEQ ID NO: 1 by amino acid substitutions at positions corresponding to amino acids 159, 192, 252, and 503 of SEQ ID NO: 1. In another embodiment, the amino acid substitutions are T159V, M192Y, Y252F, and Y503W, or conservative amino acid substitutions for any of the foregoing.

[0012] In another aspect, a polypeptide having mono-(2-hydroxyethyl)terephthalate hydrolase (MHETase) activity is provided, the polypeptide having (a) at least 70% sequence identity to SEQ ID NO:1; and (b) (i) a position corresponding to amino acid 156 of SEQ ID NO:1; (ii) a position corresponding to amino acid 159 of SEQ ID NO:1; (iii) a position corresponding to amino acid 196 of SEQ ID NO:1; (iv) a position corresponding to amino acid 197 of SEQ ID NO:1; (v) a position corresponding to amino acid 260 of SEQ ID NO:1; (vi) a position corresponding to amino acid 264 of SEQ ID NO:1; (vii) a position corresponding to amino acid 267 of SEQ ID NO:1; (viii) a position corresponding to amino acid 286 of SEQ ID NO:1; and (ix) a position corresponding to amino acid 503 of SEQ ID NO: 1 The present invention provides a polypeptide comprising an amino acid sequence that differs from SEQ ID NO:1 by an amino acid substitution at one or more positions selected from the group consisting of:

[0013] In some embodiments, the polypeptides disclosed herein have, compared to the MHETase of SEQ ID NO:1: (i) Increased recombinant expression in the host system; (ii) increased whole cell activity; and (iii) Increased thermal stability The present invention further includes one or more of the following:

[0014] The present disclosure also extends to compositions comprising the polypeptides described herein.

[0015] The present disclosure also extends to nucleic acid sequences encoding the polypeptides described herein. In certain embodiments, the nucleic acid sequences encoding the polypeptides are selected from SEQ ID NOs: 37-72 and 79-84. In preferred embodiments, the nucleic acid sequences encoding the polypeptides are selected from SEQ ID NOs: 79-84.

[0016] The present disclosure also extends to expression vectors comprising the nucleic acid sequences described herein.

[0017] The present disclosure also extends to host cells comprising the nucleic acid sequences or expression vectors described herein.

[0018] In another aspect, the disclosure provides a method for producing a polypeptide having MHETase activity, comprising: a) providing a nucleic acid sequence as described herein; b) producing the polypeptide by expressing the nucleic acid sequence in a host cell culture; and c) harvesting the polypeptide produced in (b) from the host cell culture. The present invention provides a method comprising:

[0019] In yet another aspect, a method of hydrolyzing mono-(2-hydroxyethyl) terephthalate is provided, comprising exposing mono-(2-hydroxyethyl) terephthalate to a polypeptide, composition, or host cell described herein under conditions sufficient to convert mono-(2-hydroxyethyl) terephthalate to terephthalate and ethylene glycol.

[0020] The present disclosure also extends to a method of degrading a plastic article comprising polyester, the method comprising exposing the plastic article to a polypeptide, composition or host cell described herein.

[0021] In another embodiment, the methods disclosed herein include: (i) exposing PET to polyethylene terephthalate esterase (PETase) under conditions sufficient for the PETase to catalyze the conversion of PET to produce mono-(2-hydroxyethyl) terephthalate (MHET); and (ii) exposing the MHET produced in step (a) to a polypeptide, composition or host cell described herein, either simultaneously or sequentially, under conditions sufficient for the polypeptide to catalyze the hydrolysis of MHET to yield terephthalate and ethylene glycol. Includes.

[0022] The present disclosure also extends to compositions comprising the terephthalate and / or ethylene glycol recovered by the methods disclosed herein.

[0023] In another aspect, there is provided a host cell genetically engineered to express a polypeptide described herein. [Brief description of the drawings]

[0024] [Figure 1] FIG. 1 shows the amino acid sequences of wild-type (WT) MHETase (SEQ ID NO: 1) and different consensus designs (SEQ ID NOs: 2-36). [Diagram 2] FIG. 1 shows the nucleic acid sequences of wild-type (WT) MHETase (SEQ ID NO: 37) and different consensus designs (SEQ ID NOs: 38-72). [Diagram 3] FIG. 1 shows the activity (dA465 / dt(min −1 )) of MHETase variants against an analogue of MHET (1-naphthyl terephthalate) in whole cell suspensions. [Figure 4] FIG. 1 shows the expression levels of wild-type MHETase (WT; SEQ ID NO: 1) and MHETase variants containing point mutations, including MHETase variant N156G+T159V, in soluble cell lysates by SDS-PAGE gel electrophoresis and staining with NTA-Atto550 (Sigma). [Diagram 5]FIG. 1 shows the thermal stability of purified wild-type MHETase (WT) and MHETase variants containing point mutations N156G+T159V, N156G+T159V+Y197V and N156G+T159V+YY503W as determined by circular dichroism at 222 nm (Y-axis) over the temperature range of 20-90°C (X-axis). [Figure 6] Figure 1 shows whole cell suspension FastBlue assay results from each mutagenesis round for all MHETase variants tested. Bar heights represent the average activity (dA465 / dt(min-1)) measured for each variant (n>2, individual measurements shown) and error bars represent the standard error mean of the measurement. Highlighted bars represent the variants used as parents in the next mutagenesis round. [Figure 7] FIG. 1 shows an SDS-PAGE gel of selected MHETases from each round stained using ATTO550 and imaged under UV transillumination. The expected size of the MHETase variants (approximately 64 kDa) is indicated. [Figure 8] FIG. 1 shows size exclusion chromatograms of selected MHETase variants. [Figure 9] Figure 1 shows Michaelis-Menten plots for selected MHETase variants obtained using the chromogenic assay described herein. Each point represents the average initial velocity of the reaction from three technical replicates, each incubated with 6 nM MHETase and 4 mM Fast Blue B salt. Error bars represent standard error. [Figure 10] Figure 1 shows the thermal stability of MHETase variants from three technical replicates measured by circular dichroism at 222 nm in sodium acetate pH 5.1. Data are fitted to a two-state unfolding model (lines) and error bars correspond to standard error. [Figure 11]FIG. 13 shows an HPLC assay comparing the activity of wild-type MHETase, round 5 Y252F (R5), and reversion of R5 to wild-type MHETase identity at positions 192, 156, 159, 252 and 503. [Figure 12] Figure 1 shows whole cell suspension FastBlue assay results for MHETase R5 reversion mutations. Mutations V159T, Y192M, F252Y, and W503Y were added to the background of MHETase R5 (MHETase Y252F in round 5). Bar heights represent the average activity measured for each variant (n > 2) and error bars represent the standard error of measurement. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For purposes of this invention, the following terms are defined below.

[0026] The articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article, unless expressly stated otherwise. By way of example, "an element" means one element or more than one element.

[0027] As used herein, the term "about" refers to a quantity, level, value, dimension, size or amount that varies by as much as 10% (e.g., 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%) from a reference quantity, level, value, dimension, size or amount.

[0028] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" are to be understood as implying the inclusion of a stated step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements.

[0029] The present disclosure is based, at least in part, on the inventors' unexpected discovery that certain modifications can be made to the amino acid sequence of mono-(2-hydroxyethyl)terephthalate hydrolase (MHETase) that advantageously enhance its MHETase activity. Certain modifications also unexpectedly improved expression of the modified MHETase when expressed in a host cell. Certain modifications surprisingly improved whole cell activity of the modified MHETase when expressed in a host cell. Certain modifications also unexpectedly improved thermostability of the enzyme. The inventors have also unexpectedly discovered that substitutions can be made to amino acid residues that are outside the active site of wild-type MHETase to improve the activity and expression of the MHETase compared to wild-type MHETase. Thus, in one aspect disclosed herein, there is provided a polypeptide having mono-(2-hydroxyethyl)terephthalate hydrolase (MHETase) activity, comprising an amino acid sequence that (i) has at least 70% sequence identity to SEQ ID NO:1 and (ii) differs from SEQ ID NO:1 by an amino acid substitution at one or more positions that do not otherwise contact a polyester substrate of the MHETase, wherein the MHETase activity of the polypeptide is greater than the MHETase activity of the MHETase of SEQ ID NO:1.

[0030] In another aspect disclosed herein, there is provided a polypeptide having mono-(2-hydroxyethyl)terephthalate hydrolase (MHETase) activity, the polypeptide comprising an amino acid sequence that (i) has at least 70% sequence identity to SEQ ID NO:1 and (ii) differs from SEQ ID NO:1 by amino acid substitutions at one or more positions selected from the group consisting of positions corresponding to amino acids 156-396, 398-410, and 425-603 of SEQ ID NO:1.

[0031] By "at least 70%" it is meant that the polypeptide shares at least 70%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 92%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, or more preferably 99% sequence identity to SEQ ID NO: 1. Since the polypeptides described herein are variants of the naturally occurring (wild type) MHETase of SEQ ID NO: 1, it should be understood in this context that "at least 70%" does not include 100% sequence identity across the entire sequence of SEQ ID NO: 1 (residues 1-603 or residues 18-603). In some embodiments, the polypeptides may include amino acid insertions and / or deletions, for example amino acid insertions and / or deletions at the N-terminus and / or C-terminus, provided that the modified polypeptide has greater MHETase activity compared to the MHETase of SEQ ID NO: 1 described herein.

[0032] In another aspect, a polypeptide having mono-(2-hydroxyethyl)terephthalate hydrolase (MHETase) activity is provided, the polypeptide having (a) at least 70% sequence identity to SEQ ID NO:1; and (b) (i) a position corresponding to amino acid 156 of SEQ ID NO:1; (ii) a position corresponding to amino acid 159 of SEQ ID NO:1; (iii) a position corresponding to amino acid 192 of SEQ ID NO:1; and (iv) a position corresponding to amino acid 503 of SEQ ID NO:1 The present invention provides a polypeptide comprising an amino acid sequence that differs from SEQ ID NO:1 by an amino acid substitution at one or more positions selected from the group consisting of:

[0033] In one embodiment, the amino acid sequence of the polypeptide differs from SEQ ID NO:11 by an amino acid substitution at a position corresponding to amino acid 156 of SEQ ID NO:1.

[0034] In one embodiment, the amino acid substitution at the position corresponding to amino acid 156 of SEQ ID NO:1 is N156G, or a conservative amino acid substitution thereof.

[0035] In one embodiment, the amino acid sequence of the polypeptide differs from SEQ ID NO:1 by an amino acid substitution at a position corresponding to amino acid 159 of SEQ ID NO:1.

[0036] In one embodiment, the amino acid substitution at the position corresponding to amino acid 159 of SEQ ID NO:1 is T159V, or a conservative amino acid substitution thereof.

[0037] In one embodiment, the amino acid sequence of the polypeptide differs from SEQ ID NO:1 by an amino acid substitution at a position corresponding to amino acid 252 of SEQ ID NO:1.

[0038] In one embodiment, the amino acid substitution at the position corresponding to amino acid 156 of SEQ ID NO:1 is Y252F, or a conservative amino acid substitution thereof.

[0039] In one embodiment, the amino acid sequence of the polypeptide differs from SEQ ID NO:1 by an amino acid substitution at a position corresponding to amino acid 503 of SEQ ID NO:1.

[0040] In one embodiment, the amino acid substitution at the position corresponding to amino acid 156 of SEQ ID NO:1 is Y503W, or a conservative amino acid substitution thereof.

[0041] In certain embodiments, the amino acid sequence of the polypeptide differs from SEQ ID NO: 1 by amino acid substitutions at positions corresponding to amino acids 159, 252, and 503 of SEQ ID NO: 1. In preferred embodiments, the amino acid substitutions are T159V, Y252F, and Y503W, or conservative amino acid substitutions for any of the foregoing.

[0042] In certain embodiments, the amino acid sequence of the polypeptide differs from SEQ ID NO: 1 by amino acid substitutions at positions corresponding to amino acids 159, 192, 252, and 503 of SEQ ID NO: 1. In preferred embodiments, the amino acid substitutions are T159V, M192Y, Y252F, and Y503W, or conservative amino acid substitutions for any of the foregoing.

[0043] In certain embodiments, the amino acid sequence of the polypeptide differs from SEQ ID NO: 1 by amino acid substitutions at positions corresponding to amino acids 159, 192, and 503 of SEQ ID NO: 1. In preferred embodiments, the amino acid substitutions are T159V, M192Y, and Y503W, or conservative amino acid substitutions for any of the foregoing.

[0044] In certain embodiments, the amino acid sequence of the polypeptide differs from SEQ ID NO: 1 by amino acid substitutions at positions corresponding to amino acids 156, 159, and 503 of SEQ ID NO: 1. In preferred embodiments, the amino acid substitutions are N156G, T159V, and Y503W, or conservative amino acid substitutions for any of the foregoing.

[0045] In certain embodiments, the amino acid sequence of the polypeptide differs from SEQ ID NO: 1 by amino acid substitutions at positions corresponding to amino acids 156, 159, and 503 of SEQ ID NO: 1. In preferred embodiments, the amino acid substitutions are N156G, T159V, and Y503W, or conservative amino acid substitutions for any of the foregoing.

[0046] In another aspect, a polypeptide having mono-(2-hydroxyethyl)terephthalate hydrolase (MHETase) activity is provided, the polypeptide having (a) at least 70% sequence identity to SEQ ID NO:1; and (b) (i) a position corresponding to amino acid 156 of SEQ ID NO:1; (ii) a position corresponding to amino acid 159 of SEQ ID NO:1; (iii) a position corresponding to amino acid 196 of SEQ ID NO:1; (iv) a position corresponding to amino acid 197 of SEQ ID NO:1; (v) a position corresponding to amino acid 260 of SEQ ID NO:1; (vi) a position corresponding to amino acid 264 of SEQ ID NO:1; (vii) a position corresponding to amino acid 267 of SEQ ID NO:1; (viii) a position corresponding to amino acid 286 of SEQ ID NO:1; and (ix) a position corresponding to amino acid 503 of SEQ ID NO: 1 and wherein the amino acid sequence differs from SEQ ID NO:1 by an amino acid substitution at one or more positions selected from the group consisting of: has greater whole cell MHETase activity compared to the polypeptide of SEQ ID NO:1; Polypeptides are provided.

[0047] In one embodiment, the amino acid sequence of the polypeptide differs from SEQ ID NO:1 by an amino acid substitution at a position corresponding to amino acid 156 of SEQ ID NO:1.

[0048] In one embodiment, the amino acid substitution at the position corresponding to amino acid 156 of SEQ ID NO:1 is N156G, or a conservative amino acid substitution thereof.

[0049] In one embodiment, the amino acid sequence of the polypeptide differs from SEQ ID NO:1 by an amino acid substitution at a position corresponding to amino acid 159 of SEQ ID NO:1.

[0050] In one embodiment, the amino acid substitution at the position corresponding to amino acid 159 of SEQ ID NO:1 is T159V, or a conservative amino acid substitution thereof.

[0051] In one embodiment, the amino acid sequence of the polypeptide differs from SEQ ID NO:1 by an amino acid substitution at a position corresponding to amino acid 196 of SEQ ID NO:1.

[0052] In one embodiment, the amino acid substitution at the position corresponding to amino acid 196 of SEQ ID NO:1 is S196A, or a conservative amino acid substitution thereof.

[0053] In one embodiment, the amino acid sequence of the polypeptide differs from SEQ ID NO:1 by an amino acid substitution at a position corresponding to amino acid 197 of SEQ ID NO:1.

[0054] In one embodiment, the amino acid substitution at the position corresponding to amino acid 197 of SEQ ID NO:1 is Y197V, or a conservative amino acid substitution thereof.

[0055] In one embodiment, the amino acid sequence of the polypeptide differs from SEQ ID NO:1 by an amino acid substitution at a position corresponding to amino acid 260 of SEQ ID NO:1.

[0056] In one embodiment, the amino acid substitution at the position corresponding to amino acid 260 of SEQ ID NO:1 is S260A, or a conservative amino acid substitution thereof.

[0057] In one embodiment, the amino acid sequence of the polypeptide differs from SEQ ID NO:1 by an amino acid substitution at a position corresponding to amino acid 264 of SEQ ID NO:1.

[0058] In one embodiment, the amino acid substitution at the position corresponding to amino acid 264 of SEQ ID NO:1 is S264L, or a conservative amino acid substitution thereof.

[0059] In one embodiment, the amino acid sequence of the polypeptide differs from SEQ ID NO:1 by an amino acid substitution at a position corresponding to amino acid 267 of SEQ ID NO:1.

[0060] In one embodiment, the amino acid substitution at the position corresponding to amino acid 267 of SEQ ID NO:1 is S267A, or a conservative amino acid substitution thereof.

[0061] In one embodiment, the amino acid sequence of the polypeptide differs from SEQ ID NO:1 by an amino acid substitution at a position corresponding to amino acid 286 of SEQ ID NO:1.

[0062] In one embodiment, the amino acid substitution at the position corresponding to amino acid 286 of SEQ ID NO:1 is S286A, or a conservative amino acid substitution thereof.

[0063] In one embodiment, the amino acid sequence of the polypeptide differs from SEQ ID NO:1 by an amino acid substitution at a position corresponding to amino acid 503 of SEQ ID NO:1.

[0064] In one embodiment, the amino acid substitution at the position corresponding to amino acid 503 of SEQ ID NO:1 is Y503W, or a conservative amino acid substitution thereof.

[0065] The present disclosure also contemplates combinations of amino acid substitutions at two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) positions corresponding to positions in SEQ ID NO:1 as described herein. In certain embodiments, a polypeptide includes a combination of amino acid substitutions at at least two, preferably at least three, preferably at least four, preferably at least five, preferably at least six, preferably at least seven, preferably at least eight, preferably at least nine, or more preferably at least ten positions corresponding to positions in SEQ ID NO:1 as described herein.

[0066] In one embodiment, the amino acid sequence of the polypeptide differs from SEQ ID NO:1 by amino acid substitutions at positions corresponding to amino acids 156, 159, and 197 of SEQ ID NO:1.

[0067] In certain embodiments, the amino acid substitutions are N156G, T159V and Y197V, or conservative amino acid substitutions for any of the foregoing.

[0068] The present disclosure also extends to compositions comprising the polypeptides described herein.

[0069] The present disclosure also extends to nucleic acid sequences encoding the polypeptides described herein.

[0070] The present disclosure also extends to expression vectors comprising the nucleic acid sequences described herein.

[0071] The present disclosure also extends to host cells comprising the nucleic acid sequences or expression vectors described herein.

[0072] In another aspect, the disclosure provides a method for producing a polypeptide having MHETase activity, comprising: a) providing a nucleic acid sequence as described herein; b) producing the polypeptide by expressing the nucleic acid sequence in a host cell culture; and c) harvesting the polypeptide produced in (b) from the host cell culture. The present invention provides a method comprising:

[0073] In yet another aspect, a method of hydrolyzing mono-(2-hydroxyethyl) terephthalate is provided, comprising exposing mono-(2-hydroxyethyl) terephthalate to a polypeptide, composition, or host cell described herein under conditions sufficient to convert mono-(2-hydroxyethyl) terephthalate to terephthalate and ethylene glycol.

[0074] The present disclosure also extends to a method of degrading a plastic article comprising polyester, the method comprising exposing the plastic article to a polypeptide, composition or host cell described herein.

[0075] In some embodiments, the polyester is selected from the group consisting of polylactic acid (PLA), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polyethylene terephthalate (PET), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), polycaprolactone (PCL), polyethylene adipate) (PEA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), and combinations of any of the foregoing. In some embodiments, the polyester is polyethylene terephthalate (PET).

[0076] In another embodiment, the methods disclosed herein include: (a) exposing PET to polyethylene terephthalate esterase (PETase) under conditions sufficient for the PETase to catalyze the conversion of PET to produce mono-(2-hydroxyethyl) terephthalate (MHET); and (b) exposing the MHET produced in step (a) to a polypeptide according to any one of claims 1 to 21, a composition according to claim 22, or a host cell according to claim 25, either simultaneously or sequentially, under conditions sufficient for the polypeptide to catalyze the hydrolysis of MHET to yield terephthalate and ethylene glycol. Includes.

[0077] In certain embodiments, the method further comprises recovering the terephthalate and / or ethylene glycol produced in step (b).

[0078] The present disclosure also extends to compositions comprising the terephthalate and / or ethylene glycol recovered by the methods disclosed herein.

[0079] In another aspect, there is provided a host cell genetically engineered to express a polypeptide described herein.

[0080] As used herein, the terms "peptide", "polypeptide", "protein" and "enzyme" are understood to refer to a chain of amino acids linked by peptide bonds, regardless of the number of amino acids forming said chain. Amino acids are generally represented by their one-letter or three-letter abbreviations 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).

[0081] The term "hydrolase" refers to an enzyme belonging to the class of hydrolases, classified as EC 3 according to the Enzyme Nomenclature, that catalyzes the hydrolysis of peptide bonds in peptides or proteins to generate shorter peptides.

[0082] The terms "wild-type" or "parent" are used interchangeably herein to refer to a naturally occurring isoform of a polypeptide, i.e., as it occurs in nature. In this disclosure, a wild-type polypeptide refers to mono-(2-hydroxyethyl)terephthalate hydrolase (EC 3.1.1.102; UniProt Accession No. A0A0K8P8E7) having the amino acid sequence set forth in SEQ ID NO:1.

[0083] Two recently discovered bacterial enzymes that specifically degrade polyethylene terephthalate (PET), as described by Palm et al. (2019, Nat. Comms. 10:1717), represent promising solutions for otherwise environmentally burdensome polyester-containing products. First, Ideonella sakaiensis PETase, a structurally well-characterized α / β-hydrolase fold enzyme, converts PET to mono-(2-hydroxyethyl) terephthalate (MHET). The second essential enzyme, MHETase, hydrolyzes MHET to the PET extracts terephthalate and ethylene glycol (Palm et al. (2019, Nat. Comm., 10:1717); Sagong et al. (2020, ACS Catal. 10:4805); and Yoshida et al. (2020, Science, 352(6278):1196).

[0084] The amino acid and nucleic acid sequences of wild-type MHETase will be familiar to those skilled in the art, an illustrative example of which is SEQ ID NO:1.

[0085] The terms "mutant" and "variant" may be used interchangeably herein to refer to a polypeptide comprising an amino acid sequence derived from SEQ ID NO: 1, further comprising a modification or alteration (e.g., substitution, insertion and / or deletion) at one or more (e.g., several) positions compared to the polypeptide of SEQ ID NO: 1, and having enhanced MHETase activity. Such variants may be obtained by a variety of techniques well known in the art, illustrative examples of which include site-directed mutagenesis, random mutagenesis and synthetic oligonucleotide construction. The terms "modification", "alteration", "substitution" and the like, when used herein in reference to an amino acid residue or position, generally mean that the amino acid at a particular position has been altered compared to that of the wild-type or parent polypeptide.

[0086] Suitable substitutions include replacing one amino acid residue with another, which is selected from the 20 standard naturally occurring amino acid residues, the less commonly occurring amino acid residues (e.g., hydroxyproline, hydroxylysine, allohydroxylysine, 6-N-methyllysine, N-ethylglycine, N-methylglycine, N-ethylasparagine, alloisoleucine, N-methylisoleucine, N-methylvaline, pyroglutamine, aminobutyric acid, ornithine, norleucine, norvaline), and often synthetically produced non-naturally occurring amino acid residues (e.g., cyclohexyl-alanine).Preferably, the substitution includes replacing one amino acid residue with another, which is selected from the 20 standard naturally occurring amino acid residues (G, P, A, V, L, I, M, C, F, Y, W, H, K, R, Q, N, E, D, S, and T). Modifications or alterations may be identified herein using the following terminology: Y197V indicates a substitution of valine (V) for the amino acid residue tyrosine (Y) at position 197 of the parent polypeptide sequence. Y197V / I / M indicates a substitution of one of the following amino acids: valine (V), isoleucine (I) or methionine (M) for the amino acid residue tyrosine (Y) at position 197 of the parent polypeptide sequence. The substitution may be a conservative or non-conservative substitution. Examples of conservative substitutions will be familiar to those skilled in the art. Illustrative examples of these include substitutions within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine, asparagine and threonine), hydrophobic amino acids (methionine, leucine, isoleucine, cysteine ​​and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine and serine).

[0087] Unless otherwise specified, positions disclosed in this application are numbered by reference to the amino acids set forth in SEQ ID NO: 1. In this context, the term "corresponding to," when used in reference to an amino acid position, is intended to mean an amino acid position within a polypeptide sequence when that position is aligned with the equivalent or corresponding position in the sequence set forth in SEQ ID NO: 1.

[0088] As used herein, the term "sequence identity" or "identity" refers to the number of matches (identical amino acid residues) between two polypeptide sequences (or the fraction expressed as a percentage %). In a preferred embodiment, sequence identity is determined by aligning and comparing sequences to maximize overlap and identity while minimizing sequence gaps. Depending on the length of the two sequences, sequence identity can be determined using any of a number of mathematical global or local alignment algorithms known to those skilled in the art. Sequences of similar length can be aligned using a global alignment algorithm (e.g., the Needleman and Wunsch algorithm; Needleman and Wunsch, 1970), which optimally aligns sequences over their entire length, while sequences of significantly different lengths are preferably aligned using a local alignment algorithm (e.g., the Smith and Waterman algorithm (Smith and Waterman, 1981) or the Altschul algorithm (Altschul et al., 1997; Altschul et al., 2005)). Alignment for the purpose of determining percent amino acid sequence identity can be achieved by any means available to those skilled in the art, and illustrative examples of such means include publicly available computer software, for example, available at http: / / blast.ncbi.nlm.nih.gov / or http: / / www.ebi.ac.uk / Tools / emboss / . Those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the entire length of the sequences being compared. As used herein, percent sequence identity generally refers to the value generated using pairwise sequence alignment to create an optimal global alignment of two sequences (for example, using the Needleman-Wunsch algorithm), with all search parameters set to default values, for example, score matrix=BLOSUM62, gap start=10, gap extension=0.5, end gap penalty=false, end gap start=10 and end gap extension=0.5.

[0089] The term "recombinant," as used herein, generally refers to a nucleic acid construct, vector, polypeptide, or cell that has been produced by genetic engineering.

[0090] The term "expression," as used herein, generally refers to any step involved in the production of a polypeptide, such as by transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0091] The term "expression cassette" refers to a nucleic acid construct that comprises a coding region and, preferably, a regulatory region operably linked to the coding region.

[0092] The term "expression vector" generally refers to a DNA or RNA molecule that contains an expression cassette. Expression vectors can be linear or circular double-stranded DNA molecules.

[0093] The term "polymer" as used herein generally refers to a chemical compound or mixture of compounds whose structure is composed of multiple monomers (repeating units) linked by covalent chemical bonds. Within the context of the present invention, the term polymer includes natural or synthetic polymers composed of a single type of repeating unit (i.e., homopolymers) or natural or synthetic polymers composed of a mixture of different repeating units (i.e., copolymers or heteropolymers).

[0094] As used herein, the terms "polyester-containing material", "polyester-containing product" and the like are understood to refer to products, such as plastic products, that contain at least one polyester in crystalline, semi-crystalline or completely amorphous form. Polyester-containing material can refer to any article made from at least one plastic material, such as plastic sheets, tubes, rods, profiles, mouldings, films, block blocks, fibres, fabrics, etc., that contain at least one polyester and possibly other substances or additives, such as plasticizers, inorganic or organic fillers. In an embodiment, the polyester-containing material is a fabric or textile that comprises at least one polyester-containing fibre. In another embodiment, the polyester-containing material is a plastic compound or blend, in molten or solid state, suitable for making plastic products.

[0095] Suitable polyesters will be familiar to those skilled in the art, and illustrative examples thereof include polylactic acid (PLA), polyethylene terephthalate (PET), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polyhydroxyalkanoates (PHAs), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), polycaprolactone (PCL), and poly(ethylene adipate) (PEA). Thus, in one embodiment, the polyester is selected from the group consisting of polylactic acid (PLA), polyethylene terephthalate (PET), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), polycaprolactone (PCL), poly(ethylene adipate) (PEA), and combinations of any of the foregoing.

[0096] As described elsewhere herein, the inventors have developed variants of the naturally occurring MHETase of SEQ ID NO: 1, which exhibit increased MHETase activity compared to the parent polypeptide of SEQ ID NO: 1. The MHETase variants also exhibit enhanced expression in recombinant host cell systems. More specifically, the inventors have developed novel MHETases with superior properties for use in industrial processes. Aiming to improve the activity of hydrolases, in particular MHETases, in situations where industrial production of degradable plastic products may be performed and / or environmental degradation of plastic products may be achieved, the inventors have developed novel MHETases derived from the wild-type MHETase of SEQ ID NO: 1, which unexpectedly exhibit high MHETase activity compared to the parent hydrolase. The MHETase variants disclosed herein are particularly suitable for the degradation of plastic products, in particular plastic products containing PET. Furthermore, the inventors have surprisingly discovered that amino acid residues in the structure of the protein that would not otherwise be expected to come into contact with polyester substrates can be advantageously modified to enhance MHETase activity. Thus, in another aspect disclosed herein, there is provided a polypeptide having mono-(2-hydroxyethyl)terephthalate hydrolase (MHETase) activity, comprising an amino acid sequence that (i) has at least 70% sequence identity to SEQ ID NO:1 and (ii) differs from SEQ ID NO:1 by an amino acid substitution at one or more positions that do not contact the polyester substrate of the MHETase, wherein the MHETase activity of the polypeptide is greater than the MHETase activity of the MHETase of SEQ ID NO:1. The term "contact" in this context generally refers to direct contact with the polyester substrate made by the amino acid residues of the MHETase of SEQ ID NO:1. The amino acid residues of SEQ ID NO:1 that contact the polyester substrate will be familiar to those skilled in the art. Those residues are also described in Sagong et al. (2020, ACS Catal. 10:4805) and include R411, S416 and F424 of SEQ ID NO:1.In certain embodiments, the polypeptide comprises an amino acid sequence that differs from SEQ ID NO: 1 by an amino acid substitution at one or more positions outside the active site of the MHETase of SEQ ID NO: 1. The term "active site" generally refers to the region of SEQ ID NO: 1 that is capable of contacting and hydrolyzing the polyester substrate (i.e., MHET). The amino acid positions of SEQ ID NO: 1 that lie outside the active site of the MHETase of SEQ ID NO: 1 will be familiar to those of skill in the art.

[0097] In the context of the present disclosure, references to increased or enhanced MHETase activity may include one or more of the following: increased ability of the polypeptide to hydrolyze MHET compared to the MHETase of SEQ ID NO:1; increased recombinant expression in a host cell compared to the MHETase of SEQ ID NO:1; increased whole cell activity compared to the MHETase of SEQ ID NO:1; and increased thermostability compared to the MHETase of SEQ ID NO:1.

[0098] In certain embodiments, the polypeptides disclosed herein comprise increased recombinant expression of MHETase in a host cell compared to the MHETase of SEQ ID NO:1.

[0099] In another embodiment, the polypeptides disclosed herein comprise increased thermostability compared to the MHETase of SEQ ID NO:1.

[0100] In another embodiment, the polypeptides disclosed herein comprise increased whole cell activity compared to the MHETase of SEQ ID NO: 1. As used herein, the term whole cell activity generally refers to the ability of an MHETase to hydrolyze MHET when expressed in a host cell system.

[0101] In further embodiments, the polypeptides disclosed herein comprise increased thermostability and increased whole cell activity compared to the MHETase of SEQ ID NO:1.

[0102] In further embodiments, the polypeptides disclosed herein comprise increased recombinant expression of MHETase in a host cell, increased thermostability and increased whole cell activity compared to the MHETase of SEQ ID NO:1.

[0103] In certain embodiments, the MHETase activity of the polypeptides described herein is similar to the MHETase activity of the MHETase of SEQ ID NO: 1. In certain embodiments, the MHETase activity of the polypeptides described herein is increased by at least about 5%, preferably at least about 10%, preferably at least about 20%, preferably at least about 30%, preferably at least about 40%, preferably at least about 50%, preferably at least about 100%, preferably at least about 200%, preferably at least about 300%, preferably at least about 400%, preferably at least about 500%, preferably at least about 600%, preferably at least about 700%, preferably at least about 800%, preferably at least about 900%, or more preferably at least about 1,000% or more compared to the MHETase of SEQ ID NO: 1. Suitable methods for determining or measuring the MHETase activity of a polypeptide will be familiar to those of skill in the art. Illustrative examples thereof are provided elsewhere herein. Other illustrative examples are described in Palm et al. (2019, Nat. Comm., 10:1717), Sagong et al. (2020, ACS Catal. 10:4805), and Yoshida et al. (2020, Science, 352(6278):1196), the contents of which are incorporated by reference in their entireties. In one embodiment, the MHETase activity is increased by at least about 5%, preferably at least about 10%, preferably at least about 20%, preferably at least about 30%, preferably at least about 40%, preferably at least about 50%, preferably at least about 100%, preferably at least about 200%, preferably at least about 300%, preferably at least about 400%, preferably at least about 500%, preferably at least about 600%, preferably at least about 700%, preferably at least about 800%, preferably at least about 900%, or more preferably at least about 1,000% or more compared to the MHETase of SEQ ID NO:1, as determined by a chromogenic assay using 1-naphthyl terephthalate (1NT) as a substrate.

[0104] MHETase activity can be assigned an absolute value or can be assigned a value relative to the MHETase activity of a comparator (e.g., the MHETase of SEQ ID NO: 1). In one embodiment, MHETase activity is measured as the rate of monomers and / or oligomers released (e.g., in mg) per time and per mg of enzyme under suitable conditions of temperature, pH and buffer.

[0105] MHETase activity can be measured or assayed using purified enzyme, or it can be measured as a function of the activity of the enzyme when recombinantly expressed in a host cell system (also referred to herein as cellular catalytic activity or whole cell activity).

[0106] Alternatively, the polypeptides described herein exhibit an increase or enhancement of at least about 5%, preferably at least about 10%, preferably at least about 20%, preferably at least about 30%, preferably at least about 40%, preferably at least about 50%, preferably at least about 100%, preferably at least about 200%, preferably at least about 300%, preferably at least about 400%, preferably at least about 500%, preferably at least about 600%, preferably at least about 700%, preferably at least about 800%, preferably at least about 900%, or more preferably at least about 1,000% in recombinant expression in a host cell compared to the MHETase of SEQ ID NO:1.

[0107] Advantageously, the polypeptides described herein exhibit MHETase activity at least in the temperature range of about 10°C to about 60°C, preferably about 20°C to about 680°C, preferably about 30°C to about 60°C, more preferably about 40°C to about 60°C, even more preferably about 40°C to about 50°C, and even more preferably at about 45°C. In certain embodiments, the polypeptides described herein exhibit MHETase activity at a temperature of about 10°C to about 60°C, preferably about 20°C to about 60°C, preferably about 30°C to about 60°C, more preferably about 40°C to about 60°C, even more preferably about 40°C to about 50°C, or even more preferably at about 45°C. In certain embodiments, MHETase activity is measurable at a temperature between about 40°C to about 60°C, preferably between about 40°C to about 50°C, or even more preferably at about 45°C. In another particular embodiment, polyester degrading activity can also be measured at temperatures between about 10°C and about 30°C, preferably between about 15°C and about 28°C, which correspond to the average temperature in the natural environment (room temperature).

[0108] In one embodiment, the polypeptide comprises an MHETase activity at a temperature of about 10°C to about 60°C, preferably about 20°C to about 60°C, preferably about 30°C to about 60°C, more preferably about 40°C to about 60°C, even more preferably about 40°C to about 50°C, or even more preferably at about 45°C, compared to the MHETase of SEQ ID NO:1 at the same temperature, of at least about 5%, preferably up to at least about 10%, preferably up to at least about 20%, preferably up to at least about 30%, preferably up to at least about 40%, preferably up to at least about 50%, preferably up to at least about 100%, preferably up to at least about 200%, preferably up to at least about 300%, preferably up to at least about 400%, preferably up to at least about 500%, preferably up to at least about 600%, preferably up to at least about 700%, preferably up to at least about 800%, preferably up to at least about 900%, or more preferably up to at least about 1,000% or more.

[0109] In another specific embodiment, the polypeptides described herein have increased MHETase activity compared to the polypeptide of SEQ ID NO:1 at temperatures between about 10°C and about 60°C, preferably between about 20°C and about 60°C, preferably between about 30°C and about 60°C, preferably between about 40°C and about 60°C, preferably between about 40°C and about 50°C, or more preferably at about 45°C. In certain embodiments, the polypeptides described herein have an MHETase activity at a temperature between about 20° C. and about 60° C. of at least about 5%, preferably up to at least about 10%, preferably up to at least about 20%, preferably up to at least about 30%, preferably up to at least about 40%, preferably up to at least about 50%, preferably up to at least about 100%, preferably up to at least about 200%, preferably up to at least about 300%, preferably up to at least about 400%, preferably up to at least about 500%, preferably up to at least about 600%, preferably up to at least about 700%, preferably up to at least about 800%, preferably up to at least about 900%, or more preferably up to at least about 1,000% or more, compared to the MHETase activity of SEQ ID NO:1 at the same temperature.

[0110] In another embodiment, the polypeptides described herein have increased MHETase activity compared to the polypeptide of SEQ ID NO:1 at temperatures between about 10°C and about 30°C, preferably between about 15°C and about 30°C, even more preferably between about 20°C and about 30°C, or even more preferably at about 28°C. In one embodiment, the polypeptides described herein have an MHETase activity at a temperature between about 10°C and about 30°C of at least about 5%, preferably up to at least about 10%, preferably up to at least about 20%, preferably up to at least about 30%, preferably up to at least about 40%, preferably up to at least about 50%, preferably up to at least about 100%, preferably up to at least about 200%, preferably up to at least about 300%, preferably up to at least about 400%, preferably up to at least about 500%, preferably up to at least about 600%, preferably up to at least about 700%, preferably up to at least about 800%, preferably up to at least about 900%, or more preferably up to at least about 1,000% or more, compared to the MHETase activity of SEQ ID NO:1 at the same temperature.

[0111] In one embodiment, the polypeptides described herein exhibit measurable MHETase activity at least in the pH range of 5 to 11, preferably in the pH range of 6 to 10, more preferably in the pH range of 6.5 to 9, and even more preferably in the pH range of 7 to 8.

[0112] Advantageously, the thermal stability of the polypeptides described herein is not significantly impaired compared to the polypeptide of SEQ ID NO:1. In some embodiments, the thermal stability of the polypeptides described herein is improved compared to the thermal stability of SEQ ID NO:1. The term "improved thermal stability" or "increased thermal stability" as used herein refers to an increase in the ability of an enzyme to undergo changes in its chemical and / or physical structure at higher temperatures, more specifically at temperatures between 40°C and 60°C, compared to the polypeptide of SEQ ID NO:1. In certain embodiments, the polypeptides described herein have an increased half-life at temperatures between 40°C and 60°C, compared to the polypeptide of SEQ ID NO:1. The polypeptides described herein may exhibit a higher or equivalent melting temperature (Tm) compared to the polypeptide of SEQ ID NO:1. In some embodiments, the polypeptides described herein exhibit improved thermal stability at temperatures between 40°C and 68°C, compared to the polypeptide of SEQ ID NO:1.

[0113] The thermal stability of a polypeptide can be evaluated by any suitable means known to those skilled in the art. For example, the thermal stability can be assessed by measuring the residual MHETase activity of the polypeptide after incubation at different temperatures. The ability to perform multiple rounds of MHETase-mediated hydrolysis at different temperatures can also be evaluated. Differential scanning fluorimetry (DSF) can also be used to assess the thermal stability of a polypeptide. Circular dichroism can also be used to measure the thermal stability of the polypeptides described herein, including their melting temperature (Tm). The term "melting temperature (Tm)" is taken to mean that the temperature at which a given protein is 50% denatured.

[0114] In certain embodiments, the polypeptides described herein exhibit a melting temperature (Tm) of about 45°C to about 68°C, preferably about 50°C to about 65°C, preferably about 52°C to about 63°C. In certain embodiments, the polypeptides described herein exhibit a melting temperature (Tm) lower than the melting temperature (Tm) exhibited by the polypeptide of SEQ ID NO: 1. In one embodiment, the polypeptides described herein exhibit a melting temperature (Tm) higher than the melting temperature (Tm) exhibited by the polypeptide of SEQ ID NO: 1. In certain embodiments, the polypeptides described herein exhibit a melting temperature (Tm) of about 52°C to about 64°C, preferably about 55°C to about 63°C, more preferably about 63°C.

[0115] The present disclosure also extends to a polynucleotide comprising a nucleic acid sequence encoding a MHETase polypeptide as described herein. In one aspect of this disclosure, a polynucleotide comprising a nucleic acid sequence encoding a polypeptide as described herein is provided. In one embodiment, the nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 38-72 and 79-84. In another embodiment, the nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 79-84. As used herein, the terms "nucleic acid", "nucleic acid sequence", "polynucleotide", "oligonucleotide" and "nucleotide sequence" are used interchangeably and refer to a sequence of deoxyribonucleotides and / or ribonucleotides. The nucleic acid may be DNA (cDNA or gDNA), RNA or a mixture of the two. It may be in single-stranded form or in double-stranded form or a mixture of the two. It may be of recombinant, artificial and / or synthetic origin and it may contain modified nucleotides, including, for example, modified bonds, modified purine or pyrimidine bases, or modified sugars. The nucleic acids of the invention may be in isolated or purified form and may be produced, isolated and / or manipulated by techniques known per se in the art, such as cloning and expression of cDNA libraries, amplification, enzymatic synthesis or recombinant techniques. Nucleic acids may also be synthesized in vitro by well-known chemical synthesis techniques, for example as described in Belousov (1997) Nucleic Acids Res. 25:3440-3444.

[0116] The nucleic acid sequences disclosed herein can be suitably codon-optimized. Suitable methods for codon optimization will be familiar to those skilled in the art. An illustrative example thereof is described in the reference manual of Sambrook et al. (Sambrook et al., 2001).

[0117] The nucleic acid sequences described herein can be suitably deduced from the amino acid sequences of the polypeptides described herein, and the codon usage can be adapted depending on the host cell in which the nucleic acid is to be transcribed.

[0118] In some embodiments, the nucleic acid sequences described herein may suitably include additional nucleotide sequences, such as regulatory regions, i.e., promoters, enhancers, silencers, terminators, signal peptides, etc., that may be used to cause or regulate expression of the polypeptide in a selected host cell or system. Alternatively, or in addition, the nucleic acid sequences described herein may further include additional nucleotide sequences encoding fusion proteins, such as maltose binding protein (MBP) or glutathione S-transferase (GST), that may be used to favor expression and / or solubility of the polypeptide.

[0119] As mentioned elsewhere herein, the present disclosure also extends to expression vectors and expression cassettes comprising the nucleic acid sequences described herein, optionally operably linked to one or more control sequences that direct the expression of the nucleic acid sequence in a suitable host cell. Generally, an expression vector or cassette comprises a nucleic acid sequence described herein operably linked to a control sequence, such as a transcription promoter and / or a transcription terminator. The control sequence may include a promoter recognized by a host cell or an in vitro expression system for expression of a nucleic acid encoding a polypeptide described herein. A promoter will generally include a transcription control sequence that mediates expression of the polypeptide. A promoter may be any polynucleotide that exhibits transcriptional activity in a host cell, including mutant, truncated and hybrid promoters, and may suitably be derived from a gene encoding an extracellular or intracellular polypeptide, either homologous or heterologous to the host cell. A control sequence may also be a transcription terminator recognized by a host cell to terminate transcription. The terminator is generally operably linked to the 3' end of the nucleic acid sequence encoding the polypeptide. Any terminator that is functional in the host cell can be used in this context. Generally, the expression vector or cassette contains a nucleic acid sequence described herein operably linked to a transcription promoter and a transcription terminator.

[0120] The term "vector" generally refers to a DNA molecule used as a vector to transfer recombinant genetic material into a host cell. Suitable vectors include plasmids, bacteriophages, viruses, fosmids, cosmids, and artificial chromosomes. A vector is generally a DNA sequence that contains an insert (heterologous nucleic acid sequence, transgene) and a larger sequence that serves as the "backbone" of the vector. The purpose of a vector that transfers genetic information to a host is generally to isolate, propagate, or express the insert in a target cell. Expression vectors (also called expression constructs) are particularly suitable for the expression of heterologous sequences in target cells and generally have a promoter sequence that drives the expression of a heterologous sequence that codes for a polypeptide.

[0121] In general, the regulatory elements used in expression vectors include transcription promoters, ribosome binding sites, terminators, and optionally present operators. Expression vectors may further include an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, and the possibility of high copy number. Suitable expression vectors will be familiar to those skilled in the art. Illustrative examples thereof include cloning vectors, modified cloning vectors, plasmids, and viruses. Expression vectors that can provide suitable levels of polypeptide expression in different hosts are also well known in the art. The choice of vector will generally depend on the compatibility of the vector with the host cell into which it will be introduced. In one embodiment, the vector is the bacterial expression vector pET-28a(+) (SEQ ID NO: 85).

[0122] The present disclosure also extends to host cells comprising the nucleic acid sequences described herein. The host cells can be transformed, transfected or transduced in a transient or stable manner. The nucleic acid, expression cassette or vector is introduced into the host cell so that the nucleic acid, cassette or vector is maintained as a chromosomal integrant or as a self-replicating extrachromosomal vector. The term "host cell" encompasses any progeny of a parent host cell that is not identical to the parent host cell due to mutations that occur during replication. The host cell can be any cell useful for producing the variants of the invention, for example, a prokaryotic or eukaryotic organism. The prokaryotic host cell can be any gram-positive or gram-negative bacteria. The host cell can also be a eukaryotic cell, for example, a yeast, fungal, mammalian, insect or plant cell. In certain embodiments, the host cell is selected from the group of Escherichia coli, Pseudomonas, Bacillus, Streptomyces, Trichoderma, Aspergillus, Saccharomyces, Pichia, Thermus, or Yarrowia.

[0123] The nucleic acids, expression cassettes or expression vectors according to the invention can be introduced into a host cell by any suitable method known to those skilled in the art, illustrative examples of which include electroporation, conjugation, transduction, competent cell transformation, protoplast transformation, protoplast fusion, biolistic "gene gun" transformation, PEG-mediated transformation, lipid-assisted transformation or transfection, chemically mediated transfection, lithium acetate-mediated transfection and liposome-mediated transfection.

[0124] In certain embodiments, the host cell is a genetically modified host cell or microorganism. In this regard, the host cell or microorganism may be genetically modified to enhance the expression and / or activity of a polypeptide expressed in the host cell or microorganism. For example, the polypeptides described herein may be used to complement wild-type strains of fungi or bacteria known to be capable of MHETase activity to improve and / or increase the MHETase activity of the strain.

[0125] The present disclosure provides a method for producing a polypeptide having MHETase activity, comprising: a) providing a nucleic acid sequence as described herein; b) producing the polypeptide by expressing the nucleic acid sequence in a host cell culture; and c) harvesting the polypeptide produced in (b) from the host cell culture. The present invention also extends to a method including:

[0126] The present invention also extends to an in vitro method of producing a polypeptide as described herein, comprising the steps of: (a) contacting a nucleic acid, cassette or vector of the invention with an expression system in vitro; and (b) recovering the produced polypeptide. In vitro expression systems are well known to those skilled in the art and are commercially available.

[0127] Suitable host cells will be familiar to those skilled in the art. Illustrative examples include recombinant Bacillus, E. coli, Pseudomonas, Aspergillus, Trichoderma, Streptomyces, Saccharomyces, Pichia, Thermus, or Yarrowia. In one embodiment, the host cell is E. coli.

[0128] The host cells can be cultured in a nutrient medium suitable for the production of the polypeptide, using methods that will be known to those skilled in the art. Suitable examples include the culture of the host cells by shake flask culture, or by small- or large-scale fermentation (including continuous, batch, fed-batch or solid-state fermentation) in laboratory or industrial fermenters carried out in a suitable medium and under conditions that allow the expression and / or isolation of the enzyme. The culture will generally be carried out in a suitable nutrient medium prepared according to a published composition from a commercial supplier (e.g., in the catalogue of the American Type Culture Collection), or any other culture medium suitable for cell growth. If the polypeptide is expressed and / or secreted into the culture medium, the polypeptide can be used in the form of a culture / supernatant mixture or in the form of a crude cell lysate. Alternatively, the polypeptide can be directly recovered from the culture supernatant. Conversely, the polypeptide can be recovered from a cell lysate or after permeabilization of the host cell membrane. The polypeptide can be recovered using any suitable method known to those skilled in the art, illustrative examples of which include collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. If desired, the polypeptide can be partially or completely purified by a variety of procedures known in the art, including, but not limited to, thermal shock, chromatography (e.g., ion exchange, affinity, hydrophobic, 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.

[0129] The polypeptides can be used in purified form, either alone or in combination with additional enzymes (e.g., PETase), to catalyze enzymatic reactions involved in the degradation and / or recycling of polyester-containing materials, such as polyester-containing plastic products. The polypeptides described herein can be in soluble form or on a solid phase. In particular, they can be bound to cell membranes or lipid vesicles, or to synthetic supports, such as glass, plastic, polymers, fibers, membranes, for example in the form of beads, columns, plates, etc.

[0130] The disclosure also extends to compositions comprising the polypeptides, nucleic acids or host cells described herein.

[0131] The composition may be liquid or dry, e.g., in powder form. In some embodiments, the composition is a lyophilizate. For example, the composition may include the polypeptide, the nucleic acid, and / or the host cell, and, if necessary, excipients and / or reagents, etc. Suitable excipients may include buffers commonly used in biochemistry, agents for adjusting pH, preservatives, e.g., sodium benzoate, sodium sorbate, or sodium ascorbate, conservatives, protective or stabilizing agents, e.g., starch, dextrin, gum arabic, salts, sugars, e.g., sorbitol, trehalose, or lactose, glycerol, polyethylene glycol, polyethylene glycol, polyethene glycol, propylene glycol, divalent ions, e.g., calcium, sequestrants, e.g., EDTA, reducing agents (e.g., beta-mercaptoethanol, dithiothreitol, ascorbic acid, tris(2-carboxyethyl)phosphine), amino acids, carriers, e.g., solvents or aqueous solutions, etc.

[0132] In an embodiment, the composition comprises a polypeptide as described herein (the polypeptide may be present in the composition in isolated or at least partially purified form). In an embodiment, the composition comprises a polypeptide as described herein in an amount of about 0.1% to about 99.9% by weight, preferably about 0.1% to about 50% by weight, preferably about 0.1% to about 30% by weight, preferably about 0.1% to about 5% by weight, based on the total weight of the composition. In a preferred embodiment, the composition comprises a polypeptide as described herein in an amount of about 0.1% to about 5% by weight, based on the total weight of the composition. In another embodiment, the composition comprises a polypeptide as described herein in an amount of about 0.1% to about 0.2% by weight, based on the total weight of the composition. The amount of the polypeptide in the composition can be suitably adapted by the skilled person depending, for example, on the nature and / or amount of the polyester-containing material to be degraded (hydrolyzed) and / or the presence or absence of any additional enzymes / polypeptides in the composition.

[0133] The compositions described herein may further comprise additional polypeptides that exhibit enzymatic activity, including but not limited to MHETase.

[0134] In some embodiments, the polypeptides described herein are solubilized in an aqueous medium together with one or more excipients, such as excipients that can suitably stabilize or protect the polypeptide from degradation. For example, the polypeptides described herein can be solubilized in water and then mixed with an excipient, such as glycerol, sorbitol, dextran, starch, glycols, such as propanediol, salts, etc. The resulting mixture can then be dried to obtain a powder. Methods for drying such mixtures are well known to those skilled in the art and include, but are not limited to, lyophilization, freeze drying, spray drying, supercritical drying, downdraft evaporation, thin layer evaporation, centrifugal evaporation, conveyor drying, fluidized bed drying, drum drying, or any combination thereof.

[0135] In an embodiment, the composition comprises at least one host cell expressing a polypeptide as described herein, or an extract thereof. By "cell extract" is meant any fraction obtained from a cell that is essentially free of living cells, such as cell supernatant, cell debris, cell wall, DNA extract, enzyme or enzyme preparation, or any preparation obtained from a cell by chemical, physical and / or enzymatic treatment. A preferred extract is an enzymatically active extract. The composition may comprise one or several host cells or extracts thereof that contain a polypeptide as described herein, and optionally one or several additional cells.

[0136] As noted elsewhere herein, the inventors have surprisingly discovered that the polypeptides described herein (MHETase variants) have greater MHETase activity compared to the wild-type MHETase of SEQ ID NO: 1. Thus, disclosed herein is a method of hydrolyzing mono-(2-hydroxyethyl) terephthalate comprising exposing mono-(2-hydroxyethyl) terephthalate to a polypeptide, composition or host cell described herein under conditions sufficient to convert mono-(2-hydroxyethyl) terephthalate to terephthalate and ethylene glycol. The disclosure also extends to a method of degrading a plastic article comprising polyester comprising exposing the plastic article to a polypeptide, composition or host cell described herein.

[0137] The disclosure extends to the use of the polypeptides, compositions or host cells described herein for degrading polyesters under aerobic or anaerobic conditions, and / or for recycling polyester-containing materials as plastic products made of or containing polyesters, and / or for producing biodegradable plastic products containing polyesters. Such methods and uses are particularly useful for the degradation of plastic products, including PET.

[0138] Advantageously, the polyester(s) of the polyester-containing material are depolymerized to monomers and / or oligomers. In one embodiment, at least one polyester is degraded to produce repolymerizable monomers and / or oligomers, which are advantageously recovered or recovered for further use.

[0139] In some embodiments, the polyester(s) of the polyester-containing material are completely degraded.

[0140] As noted elsewhere herein, the plastic article may comprise at least one polyester selected from the group consisting of polylactic acid (PLA), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polyethylene terephthalate (PET), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), polycaprolactone (PCL), poly(ethylene adipate) (PEA), and combinations of any of the foregoing.

[0141] The time required to degrade the polyester-containing material may vary depending on the polyester-containing material itself (i.e., the nature and origin of the plastic product, its composition, shape, etc.), the type and amount of polypeptide used, and various process parameters (i.e., temperature, pH, additional agents, etc.) A person skilled in the art can easily adapt the process parameters to the polyester-containing material.

[0142] Advantageously, the degradation process is carried out at a temperature of about 10°C to about 60°C, preferably about 20°C to about 60°C, preferably about 30°C to about 60°C, more preferably about 40°C to about 60°C, even more preferably about 40°C to about 50°C, or even more preferably at about 45°C. The temperature is generally below the inactivation temperature, which corresponds to the temperature at which the polypeptide is inactivated and / or the recombinant microorganism does not synthesize, produce or release the polyesters described herein. In an embodiment, the temperature is maintained below the glass transition temperature (Tg) of the polyester in the polyester-containing material. In an embodiment, the degradation process or method is carried out at a temperature of about 10°C to about 60°C, preferably about 20°C to about 60°C, preferably about 30°C to about 60°C, more preferably about 40°C to about 60°C, even more preferably about 40°C to about 50°C, or even more preferably at about 45°C. The process or method may suitably be carried out continuously at a temperature which allows the polypeptide to be used and / or recycled several times.

[0143] Advantageously, the degradation process or method is carried out at a pH comprised between 5 and 11, preferably between 6 and 10, more preferably between 6.5 and 9, and even more preferably between 7 and 8.

[0144] In certain embodiments, the polyester-containing material can be pretreated prior to contacting with the polypeptide to physically alter its structure in a manner that increases the contact surface between the polyester and the enzyme.

[0145] The monomers resulting from the depolymerization or decomposition process or method can be suitably recovered either sequentially or continuously. Depending on the starting polyester-containing material, a single type of monomer or several different types of monomers can be recovered.

[0146] The recovered monomer can be further purified and conditioned in a repolymerizable form using any suitable purification method. Illustrative examples of suitable purification methods include stripping processes, separation with aqueous solutions, steam selective condensation, filtration and concentration of post-bioprocessing medium, separation, distillation, vacuum evaporation, extraction, electrodialysis, adsorption, ion exchange, precipitation, crystallization, concentration and dehydration and precipitation by addition of acid, nanofiltration, acid catalyzed treatment, semi-continuous or continuous mode distillation, solvent extraction, evaporative concentration, evaporative crystallization, liquid / liquid extraction, hydrogenation, azeotropic distillation processes, adsorption, column chromatography, vacuum simple distillation, and microfiltration, either combined or uncombined.

[0147] The repolymerizable monomers can be used to synthesize new polyesters. Advantageously, polyesters of the same nature are repolymerized. However, it is possible to mix the recovered monomers with other monomers, for example to synthesize new copolymers. Alternatively, the recovered monomers can be used as intermediates to produce new chemical compounds of interest.

[0148] The present disclosure also extends to a plastic compound comprising the polypeptide, a composition containing said polypeptide, and / or a host cell expressing said polypeptide, or an extract thereof.

[0149] The present disclosure also extends to a masterbatch composition comprising a polypeptide, a composition containing said polypeptide and / or a host cell expressing said polypeptide or an extract thereof.

[0150] Advantageously, such plastic compounds or masterbatch compositions described herein can be used in the production of polyester-containing materials and / or plastic articles that will comprise the polypeptides described herein.

[0151] In certain embodiments, the resulting plastic compound, masterbatch composition or plastic article is a biodegradable plastic compound, masterbatch composition or plastic article that complies with at least one of the relevant standards and / or labels known to those skilled in the art, such as EN Standard 13432, ASTM Standard D6400, OK Biodegradation Soil (Label Vincotte), OK Biodegradation Water (Label Vincotte), OK Compost (Label Vincotte), OK Home Compost (Label Vincotte), etc.

[0152] Advantageously, the degradation process of the polyester-containing material (i.e. plastic compound, masterbatch composition or plastic product) is carried out at a temperature comprised between 10°C and 50°C, preferably between 15°C and 40°C, more preferably between 20°C and 30°C, more preferably at 28°C, + / - 2°C.

[0153] Alternatively, the degradation process of the polyester-containing material (i.e. the plastic compound, masterbatch composition or plastic article) is carried out at a temperature comprised between 50°C and 60°C, more preferably at 55°C, + / - 2°C.

[0154] The MHETase variants disclosed herein are suitable for a variety of applications, including industrial applications, illustrative examples of which include detergent additives, feed compositions (including for animal feed), textile production, electronics and biomedical applications, For example, the MHETase variants disclosed herein can be utilized in textile production, where they can be used as exonucleases to suitably modify the properties of textile fibers.

[0155] The present invention will now be described with reference to the following examples which illustrate certain preferred aspects of the invention. However, it will be understood that the specificity of the following description of the invention does not go beyond the generality of the preceding description of the invention. EXAMPLES

[0156] Materials and Methods Construction, expression and purification of recombinant MHETase A. Consensus Design and Construction 5,000 sequences were collected by BLAST+ using wild-type I. sakasiensis MHETase (SEQ ID NO: 1) as the seed sequence and an E-value threshold of 10-5 was used.

[0157] [ka]

[0158] 315 non-redundant sequences showed high similarity to MHETase and their sequences were imported from the UniProt database. Peptide transport signals were identified and detected using SignalP4.0. All sequences were aligned using the PROMALS3D library-based sequence alignment algorithm and available MHETase structures (6QGB), and then the final curated alignment was manually refined. A number of different thresholds (95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, and 50%) were used to build the consensus of the alignment at each amino acid position. A truncated codon-optimized version of the MHETase gene missing the first 19 amino acids at the N'-terminus (GenBank accession number A0A0K8P8E7), as well as 10 consensus designs, were commercially synthesized and cloned into pET-28a(+) (Twist Bioscience).

[0159] B. Protein Expression and Purification Plasmids were transformed by electroporation into electrocompetent E. coli SHuffle T7 Express cells (New England Biolabs), plated onto lysogeny broth (LB) agar supplemented with 100 μg / mL ampicillin, and incubated overnight at 37°C. A single colony was used to inoculate 10 ml of LB supplemented with 100 μg / mL ampicillin (LBA) and incubated overnight at 30°C. This culture was added to 1 L of LBA and incubated at 30°C until the OD600 reached 1.0. Isopropyl β-D-1-thiogalactopyranoside was added to a final concentration of 1 mM, and the culture was transferred to an 18°C ​​incubation for 16 h.

[0160] Cells were harvested by centrifugation at 5000×g for 15 min at 4° C. and resuspension in lysis buffer (500 mM NaCl, 30 mM imidazole, 0.5 mg / mL lysozyme, 1% (v / v) TritonX-100, 1 U / ml Turbonuclease (Sigma), 0.5 mM thidiothreitol (DTT) and 25 mM HEPES pH 7.5). The cell suspension was lysed by two rounds of sonication at 50% power and a pulse time of 5 min, and the soluble cell lysate was separated from the insoluble cell debris by centrifugation at 32,000×g for 45 min at 4° C. The lysate was passed through a 0.45 μm pore size filter and then purified by nickel-charged IMAC using 5 mL of HisTrap HP (GE Healthcare Life Sciences) equilibrated in lysis buffer and eluted with elution buffer (500 mM NaCl, 500 mM imidazole, 0.5 mM dithiothreitol (DTT) and 25 mM HEPES pH 7.5). The eluate with MHETase was collected, concentrated and filtered through a 0.2 μm filter. The final product was further purified using a HiLoad 26 / 600 Superdex 200 (GE Healthcare Life Sciences) equilibrated in SEC buffer (150 mM NaCl, 25 mM HEPES pH 7.5).

[0161] C. Chromogenic Assay 50 μL of cell suspension of expressed protein was diluted with 150 μL of reaction buffer (90 mM NaCl, 45 mM sodium phosphate pH 7.5). The reaction was initiated by the addition of 50 μL of 10 mM 1NT (1-naphthyl terephthalate) and 5 mM Fast Blue B salt dye in 100% (v / v) DMSO. Absorbance at 465 nm was monitored for 30 min using an Epoch microplate spectrophotometer (BioTek).

[0162] For kinetic determinations, homogeneous MHETase at a final concentration of 7.5 nM was used instead of soluble cell lysate while the concentration of 1NT was varied from 1 mM to 7.8 μM. The concentration of Fast Blue B salt remained constant. Absorbance was converted to product concentration by using a calibration curve of 1 mM to 7.8 μM 1-naphthol.

[0163] D. SDS-PAGE Atto550 The method was adapted from Raducanu et al. (2020, Journal of Biological Chemistry 295(34):12214-12223). 1 mL of cell pellet was resuspended in 1x BugBuster (Merck-Millipore) diluted in SEC buffer and incubated at room temperature for 10 min. The mixture was centrifuged at 15,000 × g for 10 min and 5 μL of soluble cell lysate was run on an SDS-PAGE gel for 60 min at 140 V. The gel was microwaved for 30 s in Milli-Q water (MQ) and then for 2 min in fixation solution (40% (v / v) methanol, 10% (v / v) acetic acid in MQ). The now fixed protein gel was again microwaved for 10 min in MQ and then incubated in a 1:3000 dilution of NTA-Atto550 dye in PBS buffer in a dark environment on a shaker for 1 h. The gel was then transferred to a warm MQ container for an additional 30 min of shaking. The gel was imaged using the ChemiDoc MP Imaging System (BIO-RAD) using the DyLight 550 fluorophore option.

[0164] E. Circular dichroism and temperature stability Measurements of circular dichroism spectra of MHETase were performed in 1 mm quartz cuvettes on an Applied Photophysics Chirascan Spectrometer. The homogeneous enzyme was diluted to 0.2 mg / mL in 25 mM sodium acetate pH 7.5. CD spectra were measured between 200 nm and 260 nm at 20°C, using a 1 nm bandwidth and a scan speed of 0.5 seconds to allow adaptive sampling. Spectra were measured in triplicate and a buffer blank was subtracted from the results. For assessment of thermal melting, CD at 222 nm was recorded while the temperature of the solution was increased from 20°C to 90°C at 1°C / min. A standard sigmoidal curve was fitted to the thermal melting curve to determine the Tm.

[0165] F. Assessment of MHETase activity by high performance liquid chromatography (HPLC) The HPLC assay was adapted from Palm et al. (2019). Homogeneous MHETase was diluted in reaction buffer to a final concentration of 7.5 nM (80 μL). The reaction was initiated by adding 20 μL of 1 mM MHET dissolved in 100% DMSO. The reaction was quenched after set time points (0, 10, 30 and 60 min) by adding 100 μL of quenching buffer (160 mM sodium phosphate pH 2) and heated to 80 °C for 10 min. A volume of 10 μL of the reaction mix was loaded onto an Agilent ZORBAX SB-C18, 3.5 um, 4.6 × 150 mm column. TPA and MHET were separated using a flow rate of 1 mL / min at 30 °C, equilibrated with 50% phosphate buffer (20 mM sodium phosphate pH 2.0) and 50% acetonitrile over a 7 min run time. TPA and MHET were detected at 240 nm and quantified against a calibration curve.

[0166] Example 1 Variant MHETase A consensus-based design was performed using aligned sequences of MHETase and its closest relatives. From this design, a number of different combinatorial MHETase sequences were constructed using different consensus thresholds: 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55% and 50%. For example, the consensus design at the 95% threshold will show all of the differences found in 95% of the aligned sequences but not in the WT MHETase. The amino acid sequence of WT MHETase (SEQ ID NO: 1) and the different consensus designs (SEQ ID NOs: 2-36) are shown in FIG. 1. The nucleic acid sequence of WT MHETase and the different consensus designs (SEQ ID NOs: 37-72) are shown in FIG. 2. The different consensus designs resulting from thresholds of 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55% and 50% are referred to herein as Round 1 consensuses A, B, C, D, E, F, G, H, I and J, respectively.

[0167] MHETase activity / expression was measured using the chromogenic assay described above using whole cell suspensions.

[0168] As shown in FIG. 3, variant "Round 1 Consensus A" (SEQ ID NO: 73) showed higher whole cell activity than WT MHETase or other consensus designs. Round 1 Consensus A contained two amino acid substitutions compared to WT, namely N156G and T159V. As an initial study, point mutations were added to this variant based on other consensus residues identified by multiple sequence alignment to identify mutations that further stabilize / improve MHETase activity and / or expression. The remaining variants (mutants; SEQ ID NOs: 3-36) contain one of the following point mutations compared to the Round 1 Consensus A sequence (this nomenclature refers to the amino acid positions in wild-type MHETase, SEQ ID NO: 1): T68V, A78P, E110A, M117L, E128Q, S131G, N134D, A161G, G156N, D191L, M192Y, S196A, Y197V, G204A, A207L, A216P, E226N, L234A, S235A, P255V, G258A, S260A, T264L, T265L, N284L, L295V, S296A, T355R, A377P, S463L, A493P, Y503W, E594A and N496S

[0169] Also shown in FIG. 3, the point mutations S196A, Y197V, S235A, P255V, S260A, S286A, Y503W, when added to the round 1 consensus A sequence, showed higher whole cell activity when expressed in E. coli.

[0170] Soluble cell lysates were run by SDS-PAGE and stained with NTA-Atto550 to identify the expression rate of the enzyme. MHETase migration at 64 kDa on a gel is shown in Figure 4. The gel shows that the majority of MHETase variants (including the control) are not visible even under this more specific staining method. This result is consistent with low soluble protein expression. Round 1 consensus A and variants containing point mutations S196A, Y197V, S235A, P255V, S260A, S286A or Y503W on the variant B backbone appeared to show increased heterologous expression over wild type (WT) in E. coli.

[0171] The stability of selected generated variants was also tested using purified protein variants. As shown in Figure 5, the thermal melting (T m ) is approximately 59 o C, whereas introduction of the Y197V point mutation into round 1 consensus A resulted in T m 52 o was lowered to C.

[0172] One of the key limitations to the use of wild-type MHETase in industrial applications is its low expression in industrial strains. Increasing the total cell activity (a combination of catalytic efficiency and expression yield of active protein), as demonstrated by the variants disclosed herein, advantageously reduces the costs of production and use.

[0173] Example 2 Engineering MHETase with improved activity Since round 1 consensus A was found to have improved whole cell MHETase activity (Figure 3) with little change to thermostability (Figure 5), round 1 consensus A (containing N156G and T159V point mutations) was selected as the basis for further engineering to generate variants with improved thermostability. In this new engineering process, point mutations were added to round 1 consensus A to generate a library of mutants (round 2) and screened for whole cell activity. This process entails selecting the most promising variants and iteratively introducing single point mutations over several design rounds.

[0174] In round 2, the following point mutations were introduced into the round 1 consensus A backbone (nomenclature refers to the amino acid positions in wild-type MHETase, SEQ ID NO:1): T593D, P543A, Y503W, S463L, P449A, T355R, G301A, S296A, H293Q, S286A, N284L, S267A, T265L, T264L, S26 0A, P255V, S235A, K218R, A216P, A207L, Y197B, S196A, D191L, L190I, E110A, Y107Q, A99N, A81P, A78P, T68V

[0175] The best performing variant selected from round 2 is "Round 2 Y503W" (containing N156G, T159V and Y503W point mutations; SEQ ID NO: 74).

[0176] In round 3, the following point mutations were introduced into "round 2 Y503W": E594A, A493P, A377P, S286A, S267A, T264L, S260A, S196A, M192Y

[0177] The best performing variant selected from round 3 is "Round 3 M192Y" (containing N156G, T159V, M192Y and Y503W point mutations; SEQ ID NO: 75).

[0178] In round 4, the following point mutations were introduced into round 3 M192Y, where "+" indicates the insertion of an amino acid at a particular position: +N564, S561A, G534A, L486V, A469P, +P467, H467M, W398K, A377P, M361F, I357L, S288T, L282D, S286A, S267A, S260A , Y252F, M233V, G231A, E230H, V208I, Q202P, V200L, T162S, A161S, G156N, V159T, L137V, G130N, R114E, L112G, A79G

[0179] The best performing variant selected from round 4 is "Round 4 G156N" (containing T159V, M192Y and Y503W point mutations; SEQ ID NO: 76).

[0180] In round 5, the following point mutations were introduced into round 4 G156N, where "+" indicates the insertion of an amino acid at a particular position: N592E, I582V, G562R, R537Q, A494V, W466F, Q461E, M361F, I357L, N316D, I283L, Y252F, V246L, Y242F, G231A, V200L, G164A, I104V, E90A, E71T

[0181] The best performing round 5 selected variant was "Round 5 Y252F" (containing T159V, M192Y, Y252F and Y503W; SEQ ID NO: 77), which showed a 16-fold increase in whole cell activity compared to wild type (Figure 6).

[0182] Expression of the highest variant from each round was qualitatively measured using SDS-PAGE stained with His-tag-specific fluorescently labeled ATTO550. Expression of WT MHETase was too low to be conclusively detected against background endogenous E. coli protein expression. However, by engineering round 2, the recombinant expression level of variant MHETase (round 2 Y503W) was improved to a level that was visibly detectable using the ATTO550 fluorescent dye (Figure 7).

[0183] The kinetic parameters of these enzymes were determined using the chromogenic assay described herein with purified proteins (Figure 8). The catalytic efficiency (kcat / KM) of all variants remained constant relative to that of wild-type MHETase. Figure 9 shows the Michaelis-Menten plot of the rate of enzyme function at various substrate concentrations. From these data, two constants -kcat and KM- were obtained, as shown in Table 1.

[0184] [Table 1]

[0185] To validate the data observed using the chromogenic assay described herein, the activity of wild-type MHETase and the best variant from round 5 (round 5 Y252F; SEQ ID NO: 77) on the natural substrate MHET was determined by high performance liquid chromatography (HPLC). HPLC was used to measure the concentrations of terephthalic acid (TPA) and MHET over time in enzymatic reactions containing wild-type MHETase or round 5 Y252F. The data supports the kDa activity observed using the chromogenic assay. cat and K M This is consistent with the decrease in , confirming that round 5 Y252F hydrolyzes MHET to TPA. (See Figure 11).

[0186] Stability studies on the consensus designs were also performed using purified protein variants. Mutations at positions 192 and 252 improve the temperature stability (Tm) compared to WT MHETase (Figure 10).

[0187] Overall, these stabilizing properties contribute to the improved expression and activity of round 5 Y252F compared to wild-type MHETase. However, despite these improvements, the specific activity of this variant remains consistent with its lower k cat The expression level of IL-1 is reduced compared to the wild type, as demonstrated by (Table 1).

[0188] Further manipulations were performed to revert some of the individual mutations of round 5 Y252F to their wild type residues. In the case of position 156, the mutation N156G was reintroduced into the MHETase R5 background to investigate the impact of reverting this residue to wild type identity in round 4. Whole cell activity determined by FastBlue assay is shown in FIG. 12.

[0189] When the mutation M192Y was reverted to the wild type residue (M) in round 5 Y252F ("R5-Y192M"; SEQ ID NO: 78), an approximately 3-fold increase in specific activity was observed compared to round 5 Y252F (Figure 11). This is more highly comparable to that of the wild type MHETase of SEQ ID NO: 1. Reversion of other point mutations in round 5 Y252F either reduced or did not significantly change the enzyme activity. The temperature stability of the MHETase variants is shown in Table 2.

[0190] [Table 2]

[0191] The disclosures of all patents, patent applications and publications cited herein are hereby incorporated by reference in their entireties.

[0192] The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the present application.

[0193] Throughout this specification, the objective has been to describe preferred embodiments of the invention, without limiting the invention to any one embodiment or particular collection of features. Thus, in light of this disclosure, those skilled in the art will appreciate that various modifications and changes can be made to the specific embodiments illustrated without departing from the scope of the invention. Such modifications and changes are intended to be included within the scope of the appended claims. [Explanation of symbols]

[0194] 1NT 1-Naphthyl terephthalate GST Glutathione S-transferase MBP Maltose binding protein MHET Mono-2-hydroxyethyl terephthalate or mono-(2-hydroxyethyl) terephthalate MHETase Mono-(2-hydroxyethyl)terephthalate hydrolase PBS Polybutylene Succinate PBAT Polybutylene Adipate Terephthalate PBD Polybutylene succinate PBSA Polybutylene Succinate Adipate PBT Polybutylene Terephthalate PCL Polycaprolactone PEA (polyethylene adipate) or poly(ethylene adipate) PEF Polyethylene furanoate PEIT Polyethylene isosorbide terephthalate PET Polyethylene terephthalate PETase Polyethylene terephthalate esterase PGA Poly(glycolic acid) PHA Polyhydroxyalkanoate PLA Polylactic Acid PLGA Poly(lactic-co-glycolic acid) PTT Polytrimethylene terephthalate TPA Terephthalic Acid

Claims

1. A polypeptide having mono-(2-hydroxyethyl) terephthalic acid hydrolase (MHETase) activity, comprising: (i) having at least 70% sequence identity to SEQ ID NO: 1, (ii) positions corresponding to amino acid position 156 of SEQ ID NO: 1; (ii) positions corresponding to amino acid position 159 of SEQ ID NO: 1; (iii) positions corresponding to amino acid position 192 of SEQ ID NO: 1; and (iv) positions corresponding to amino acid position 503 of SEQ ID NO: 1 and comprising an amino acid sequence different from SEQ ID NO: 1 by amino acid substitutions at one or more positions selected from the group consisting of, wherein the MHETase activity of the polypeptide is greater than the MHETase activity of the MHETase of SEQ ID NO:

1. A polypeptide.

2. The polypeptide according to claim 1, wherein the amino acid sequence of the polypeptide is different from SEQ ID NO: 1 by an amino acid substitution at a position corresponding to amino acid position 156 of SEQ ID NO:

1.

3. The polypeptide according to claim 2, wherein the amino acid substitution at the position corresponding to amino acid position 156 of SEQ ID NO: 1 is N156G or a conservative amino acid substitution thereof.

4. The polypeptide according to claim 1, wherein the amino acid sequence of the polypeptide is different from SEQ ID NO: 1 by an amino acid substitution at a position corresponding to amino acid position 159 of SEQ ID NO:

1.

5. The polypeptide according to claim 4, wherein the amino acid substitution at the position corresponding to amino acid position 159 of SEQ ID NO: 1 is T159V or a conservative amino acid substitution thereof.

6. The polypeptide according to claim 1, wherein the amino acid sequence of the polypeptide is different from SEQ ID NO: 1 by an amino acid substitution at a position corresponding to amino acid position 252 of SEQ ID NO:

1.

7. The polypeptide according to claim 6, wherein the amino acid substitution at the position corresponding to amino acid position 252 of SEQ ID NO: 1 is Y252F or a conservative amino acid substitution thereof.

8. The polypeptide according to claim 1, wherein the amino acid sequence of the polypeptide is different from SEQ ID NO: 1 by an amino acid substitution at a position corresponding to amino acid position 503 of SEQ ID NO:

1.

9. The polypeptide according to claim 8, wherein the amino acid substitution at the position corresponding to amino acid position 503 of SEQ ID NO: 1 is Y503W or a conservative amino acid substitution thereof.

10. The polypeptide according to claim 1, wherein the amino acid sequence of the polypeptide differs from SEQ ID NO: 1 by amino acid substitutions at positions corresponding to amino acids 159, 252, and 503 of SEQ ID NO:

1.

11. The polypeptide according to claim 1, wherein the amino acid sequence of the polypeptide differs from SEQ ID NO: 1 by amino acid substitutions at positions corresponding to amino acids 159, 192, 252, and 503 of SEQ ID NO:

1.

12. The polypeptide according to claim 1, wherein the amino acid sequence of the polypeptide differs from SEQ ID NO: 1 by amino acid substitutions at positions corresponding to amino acids 159, 192, and 503 of SEQ ID NO:

1.

13. The polypeptide according to claim 1, wherein the amino acid sequence of the polypeptide differs from SEQ ID NO: 1 by amino acid substitutions at positions corresponding to amino acids 156, 159, and 503 of SEQ ID NO:

1.

14. The polypeptide according to claim 1, wherein the amino acid sequence of the polypeptide differs from SEQ ID NO: 1 by amino acid substitutions T159V, Y252F, and Y503W, or conservative amino acid substitutions for any of the foregoing.

15. The polypeptide according to claim 1, wherein the amino acid sequence of the polypeptide differs from SEQ ID NO: 1 by amino acid substitutions T159V, M192Y, Y252F, and Y503W, or conservative amino acid substitutions for any of the foregoing.

16. The polypeptide according to claim 1, wherein the amino acid sequence of the polypeptide differs from SEQ ID NO: 1 by amino acid substitutions N156G, M192Y, and Y503W, or conservative amino acid substitutions for any of the foregoing.

17. The polypeptide according to claim 1, wherein the amino acid sequence of the polypeptide differs from SEQ ID NO: 1 by amino acid substitutions N156G, T159V, M192Y, and Y503W, or conservative amino acid substitutions for any of the foregoing.

18. A composition comprising the polypeptide according to claim 1.

19. A polynucleotide comprising a nucleic acid sequence encoding the polypeptide according to claim 1.

20. A host cell comprising the polynucleotide according to claim 19.

21. A method for producing a polypeptide having MHETase activity, comprising: (a) preparing the polynucleotide according to claim 19; (b) A step of producing a polypeptide by expressing a nucleic acid sequence in a host cell culture; and (c) A step of collecting the polypeptide produced in (b) from the host cell culture A method comprising the steps. **Claim 22** A method for hydrolyzing mono-(2-hydroxyethyl) terephthalate, the method comprising exposing the mono-(2-hydroxyethyl) terephthalate to the polypeptide according to claim 1 or the host cell according to claim 20 under conditions sufficient for the mono-(2-hydroxyethyl) terephthalate to be converted to terephthalate and ethylene glycol. **Claim 23** A method for decomposing a plastic product containing a polyester, the method comprising exposing the plastic product to the polypeptide according to claim 1 or the host cell according to claim 20, wherein the polyester is selected from the group consisting of polylactic acid (PLA), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polyethylene terephthalate (PET), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), polycaprolactone (PCL), polyethylene adipate (PEA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA), and combinations of any of the foregoing. **Claim 24** (a) A step of exposing the PET to polyethylene terephthalic acid esterase (PETase) under conditions sufficient for the PETase to catalyze the conversion of the PET to produce mono-(2-hydroxyethyl) terephthalate (MHET); and (b) A step of exposing the MHET produced in step (a) to the polypeptide according to claim 1 or the host cell according to claim 20 under conditions sufficient for the polypeptide to catalyze the hydrolysis of the MHET to produce terephthalate and ethylene glycol, either simultaneously or sequentially The method according to claim 23, comprising the steps. **Claim 25** The method according to claim 24, further comprising a step of recovering the terephthalate and / or ethylene glycol produced in step (b).

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