Enzyme variants and uses thereof

Abstract

The present disclosure generally relates to a method of hydrolyzing a monoester of terephthalic acid, the method comprising exposing the monoester of terephthalic acid to a polypeptide having MHETase activity under conditions sufficient to enable the polypeptide to convert the monoester of terephthalic acid to terephthalic acid and an alcohol, wherein the terephthalic acid monoester is not terephthalic acid mono (2-hydroxyethyl) ester.

Description

[0001] This application claims priority to Australian Provisional Application No. 2022902460, filed on August 26, 2022, entitled "Enzyme variants and uses thereof", the content of which is incorporated herein by reference in its entirety.

[0002] The present invention relates to novel enzymes, and more particularly, to recombinant enzymes that hydrolyze the ester bond of mono-esters of terephthalic acid and uses thereof.

[0003] Background

[0004] 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 an admission that any of these documents form part of the common general knowledge in Australia or in any other country.

[0005] Global industrialization has had a significant impact on the environment, particularly the increased manufacture and reliance on plastics and plastic products. Despite increasing efforts to find suitable and environmentally sustainable plastic alternatives, including their manufacture and disposal, such products remain a major problem and contribute to the vast majority of environmental pollutants. One of the main causes of this problem is polyethylene terephthalate (PET) and its waste products, which are produced in millions of tons globally each year. The environmental significance of this problem is at least partly attributed to the chemical nature of plastics (especially PET-based products) as they do not readily decompose in nature.

[0006] Methods for dealing with the problem of plastic waste products generally include incineration, landfill disposal, and mechanical decomposition. However, these methods also have significant environmental impacts. For example, the incineration of plastics produces potentially harmful by-products that are released into the atmosphere; the decomposition rate of plastics in landfills is usually very slow and there is a risk of toxic substances leaching into groundwater; while mechanical decomposition is relatively expensive and the uses of its by-products are usually limited.

[0007] Recently, chemical and biological (enzymatic) degradation of plastics has been considered as an alternative method for reducing the accumulation of plastic waste. Chemical methods involve cleaving the ester bonds in PET polymers through hydrolysis or transesterification reactions, where the resulting oligomers or monomers can be used to recycle plastic products. The widespread acceptance of chemical recycling methods is limited because they are energy and resource intensive and would be costly. Additionally, the chemical recycling process may produce oligomer or monomer products that cannot be effectively recycled into other plastic products.

[0008] Enzymatic methods include using PETase, an enzyme of the esterase class that catalyzes the hydrolysis of PET to the monomer mono-2-hydroxyethyl terephthalate (MHET) and some bis-(2-hydroxyethyl) terephthalate (BHET). MHETase is a class of enzymes of the esterase that hydrolyzes MHET to terephthalic acid ester / TPA (which can be suitably recycled as a material for manufacturing new products, including plastics) and ethylene glycol. MHETase was initially discovered together with PETase in the bacterium Ideonella sakaiensis. These two enzymes enable the bacterium to survive using plastic PET as a carbon source (Yoshida et al., (2016) Science 351:1196).

[0009] As an esterase, MHETase shows no broad substrate specificity; MHETase cannot convert gallate esters, the most closely related substrates in the tannase family. MHETase also does not hydrolyze p-nitrophenyl esters of aliphatic monocarboxylic acids, such as the widely used esterase substrate p-nitrophenyl acetate. Native MHETase also cannot hydrolyze BHET, mono(2-hydroxyethyl) isophthalate (MHEI), or mono(2-hydroxyethyl) furoate (MHEF) (which may be industrial chemicals and / or PETase degradation products due to the use of isophthalate comonomers) (Knott et al., (2020) PNAS 117:25476).

[0010] Although the enzymatic degradation of plastics is an attractive alternative to mitigate the environmental impact of plastic waste products and their disposal, it has not been widely adopted, including because of its relatively low efficiency, slow enzymatic degradation rate, and low enzyme expression levels in common industrial host strains. Therefore, there is still an urgent need for improved methods and reagents for the enzymatic degradation of plastics. Summary of the Invention

[0012] In one aspect disclosed herein, a method for hydrolyzing a terephthalic acid monoester is provided, the method comprising exposing the terephthalic acid monoester to a polypeptide having MHETase activity under conditions sufficient for the polypeptide to convert the terephthalic acid monoester to terephthalic acid and an alcohol; wherein the terephthalic acid monoester is not mono-2-hydroxyethyl terephthalate. In an embodiment, the ester is an optionally benzyl-substituted C 1 -C 10 alkyl ester. In another embodiment, the ester is an optionally benzyl-substituted C 6 -C 10 alkyl ester. In one embodiment, the ester is a C 6 alkyl ester. In one embodiment, the ester is a C 7 alkyl ester. In one embodiment, the ester is a C 8Hydrocarbyl esters. In another embodiment, the ester is C 9 Hydrocarbyl esters. In another embodiment, the ester is C 10 Hydrocarbyl esters. In an embodiment, the monoesters of terephthalic acid are selected from the group consisting of monobenzyl terephthalate (MBZT), monohexyl terephthalate, monooctyl terephthalate (MOCT), and monooctyl terephthalate (MOCT). In a preferred embodiment, the monoesters of terephthalic acid are MBZT. In another preferred embodiment, the monoesters of terephthalic acid are MOCT.

[0013] In an embodiment, the polypeptide comprises the amino acid sequence of amino acids 20-603 of SEQ ID NO:1 or an amino acid sequence having at least 70% sequence identity thereto. In an embodiment, the polypeptide comprises the amino acid sequence of amino acids 20-603 of SEQ ID NO:1. In an embodiment, the polypeptide has at least 70% sequence identity with amino acids 20-603 of SEQ ID NO:1 and differs from amino acids 20-603 of SEQ ID NO:1 by amino acid substitutions at one or more positions selected from the group consisting of: the position corresponding to amino acid position 156 of SEQ ID NO:1; the position corresponding to amino acid position 159 of SEQ ID NO:1; the position corresponding to amino acid position 192 of SEQ ID NO:1; the position corresponding to amino acid position 196 of SEQ ID NO:1; the position corresponding to amino acid position 197 of SEQ ID NO:1; the position corresponding to amino acid position 252 of SEQ ID NO:1; the position corresponding to amino acid position 260 of SEQ ID NO:1; the position corresponding to amino acid position 264 of SEQ ID NO:1; the position corresponding to amino acid position 267 of SEQ ID NO:1; the position corresponding to amino acid position 286 of SEQ ID NO:1; and the position corresponding to amino acid position 503 of SEQ ID NO:1.

[0014] In an embodiment, the polypeptide differs from amino acids 20-603 of SEQ ID NO:1 by amino acid substitutions at the positions corresponding to amino acid positions 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 of any of the foregoing.

[0015] In another embodiment, the polypeptide differs from amino acids 20 - 603 of SEQ ID NO:1 by amino acid substitutions at positions corresponding to amino acid positions 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 of any of the foregoing. In a preferred embodiment, the polypeptide comprises the amino acid sequence of SEQ ID NO:77.

[0016] In some embodiments, the terephthalic acid monoester is produced by hydrolysis or degradation of a terephthalic acid diester or polyethylene terephthalate (PET). In another embodiment, the terephthalic acid monoester is produced by a process comprising exposing a terephthalic acid diester to sodium hydroxide and / or contacting the terephthalic acid diester with an esterase.

[0017] In another embodiment, the terephthalic acid monoester is produced by a process comprising reacting PET with a C 6 -C 10 monohydric alcohol in a base-catalyzed transesterification reaction; and / or exposing the terephthalic acid diester to an esterase. In a preferred embodiment, the C 6 -C 10 monohydric alcohol is benzyl alcohol, octanol, or heptanol. The present disclosure also extends to compositions comprising terephthalic acid and / or alcohol recovered by the methods described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Shows the amino acid sequences of wild-type (WT) MHETase (SEQ ID NO:1) and different consensus designs (SEQ ID NO:2 - 36, 73 - 78, 86).

[0020] Figure 2 Shows the nucleic acid sequences of wild-type (WT) MHETase (SEQ ID NO:37) and different consensus designs (SEQ ID NO:38 - 72 and 79 - 85).

[0021] Figure 3 Shows the activity (dA 465 / dt (min -1 )) of MHETase variants in whole cell suspensions against an MHET analogue (1-naphthyl terephthalate).

[0022] Figure 4Shows the expression levels of wild-type MHETase 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).

[0023] Figure 5 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 a temperature range of 20°C - 90°C (X-axis).

[0024] Figure 6 Shows the results of the whole-cell suspension fast blue assay for all tested MHETase variants from each round of mutagenesis. Bar height represents the average activity measured for each variant (n≥2, showing individual measurements) (dA 465 / dt (min -1 )), and error bars represent the mean standard error of the measurements. Highlighted bars represent the variants used as parents in the next round of mutagenesis.

[0025] Figure 7 Shows SDS-PAGE gels of selected MHETase variants from each round, stained with ATTO550 and imaged under UV transillumination. The expected size of the MHETase variants (≈64 kDa) is indicated.

[0026] Figure 8 Shows the size-exclusion chromatograms of selected MHETase variants.

[0027] Figure 9 Shows Michaelis-Menten plots of selected MHETase variants obtained using the colorimetric assay described herein. Each point represents the mean initial reaction rate of three technical replicates, each incubated with 6 nM MHETase and 4 mM fast blue B salt. Error bars represent the mean standard error.

[0028] Figure 10 Shows the thermal stability of MHETase variants from three replicates measured by circular dichroism at 222 nm in sodium acetate at pH 5.1. Data were fit to a two-state unfolding model (line), and error bars correspond to the mean standard error.

[0029] Figure 11An HPLC assay showing the comparison of the activities of wild-type MHETase, the 5th round Y252F (R5), and R5 with the wild-type MHETase identity restored at positions 192, 156, 159, 252, and 503.

[0030] Figure 12 The results of the Fast Blue assay of the whole-cell suspension of the MHETase R5 revertants are shown. The mutations V159T, Y192M, F252Y, and W503Y were performed in the background of MHETase R5 (the MHETase Y252F of the 5th round). The bar height represents the average activity measured for each variant (n≥2), and the error bars represent the mean standard error.

[0031] Figure 13 The structures of mono(2-hydroxyethyl) terephthalate (MHET) and other monoesters of terephthalic acid (TPA) are shown, including monoheptyl terephthalate (MHPT), monooctyl terephthalate (MOCT), monobenzyl terephthalate (MBZT), monohexyl terephthalate (MHXT), monopentyl terephthalate (MPET), monobutyl terephthalate (MBT), monopropyl terephthalate (MPT), monoethyl terephthalate (MET), and monomethyl terephthalate (MMT). The common terephthalic acid moiety is highlighted.

[0032] Figure 14 An HPLC assay showing the activities of the 5th round Y252F of MHETase (R5; SEQ ID NO:77) against the substrates monooctyl terephthalate (MOCT) and monobenzyl terephthalate (MBZT). A) Shows the increase in TPA concentration over time when 200 nM R5 was incubated with 1.5 mM MOCT at 40 °C, compared to the control (no enzyme). B) Shows the corresponding decrease in MOCT concentration. The data indicate that all MOCT was converted to TPA within 8 minutes. C) Shows the increase in TPA concentration over time when 200 nM R5 was incubated with 1.5 mM MBZT at 40 °C, compared to the control (no enzyme). D) Shows the corresponding decrease in MBZT concentration. The data indicate that all MBZT was converted to TPA within 8 minutes.

[0033] Figure 15Shows the activity of engineered MHETase Round 5 Y252F (R5; SEQ ID NO:77) compared to an esterase from wild boar (S. scrofa), a lipase from T. lanuginosa, and a lipase from R. miehei. Shows the concentration of A) MOCT and B) TPA over time for all enzyme variants and the enzyme-free control. Concentrations were determined using high performance liquid chromatography (HPLC). All data are the concentration of substrate or product as a percentage of the initial (time 0 minutes) concentration. SEQ ID NO:77 shows complete hydrolysis of MOCT to TPA within <10 minutes, while the esterase from wild boar, the lipase from T. lanuginosa, and the lipase from R. miehei showed no activity compared to the control. DETAILED DESCRIPTION OF THE INVENTION

[0035] 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, the preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.

[0036] Unless otherwise expressly stated, the articles "a" and "an" as used herein are used to refer to one or more than one (i.e., at least one) of the grammatical objects of the article. By way of example, "an element" means one element or more than one element.

[0037] As used herein, the term "about" means a quantity, level, value, dimension, size, or amount that varies by up to 10% (e.g., varies by 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%) relative to a reference quantity, level, value, dimension, size, or amount.

[0038] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises", and "comprising" will be understood to mean including the stated step, or element, or group of steps or elements, but not excluding any other step, or element, or group of steps or elements.

[0039] The present disclosure is at least in part based on the unexpected discovery by the inventors that a polypeptide having MHETase activity can hydrolyze substrates other than MHET; namely, monoesters of terephthalic acid, into terephthalic acid and an alcohol. The inventors also found that certain modifications can be made to the amino acid sequence of MHETase to advantageously enhance its activity of hydrolyzing monoesters of terephthalic acid into terephthalic acid and an alcohol, wherein the monoesters of terephthalic acid are not mono(2-hydroxyethyl) terephthalate. The inventors also unexpectedly found that substitutions can be made to amino acid residues outside the active site of wild-type MHETase to enhance its activity of converting monoesters of terephthalic acid into terephthalic acid and an alcohol; wherein the monoesters of terephthalic acid are not mono(2-hydroxyethyl) terephthalate.

[0040] Certain modifications also unexpectedly confer enhanced or improved activity on the modified MHETase in hydrolyzing monoesters of terephthalic acid into terephthalic acid and an alcohol, wherein the monoesters of terephthalic acid are not mono(2-hydroxyethyl) terephthalate.

[0041] Accordingly, in one aspect disclosed herein, there is provided a method of hydrolyzing a monoesters of terephthalic acid, the method comprising exposing the monoesters of terephthalic acid to a polypeptide having MHETase activity under conditions sufficient to enable the polypeptide to convert the monoesters of terephthalic acid into terephthalic acid and an alcohol, wherein the monoesters of terephthalic acid are not mono(2-hydroxyethyl) terephthalate.

[0042] Monoesters of terephthalic acid will be familiar to those skilled in the art. For example, as used herein, the term monoesters of terephthalic acid refers to 1,4-disubstituted benzenes in which the substitution is a carboxylic acid functional group and an ester functional group. Monoesters of terephthalic acid include monoalkyl terephthalates. In some embodiments, the monoesters of terephthalic acid are formed by transesterification of PET with C 1 -C 10 monohydric alcohols. In certain embodiments, the monoesters of terephthalic acid are formed by transesterification of PET with C 6 -C 10 monohydric alcohols. In certain embodiments, the monoesters of terephthalic acid are formed by transesterification of PET with benzyl alcohol, hexanol, heptanol, or octanol.

[0043] In an embodiment, the polypeptide comprises the amino acid sequence of amino acids 20 - 603 of SEQ ID NO:1 or an amino acid sequence having at least 70% sequence identity thereto. In an embodiment, the polypeptide comprises the amino acid sequence of amino acids 20 - 603 of SEQ ID NO:1. In an embodiment, the polypeptide comprises (i) an amino acid sequence having at least 70% sequence identity to amino acids 20 - 603 of SEQ ID NO:1, and (ii) is different from amino acids 20 - 603 of SEQ ID NO:1 by amino acid substitutions at one or more positions that do not contact the polyester substrate of MHETase.

[0044] In an embodiment, the polypeptide comprises (i) an amino acid sequence having at least 70% sequence identity to amino acids 20 - 603 of SEQ ID NO:1, and (ii) is different from amino acids 20 - 603 of SEQ ID NO:1 by amino acid substitutions at one or more positions selected from the group consisting of positions corresponding to amino acid positions 156 to 396, 398 to 410, and 425 to 603 of SEQ ID NO:1.

[0045] "At least 70%" means 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 with SEQ ID NO:1. Since the polypeptides described herein are variants of the MHETase of the naturally occurring (wild - type) SEQ ID NO:1, it should be understood that herein, "at least 70%" does not include 100% sequence identity of the entire sequence of SEQ ID NO:1 (residues 1 - 603 or residues 18 - 603). In some embodiments, the polypeptide may comprise amino acid insertions and / or deletions such as at the N - terminus and / or C - terminus, as described herein.

[0046] In another embodiment, the polypeptide comprises (a) an amino acid sequence having at least 70% sequence identity to amino acids 20 - 603 of SEQ ID NO:1, and (b) an amino acid sequence different from amino acids 20 - 603 of SEQ ID NO:1 by amino acid substitutions at one or more positions selected from the group consisting of:

[0047] (i) the position corresponding to amino acid position 156 of SEQ ID NO:1;

[0048] (ii) the position corresponding to amino acid position 159 of SEQ ID NO:1;

[0049] (iii) the position corresponding to amino acid position 192 of SEQ ID NO:1; and

[0050] (iv) the position corresponding to amino acid position 503 of SEQ ID NO:1.

[0051] In an embodiment, the amino acid sequence of the polypeptide differs from amino acids 20 - 603 of SEQ ID NO:1 by an amino acid substitution at the position corresponding to amino acid position 156 of SEQ ID NO:1.

[0052] In an embodiment, 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.

[0053] In an embodiment, the amino acid sequence of the polypeptide differs from amino acids 20 - 603 of SEQ ID NO:1 by an amino acid substitution at the position corresponding to amino acid position 159 of SEQ ID NO:1.

[0054] In an embodiment, 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.

[0055] In an embodiment, the amino acid sequence of the polypeptide differs from amino acids 20 - 603 of SEQ ID NO:1 by an amino acid substitution at the position corresponding to amino acid position 252 of SEQ ID NO:1.

[0056] In an embodiment, 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.

[0057] In an embodiment, the amino acid sequence of the polypeptide differs from amino acids 20 - 603 of SEQ ID NO:1 by an amino acid substitution at the position corresponding to amino acid position 503 of SEQ ID NO:1.

[0058] In an embodiment, 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.

[0059] In a particular embodiment, the amino acid sequence of the polypeptide differs from amino acids 20 - 603 of SEQ ID NO:1 by amino acid substitutions at the positions corresponding to amino acid positions 159, 252 and 503 of SEQ ID NO:1. In a preferred embodiment, the amino acid substitutions are T159V, Y252F and Y503W, or conservative amino acid substitutions of any of the foregoing.

[0060] In certain embodiments, the amino acid sequence of the polypeptide differs from amino acids 20 - 603 of SEQ ID NO:1 by amino acid substitutions at positions corresponding to amino acid positions 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 of any of the foregoing.

[0061] In certain embodiments, the amino acid sequence of the polypeptide differs from amino acids 20 - 603 of SEQ ID NO:1 by amino acid substitutions at positions corresponding to amino acid positions 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 of any of the foregoing.

[0062] In certain embodiments, the amino acid sequence of the polypeptide differs from amino acids 20 - 603 of SEQ ID NO:1 by amino acid substitutions at positions corresponding to amino acid positions 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 of any of the foregoing.

[0063] In certain embodiments, the amino acid sequence of the polypeptide differs from amino acids 20 - 603 of SEQ ID NO:1 by amino acid substitutions at positions corresponding to amino acid positions 156, 159, 192, and 503 of SEQ ID NO:1. In preferred embodiments, the amino acid substitutions are N156G, T159V, M192Y, and Y503W, or conservative amino acid substitutions of any of the foregoing.

[0064] In another embodiment, the polypeptide comprises (a) an amino acid sequence having at least 70% sequence identity to amino acids 20 - 603 of SEQ ID NO:1, and (b) an amino acid sequence that differs from amino acids 20 - 603 of SEQ ID NO:1 by amino acid substitutions at one or more positions selected from the group consisting of:

[0065] (i) a position corresponding to amino acid position 156 of SEQ ID NO:1;

[0066] (ii) a position corresponding to amino acid position 159 of SEQ ID NO:1;

[0067] (iii) a position corresponding to amino acid position 196 of SEQ ID NO:1;

[0068] (iv) The position corresponding to amino acid position 197 of SEQ ID NO:1;

[0069] (v) The position corresponding to amino acid position 260 of SEQ ID NO:1;

[0070] (vi) The position corresponding to amino acid position 264 of SEQ ID NO:1;

[0071] (vii) The position corresponding to amino acid position 267 of SEQ ID NO:1;

[0072] (viii) The position corresponding to amino acid position 286 of SEQ ID NO:1; and

[0073] (ix) The position corresponding to amino acid position 503 of SEQ ID NO:1.

[0074] In an embodiment, the amino acid sequence of the polypeptide differs from amino acids 20 - 603 of SEQ ID NO:1 by an amino acid substitution at the position corresponding to amino acid position 156 of SEQ ID NO:1.

[0075] In an embodiment, 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.

[0076] In an embodiment, the amino acid sequence of the polypeptide differs from amino acids 20 - 603 of SEQ ID NO:1 by an amino acid substitution at the position corresponding to amino acid position 159 of SEQ ID NO:1.

[0077] In an embodiment, 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.

[0078] In an embodiment, the amino acid sequence of the polypeptide differs from amino acids 20 - 603 of SEQ ID NO:1 by an amino acid substitution at the position corresponding to amino acid position 196 of SEQ ID NO:1.

[0079] In an embodiment, the amino acid substitution at the position corresponding to amino acid position 196 of SEQ ID NO:1 is S196A, or a conservative amino acid substitution thereof.

[0080] In an embodiment, the amino acid sequence of the polypeptide differs from amino acids 20 - 603 of SEQ ID NO:1 by an amino acid substitution at the position corresponding to amino acid position 197 of SEQ ID NO:1.

[0081] In an embodiment, the amino acid substitution at the position corresponding to amino acid position 197 of SEQ ID NO:1 is Y197V, or a conservative amino acid substitution thereof.

[0082] In an embodiment, the amino acid sequence of the polypeptide differs from amino acids 20 - 603 of SEQ ID NO:1 by an amino acid substitution at the position corresponding to amino acid position 260 of SEQ ID NO:1.

[0083] In an embodiment, the amino acid substitution at the position corresponding to amino acid position 260 of SEQ ID NO:1 is S260A, or a conservative amino acid substitution thereof.

[0084] In an embodiment, the amino acid sequence of the polypeptide differs from amino acids 20 - 603 of SEQ ID NO:1 by an amino acid substitution at the position corresponding to amino acid position 264 of SEQ ID NO:1.

[0085] In an embodiment, the amino acid substitution at the position corresponding to amino acid position 264 of SEQ ID NO:1 is S264L, or a conservative amino acid substitution thereof.

[0086] In an embodiment, the amino acid sequence of the polypeptide differs from amino acids 20 - 603 of SEQ ID NO:1 by an amino acid substitution at the position corresponding to amino acid position 267 of SEQ ID NO:1.

[0087] In an embodiment, the amino acid substitution at the position corresponding to amino acid position 267 of SEQ ID NO:1 is S267A, or a conservative amino acid substitution thereof.

[0088] In an embodiment, the amino acid sequence of the polypeptide differs from amino acids 20 - 603 of SEQ ID NO:1 by an amino acid substitution at the position corresponding to amino acid position 286 of SEQ ID NO:1.

[0089] In an embodiment, the amino acid substitution at the position corresponding to amino acid position 286 of SEQ ID NO:1 is S286A, or a conservative amino acid substitution thereof.

[0090] In an embodiment, the amino acid sequence of the polypeptide differs from amino acids 20 - 603 of SEQ ID NO:1 by an amino acid substitution at the position corresponding to amino acid position 503 of SEQ ID NO:1.

[0091] In an embodiment, 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.

[0092] 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 an embodiment, the polypeptide comprises a combination of amino acid substitutions at at least 2, preferably at least 3, preferably at least 4, preferably at least 5, preferably at least 6, preferably at least 7, preferably at least 8, preferably at least 9, or more preferably at least 10 positions corresponding to positions in SEQ ID NO:1, as described herein.

[0093] In an embodiment, the amino acid sequence of the polypeptide differs from amino acids 20 - 603 of SEQ ID NO:1 by amino acid substitutions at positions corresponding to amino acid positions 156, 159, and 197 of SEQ ID NO:1.

[0094] In an embodiment, the amino acid substitutions are N156G, T159V, and Y197V, or conservative amino acid substitutions of any of the foregoing.

[0095] In one embodiment, the polypeptide is not an esterase from wild boar or a lipase from T. lanuginosa or Rhizomucor miehei.

[0096] The polypeptide can be used alone in purified form or in combination with other enzymes (e.g., PETase or MHETase or carboxylesterase or cutinase having PETase or MHETase or esterase activity) to catalyze the enzymatic reactions involved in the degradation and / or recycling of materials containing TPA polyesters or monoester / diesters (such as plastic products containing polyesters or monoester / diesters of TPA). The polypeptides described herein can be in soluble form, or they can be immobilized on a substrate. Suitable substrates will be familiar to those skilled in the art, and illustrative examples thereof include cell membranes, lipid vesicles, glass, plastic, polymers, filters, membranes, beads, columns, and plates.

[0097] It may be convenient to use the polypeptide immobilized on a substrate for the methods of the present invention, including when the methods of the present invention are carried out in a semi - continuous or continuous manner.

[0098] In an embodiment, the polypeptides described herein are immobilized on a substrate.

[0099] Polypeptides can be immobilized on any suitable substrate using techniques known to those skilled in the art. For example, polypeptides can be immobilized on a support resin by ion exchange, adsorption (e.g., hydrophobic adsorption), or covalent coupling.

[0100] In an embodiment, the substrate is a resin. Suitable resins will be known to those skilled in the art, and illustrative examples thereof are ion exchange resins. In one embodiment, the polypeptide is immobilized on a resin. In another embodiment, the polypeptide is immobilized on an adsorption resin. In another embodiment, the polypeptide is immobilized on a nickel affinity resin. In an embodiment, the polypeptide is immobilized on a covalent resin. In one embodiment, the polypeptide is immobilized on an ion exchange resin.

[0101] Thus, in an embodiment, the substrate is an ion exchange resin. Those skilled in the art will be familiar with the general principles of enzymatic immobilization techniques, and this principle can be advantageously applied in the context of immobilizing polypeptides on substrates according to the present invention.

[0102] Suitable ion exchange resins will generally comprise a polymer matrix or a polymer / ceramic hybrid matrix. Exemplary examples of such resins include, but are not limited to, CM Ceramic ion exchange chromatography resins.

[0103] In an embodiment, the ion exchange resin is a cation exchange resin. For the operation of the methods described herein, the polypeptide will generally be immobilized on a support resin and loaded into a column.

[0104] The present disclosure also extends to compositions comprising the polypeptides described herein.

[0105] The present disclosure also extends to nucleic acid sequences encoding the polypeptides described herein.

[0106] The present disclosure also extends to expression vectors comprising the nucleic acid sequences described herein.

[0107] The present disclosure also extends to host cells comprising the nucleic acid sequences or expression vectors described herein.

[0108] In an embodiment, monomethyl terephthalate is produced as a byproduct of the degradation, hydrolysis, or recycling of polyethylene terephthalate (PET). In an embodiment, monomethyl terephthalate is produced by the degradation or hydrolysis of dimethyl terephthalate. In another embodiment, monomethyl terephthalate is produced by a process comprising: exposing dimethyl terephthalate to sodium hydroxide, and / or contacting dimethyl terephthalate with an esterase.

[0109] In another embodiment, monomethyl terephthalate is produced by a process comprising: contacting PET with C 6 -C 10The transesterification reaction of a monohydric alcohol is carried out under base catalysis; and / or PET is contacted with an esterase. In a preferred embodiment, C 6 -C 10 The monohydric alcohol is benzyl alcohol, octanol or heptanol.

[0110] The present disclosure also extends to a composition comprising terephthalic acid and / or an alcohol recovered by the methods described herein.

[0111] The present disclosure also extends to a method for degrading a plastic product comprising a polyester, the method comprising exposing the plastic product to a polypeptide, composition or host cell described herein.

[0112] In an embodiment, 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 adipate succinate (PBSA), polybutylene terephthalate adipate (PBAT), polyethylene furandicarboxylate (PEF), polycaprolactone (PCL), polyethylene adipate (PEA), polyglycolic acid (PGA), poly(lactic-co-glycolic acid) (PLGA), and combinations of any of the foregoing. In an embodiment, the polyester is polyethylene terephthalate (PET).

[0113] In another embodiment, the method disclosed herein comprises subjecting PET to a base-catalyzed transesterification reaction with a C 6 -C 10 monohydric alcohol to produce a terephthalic acid monoester C 6 -C 10 monohydric alcohol derivative, and contacting the terephthalic acid monoester C 6 -C 10 monohydric alcohol derivative with a polypeptide under conditions sufficient to enable the polypeptide to convert the terephthalic acid monoester C 6 -C 10 monohydric alcohol derivative into terephthalic acid and an alcohol.

[0114] In one embodiment, the C 6 -C 10 monohydric alcohol is selected from hexanol, pentanol, octanol, nonanol, decanol and benzyl alcohol. In a preferred embodiment, the C 6 -C 10 monohydric alcohol is hexanol, pentanol, octanol.

[0115] The transesterification reaction carried out according to the method of the present invention utilizes a base catalyst. There is no particular limitation on the type of base catalyst that can be used.

[0116] In one embodiment, an alkali metal base is used to catalyze the transesterification reaction. Examples of suitable alkali metal bases include, but are not limited to, alkali metal hydroxides. Examples of suitable alkali metal hydroxides include, but are not limited to, lithium hydroxide, sodium hydroxide, and potassium hydroxide. In a preferred embodiment, sodium hydroxide or potassium hydroxide is used to catalyze the transesterification reaction.

[0117] The present disclosure also extends to compositions comprising terephthalic acid and / or an alcohol recovered by the methods disclosed herein.

[0118] In another aspect, a host cell genetically modified to express a polypeptide described herein is provided.

[0119] As used herein, the terms "peptide", "polypeptide", "protein", "enzyme" are understood to refer to a chain of amino acids linked by peptide bonds, regardless of the number of amino acids forming the chain. Amino acids are typically represented by their single-letter or three-letter codes according to the following nomenclature: A: alanine (Ala); C: cysteine (Cys); D: aspartic acid (Asp); E: glutamic acid (Glu); F: phenylalanine (Phe); G: glycine (Gly); H: histidine (His); I: isoleucine (Ile); K: lysine (Lys); L: leucine (Leu); M: methionine (Met); N: asparagine (Asn); P: proline (Pro); Q: glutamine (Gln); R: arginine (Arg); S: serine (Ser); T: threonine (Thr); V: valine (Val); W: tryptophan (Trp); and Y: tyrosine (Tyr).

[0120] The term "hydrolase" refers to an enzyme belonging to a class of hydrolases classified as EC 3 according to enzyme nomenclature that catalyzes the hydrolysis of peptide bonds in a peptide or protein to produce shorter peptides. As used herein, the term "esterase" generally refers to an enzyme belonging to the class of hydrolases that hydrolyze esters into acids and alcohols (enzyme class EC 3.1). As used herein, the term "MHETase" generally refers to a carboxylesterase (enzyme class EC 3.1.1.102) that hydrolyzes 2-hydroxyethyl terephthalic acid into terephthalic acid and an alcohol.

[0121] The terms "wild-type" or "parent" are used interchangeably herein to denote the naturally occurring isoform of a polypeptide; that is, as it occurs in nature. In the present disclosure, the wild-type polypeptide refers to the terephthalic acid mono(2-hydroxyethyl) ester hydrolase (EC 3.1.1.102; UniProt accession number A0A0K8P8E7) having the amino acid sequence listed in SEQ ID NO:1, or comprising amino acids 20 - 603 of SEQ ID NO:1.

[0122] As mentioned by Palm et al. (2019, Nat. Comms. 10:1717), two recently discovered bacterial enzymes that specifically degrade polyethylene terephthalate (PET) represent a promising solution to the problem of polyester-containing products that would otherwise burden the environment. First, Ideonella sakaiensis PETase, an α / β-hydrolase foldase with well-characterized structure, converts PET into mono(2-hydroxyethyl) terephthalate (MHET). The second key enzyme, MHETase, hydrolyzes MHET into the PET educts terephthalic acid 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).

[0123] Those skilled in the art will be familiar with the amino acid and nucleic acid sequences of wild-type MHETase, illustrative examples of which include SEQ ID NO:1.

[0124] The terms “mutant” and “variant” are used interchangeably herein to refer to a polypeptide that contains an amino acid sequence derived from SEQ ID NO:1 and that also contains a modification or alteration (e.g., substitution, insertion, and / or deletion) at one or more (e.g., several) positions when compared to the polypeptide of SEQ ID NO:1. Such variants can 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. As used herein, the terms “modification,” “alteration,” “substitution,” etc., in relation to an amino acid residue or position generally mean that the amino acid at a particular position has been modified compared to the amino acid of the wild-type or parental polypeptide.

[0125] Suitable substitutions can include replacement of one amino acid residue with another amino acid residue selected from: the 20 standard naturally-occurring amino acid residues, rare naturally-occurring amino acid residues (e.g., hydroxyproline, hydroxylysine, allo-hydroxylysine, 6-N-methyllysine, N-ethylglycine, N-methylglycine, N-ethylasparagine, alloisoleucine, N-methylisoleucine, N-methylvaline, pyroglutamine, aminobutyric acid, ornithine, norleucine, norvaline) and non-naturally-occurring amino acid residues typically prepared synthetically (e.g., cyclohexyl-alanine). Preferably, the substitution includes replacement of one amino acid residue with another amino acid residue selected from the 20 standard naturally-occurring amino acid residues (G, P, A, V, L, I, M, C, F, Y, W, H, K, R, Q, N, E, D, S and T). Modifications or alterations can be identified herein using the following terminology: Y197V indicates that the amino acid residue tyrosine (Y) at position 197 of the parental polypeptide sequence is replaced with valine (V). Y197V / I / M indicates that the amino acid residue tyrosine (Y) at position 197 of the parental sequence can be replaced with one of the following amino acids: valine (V), isoleucine (I) or methionine (M). Substitutions can be conservative substitutions or non-conservative substitutions. Those skilled in the art will be familiar with examples of conservative substitutions, illustrative examples of which include substitutions within the following groups: basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine, asparagine and threonine), hydrophobic amino acids (methionine, leucine, isoleucine, cysteine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine) and small amino acids (glycine, alanine and serine).

[0126] Unless otherwise indicated, the position references disclosed in this application are numbered with respect to the amino acid sequence set forth in SEQ ID NO:1. As used herein, the term "corresponding to", when used in reference to an amino acid position, is intended to mean the amino acid position in a polypeptide sequence when that position is aligned with the equivalent or corresponding position in the sequence set forth in SEQ ID NO:1.

[0127] As used herein, the term "sequence identity" or "identity" refers to the number (or proportion expressed as a percentage %) of matches (identical amino acid residues) between two polypeptide sequences. In a preferred embodiment, sequence identity is determined by comparing the sequences when they are aligned such that the overlap and identity are maximized while minimizing sequence gaps. Depending on the lengths of the two sequences, any of a number of mathematical global or local alignment algorithms known to those of skill in the art can be used to determine sequence identity. 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 the 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 the percentage amino acid sequence identity can be achieved by any means available to those of skill in the art, illustrative examples of which include publicly available computer software, such as that available at http: / / blast.ncbi.nlm.nih.gov / or http: / / www.ebi.ac.uk / Tools / emboss / . Those of skill in the art can readily determine the appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the sequences being compared. As used herein, % sequence identity generally refers to the value produced using a pairwise sequence alignment that creates the best global alignment of two sequences (e.g., using the Needleman-Wunsch algorithm), where all retrieval parameters are set to default values, e.g., scoring matrix = BLOSUM62, gap open = 10, gap extend = 0.5, end gap penalty = false, end gap open = 10, and end gap extend = 0.5.

[0128] As used herein, the term "recombinant" generally refers to a nucleic acid construct, vector, polypeptide, or cell produced by genetic engineering.

[0129] As used herein, the term "expression" generally refers to any step involved in polypeptide production, such as by transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0130] The term "expression cassette" refers to a nucleic acid construct that contains a coding region and a suitably regulatory region, to which the coding region is operably linked.

[0131] The term "expression vector" generally means a DNA or RNA molecule that contains an expression cassette. The expression vector can be a linear or circular double-stranded DNA molecule.

[0132] As used herein, the term "polymer" generally refers to a chemical compound or mixture of compounds whose structure is composed of multiple monomers (repeating units) linked by covalent chemical bonds. In the context of the present invention, the term polymer includes natural or synthetic polymers composed of a single type of repeating unit (i.e., homopolymer) or a mixture of different repeating units (i.e., copolymer or heteropolymer).

[0133] As used herein, terms such as "polyester-containing material", "polyester-containing product", etc. should be understood to refer to a product containing at least one polyester in a crystalline, semi-crystalline or completely amorphous form, such as a plastic product. A polyester-containing material can refer to any article made of at least one plastic material, such as plastic sheets, tubes, rods, profiles, shapes, films, massive blocks, fibers, textiles, etc., which contain at least one polyester, and possibly other substances or additives, such as plasticizers, mineral or organic fillers. In an embodiment, the polyester-containing material is a textile or fabric containing at least one polyester-containing fiber. In another embodiment, the polyester-containing material is a plastic compound or plastic preparation in a molten or solid state suitable for manufacturing plastic products.

[0134] Those skilled in the art will be familiar with suitable polyesters, illustrative examples of which include polylactic acid (PLA), polyethylene terephthalate (PET), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polyhydroxyalkanoates (PHA), polybutylene succinate (PBS), polybutylene adipate succinate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furandicarboxylate (PEF), polycaprolactone (PCL), and polyethylene adipate (PEA). Thus, in an 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), polyhydroxyalkanoates (PHA), polybutylene succinate (PBS), polybutylene adipate succinate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furandicarboxylate (PEF), polycaprolactone (PCL), polyethylene adipate (PEA), and combinations of any of the foregoing.

[0135] As described elsewhere herein, the inventors unexpectedly found that naturally occurring MHETase and its functional variants are capable of converting monoesters of terephthalic acid into terephthalic acid and alcohol.

[0136] The activity of this newly identified MHETase is particularly suitable for the degradation of plastic products, especially those containing PET. Additionally, the inventors surprisingly found that amino acid residues in the protein structure that were not originally expected to contact the polyester substrate can be advantageously modified to enhance the activity of MHETase in converting monoesters of terephthalic acid to terephthalic acid and an alcohol, where the monoesters of terephthalic acid are not mono(2-hydroxyethyl) terephthalate.

[0137] In embodiments, there are provided polypeptides that comprise (i) having at least 70% sequence identity to amino acids 20 - 603 of SEQ ID NO:1, and (ii) an amino acid sequence that is different from amino acids 20 - 603 of SEQ ID NO:1 by amino acid substitutions at one or more positions that do not contact the polyester substrate of MHETase, wherein the polypeptide is capable of converting a monoesters of terephthalic acid to terephthalic acid and an alcohol, where the monoesters of terephthalic acid are not mono(2-hydroxyethyl) terephthalate. In some embodiments, the ester is a C 1 -C 10 hydrocarbyl ester optionally substituted with benzyl. In another embodiment, the ester is a C 6 -C 10 hydrocarbyl ester. In one embodiment, the ester is a C 6 hydrocarbyl ester. In one embodiment, the ester is a C 7 hydrocarbyl ester. In one embodiment, the ester is a C 8 hydrocarbyl ester. In another embodiment, the ester is a C 9 hydrocarbyl ester. In another embodiment, the ester is a C 10Hydrocarbyl esters. In embodiments, the monoesters of terephthalic acid are selected from the group consisting of: monobenzyl terephthalate (MBZT), monohexyl terephthalate (MHXT), monooctyl terephthalate (MOCT), and monooctyl terephthalate (MOCT). In a preferred embodiment, the monoesters of terephthalic acid are MBZT. In another preferred embodiment, the monoesters of terephthalic acid are MOCT. As used herein, the term "contact" generally refers to the direct contact of the amino acid residues of SEQ ID NO:1 MHETase with its polyester substrate. Those skilled in the art will be familiar with the amino acid residues of SEQ ID NO:1 that contact its polyester substrate. These 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 embodiments, the polypeptide comprises an amino acid sequence that is different from amino acids 20-603 of SEQ ID NO:1 by amino acid substitutions 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 in SEQ ID NO:1 that is capable of contacting and hydrolyzing the polyester substrate (i.e., MHET). Those skilled in the art will be familiar with the amino acid positions of SEQ ID NO:1 that are outside the MHETase active site of SEQ ID NO:1.

[0138] In the context of the present disclosure, reference to increased or enhanced activity refers to the ability to convert a monoesters of terephthalic acid to terephthalic acid and an alcohol; wherein the monoesters of terephthalic acid is not mono(2-hydroxyethyl) terephthalate.

[0139] In embodiments, the polypeptides disclosed herein are capable of converting monobenzyl terephthalate (MBZT) to terephthalic acid and an alcohol, wherein the monoesters of terephthalic acid is not mono(2-hydroxyethyl) terephthalate. In embodiments, the polypeptides disclosed herein are capable of converting monobenzyl terephthalate (MBZT) to terephthalic acid and benzyl alcohol.

[0140] In embodiments, the polypeptides disclosed herein are capable of converting monohexyl terephthalate (MHXT) to terephthalic acid and an alcohol, wherein the monoesters of terephthalic acid is not mono(2-hydroxyethyl) terephthalate. In embodiments, the polypeptides disclosed herein are capable of converting monohexyl terephthalate (MHXT) to terephthalic acid and heptanol.

[0141] In embodiments, the polypeptides disclosed herein are capable of converting monooctyl terephthalate (MHPT) to terephthalic acid and an alcohol, wherein the monoesters of terephthalic acid is not mono(2-hydroxyethyl) terephthalate. In embodiments, the polypeptides disclosed herein are capable of converting monooctyl terephthalate (MHPT) to terephthalic acid and heptanol.

[0142] In an embodiment, the polypeptides disclosed herein are capable of converting monooctyl terephthalate (MOCT) into terephthalic acid and an alcohol, wherein the terephthalic acid monoester is not mono(2-hydroxyethyl) terephthalate. In an embodiment, the polypeptides disclosed herein are capable of converting monooctyl terephthalate (MOCT) into terephthalic acid and octanol.

[0143] In an embodiment, the polypeptides described herein have an activity in converting a terephthalic acid monoester into terephthalic acid and an alcohol (wherein the terephthalic acid monoester is not mono(2-hydroxyethyl) terephthalate) similar to the activity of the MHETase of SEQ ID NO:1. In an embodiment, compared to the MHETase of SEQ ID NO:1, the polypeptides described herein have an increased activity in converting a terephthalic acid monoester into terephthalic acid and an alcohol (wherein the terephthalic acid monoester is not mono(2-hydroxyethyl) terephthalate) 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% or more. Those skilled in the art will be familiar with suitable methods for determining or measuring the specific activity of a polypeptide, illustrative examples of which are described elsewhere herein. For example, the activity of a polypeptide in converting a terephthalic acid monoester into terephthalic acid and an alcohol can be detected and / or measured by detecting / measuring the amount of terephthalic acid produced. 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 herein by reference in their entirety. In an embodiment, when determined by an assay using a terephthalic acid monoester as a substrate, compared to the MHETase of SEQ ID NO:1, the activity in converting a terephthalic acid monoester into terephthalic acid and an alcohol (wherein the terephthalic acid monoester is not mono(2-hydroxyethyl) terephthalate) 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.

[0144] The activity of converting a terephthalic acid monoester to terephthalic acid and an alcohol, wherein the terephthalic acid monoester is not terephthalic acid mono(2-hydroxyethyl) ester, can be specified as an absolute value or as a value relative to a comparator (e.g., the MHETase of SEQ ID NO:1). In an embodiment, the activity of converting a terephthalic acid monoester to terephthalic acid and an alcohol (wherein the terephthalic acid monoester is not terephthalic acid mono(2-hydroxyethyl) ester) is measured as the rate of monomers and / or oligomers (e.g., in mg) released per hour and per mg of enzyme under suitable temperature, pH, and buffer conditions.

[0145] The activity of converting a terephthalic acid monoester to terephthalic acid and an alcohol, wherein the terephthalic acid monoester is not terephthalic acid mono(2-hydroxyethyl) ester, can be measured or determined using a purified enzyme. Optionally, the activity of converting a terephthalic acid monoester to terephthalic acid and an alcohol (wherein the terephthalic acid monoester is not terephthalic acid mono(2-hydroxyethyl) ester) can be measured as a function of the activity when the enzyme is recombinantly expressed in a host cell system (also referred to herein as cell catalytic activity or whole cell activity).

[0146] Advantageously, the polypeptides described herein exhibit the activity of converting a terephthalic acid monoester to terephthalic acid and an alcohol (wherein the terephthalic acid monoester is not terephthalic acid mono(2-hydroxyethyl) ester) at least in the temperature range 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, and even more preferably at about 45°C. In an embodiment, the polypeptides described herein exhibit activity at 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 an embodiment, the activity is measurable between about 40°C and about 60°C, preferably between about 40°C and about 50°C, or even more preferably at about 45°C. In another specific embodiment, the polyester degradation activity is still measurable at a temperature between about 10°C and about 30°C, preferably between about 15°C and about 28°C, corresponding to the average temperature in the natural environment (ambient temperature).

[0147] In an embodiment, the polypeptide has the activity of converting a terephthalic acid monoester into terephthalic acid and an alcohol at a temperature from about 10°C to about 60°C, preferably from about 20°C to about 60°C, preferably from about 30°C to about 60°C, more preferably from about 40°C to about 60°C, even more preferably from about 40°C to about 50°C or even more preferably at about 45°C (wherein the terephthalic acid monoester is not terephthalic acid mono(2-hydroxyethyl) ester), and the activity is 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 higher compared to the activity of converting a terephthalic acid monoester into terephthalic acid and an alcohol by SEQ ID NO:1 at the same temperature (wherein the terephthalic acid monoester is not terephthalic acid mono(2-hydroxyethyl) ester).

[0148] In another specific embodiment, when compared to the polypeptide of SEQ ID NO:1, the polypeptides described herein have enhanced activity of converting a terephthalic acid monoester into terephthalic acid and an alcohol at a temperature between about 10°C and about 60°C, preferably between about 20°C and about 60°C, preferably from about 30°C to 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, wherein the terephthalic acid monoester is not terephthalic acid mono(2-hydroxyethyl) ester. In an embodiment, the polypeptides described herein have the activity of converting a terephthalic acid monoester into terephthalic acid and an alcohol (wherein the terephthalic acid monoester is not terephthalic acid mono(2-hydroxyethyl) ester) between about 20°C and about 60°C, and the activity is 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 higher compared to the activity of converting a terephthalic acid monoester into terephthalic acid and an alcohol by SEQ ID NO:1 at the same temperature (wherein the terephthalic acid monoester is not terephthalic acid mono(2-hydroxyethyl) ester).

[0149] In another embodiment, compared to the polypeptide of SEQ ID NO:1, the polypeptides described herein have increased activity for converting monoesters of terephthalic acid to terephthalic acid and alcohol at a temperature 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, wherein the monoester of terephthalic acid is not mono(2-hydroxyethyl) terephthalate. In an embodiment, the polypeptides described herein have activity for converting monoesters of terephthalic acid to terephthalic acid and alcohol (wherein the monoester of terephthalic acid is not mono(2-hydroxyethyl) terephthalate) at a temperature between about 10°C and about 30°C, which is 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 higher compared to the activity of SEQ ID NO:1 for converting monoesters of terephthalic acid to terephthalic acid and alcohol (wherein the monoester of terephthalic acid is not mono(2-hydroxyethyl) terephthalate) at the same temperature.

[0150] In an embodiment, the polypeptides described herein exhibit measurable activity for converting monoesters of terephthalic acid to terephthalic acid and alcohol 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, even more preferably in the pH range of 7 to 8, wherein the monoester of terephthalic acid is not mono(2-hydroxyethyl) terephthalate.

[0151] The present disclosure also extends to polynucleotides comprising nucleic acid sequences encoding the MHETase polypeptides described herein. In aspects disclosed herein, polynucleotides are provided that comprise nucleic acid sequences encoding the polypeptides described herein. In an 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 can be DNA (cDNA or gDNA), RNA, or a mixture of both. The nucleic acid can be in single-stranded form, double-stranded form, or a mixture of both. The nucleic acid can be of recombinant, artificial, and / or synthetic origin, and the nucleic acid can contain modified nucleotides, including, for example, modified linkages, modified purine or pyrimidine bases, or modified sugars. The nucleic acids of the invention can be in isolated or purified form and are prepared, isolated, and / or manipulated by techniques known per se in the art, such as cloning and expression of cDNA libraries, amplification, enzymatic synthesis, or recombinant techniques. The nucleic acids can also be synthesized in vitro by well-known chemical synthesis techniques, as described, for example, in Belousov (1997) Nucleic Acids Res. 25:3440 - 3444.

[0152] The nucleic acid sequences disclosed herein can be suitably codon-optimized. Those skilled in the art will be familiar with suitable methods for codon optimization, illustrative examples of which are described in the reference manual Sambrook et al. (Sambrook et al., 2001).

[0153] 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 adjusted according to the host cell in which the nucleic acid will be transcribed.

[0154] In some embodiments, the nucleic acid sequences described herein can suitably comprise additional nucleotide sequences that can be used to cause or regulate the expression of the polypeptide in a selected host cell or system, such as regulatory regions, i.e., promoters, enhancers, silencers, terminators, signal peptides, etc. Optionally or additionally, the nucleic acid sequences described herein can also comprise additional nucleotide sequences encoding fusion proteins, such as maltose-binding protein (MBP) or glutathione S-transferase (GST), which can be used to facilitate polypeptide expression and / or solubility.

[0155] As described elsewhere herein, the present disclosure also extends to expression vectors and expression cassettes comprising the nucleic acid sequences described herein, which nucleic acid sequences are optionally operably linked to one or more control sequences that direct the expression of the nucleic acid in a suitable host cell. Generally, the expression vector or expression cassette comprises the nucleic acid sequences described herein operably linked to control sequences such as a transcriptional promoter and / or a transcriptional terminator. The control sequences can include a promoter recognized by the host cell or an in vitro expression system for expressing the nucleic acid encoding the polypeptides described herein. The promoter will generally comprise transcriptional control sequences that mediate the expression of the polypeptide. The promoter can be any polynucleotide that exhibits transcriptional activity in the host cell, including mutant, truncated, and hybrid promoters, and can be suitably obtained from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell. The control sequence can also be a transcriptional terminator, which is recognized by the host cell to terminate transcription. The terminator is generally operably linked to the 3'-end of the nucleic acid encoding the polypeptide. Any terminator that functions in the host cell can be used herein. Generally, the expression vector or expression cassette comprises the nucleic acid sequences described herein operably linked to a transcriptional promoter and a transcriptional terminator.

[0156] The term "vector" generally refers to a DNA molecule that serves as a vehicle to transfer recombinant genetic material into a host cell. Suitable vectors include plasmids, phages, viruses, fosmids, cosmids, and artificial chromosomes. A vector is generally a DNA sequence that comprises an insert (heterologous nucleic acid sequence, transgene) and a larger sequence that serves as the "backbone" of the vector. The purpose of the vector in transferring genetic information to the host is generally to isolate, propagate, or express the insert in the target cell. An expression vector (also referred to as an expression construct) is specifically adapted for the expression of a heterologous sequence in a target cell and generally has a promoter sequence that drives the expression of the heterologous sequence encoding a polypeptide.

[0157] Generally, the regulatory elements used in an expression vector include a transcriptional promoter, a ribosome binding site, a terminator, and optionally an operator gene. The expression vector can also comprise an origin of replication for autonomous replication in the host cell, a selectable marker, a limited number of useful restriction enzyme sites, and the potential for high copy number. Those skilled in the art will be familiar with suitable expression vectors, illustrative examples of which include cloning vectors, modified cloning vectors, plasmids, and viruses. Expression vectors that can provide appropriate 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 the vector is to be introduced. In one embodiment, the vector is the bacterial expression vector pET-28a(+) (SEQ ID NO:85).

[0158] 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 in such a way 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 parental host cell that is not identical to the parental host cell due to mutations that occur during replication. The host cell can be any cell used to produce the variants of the invention, e.g., prokaryotic or eukaryotic. The prokaryotic host cell can be any Gram-positive or Gram-negative bacterium. The host cell can also be a eukaryotic cell such as a yeast cell, a fungal cell, a mammalian cell, an insect cell, or a plant cell. In certain embodiments, the host cell is selected from the group consisting of Escherichia coli, Pseudomonas, Bacillus, Streptomyces, Trichoderma, Aspergillus, yeast, Pichia, Thermus, or Yarrowia.

[0159] The nucleic acid, expression cassette, or expression vector according to the invention can be introduced into the 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 transformation, and liposome-mediated transformation.

[0160] In an embodiment, the host cell is a genetically modified host cell or microorganism. Herein, the host cell or microorganism can be genetically modified to enhance the expression and / or activity of the polypeptide it expresses. For example, the polypeptides described herein can be used to supplement wild-type strains of fungi or bacteria known to have MHETase activity and / or capable of converting monoesters of terephthalic acid to terephthalic acid and alcohols, where the monoesters of terephthalic acid are not mono(2-hydroxyethyl) terephthalate, to increase and / or enhance the activity of the strain to convert monoesters of terephthalic acid to terephthalic acid and alcohols, where the monoesters of terephthalic acid are not mono(2-hydroxyethyl) terephthalate.

[0161] The present disclosure also extends to a method for producing a polypeptide having activity to convert monoesters of terephthalic acid to terephthalic acid and alcohols, where the monoesters of terephthalic acid are not mono(2-hydroxyethyl) terephthalate, the method comprising:

[0162] a) providing a nucleic acid sequence as described herein;

[0163] b) expressing the nucleic acid sequence in a host cell culture to produce a polypeptide; and

[0164] c) recovering the polypeptide produced in (b) from the host cell culture.

[0165] The disclosure of the present invention also extends to an in vitro method of producing the polypeptides described herein, the method comprising: (a) contacting a nucleic acid, cassette or vector of the present invention with an in vitro expression system; and (b) recovering the produced polypeptide. In vitro expression systems are well known to those skilled in the art and are commercially available.

[0166] Those skilled in the art will be familiar with suitable host cells, illustrative examples of which include recombinant Bacillus, recombinant Escherichia coli, recombinant Pseudomonas, recombinant Aspergillus, recombinant Trichoderma, recombinant Streptomyces, recombinant Saccharomyces, recombinant Pichia, recombinant Thermus or recombinant Yarrowia. In an embodiment, the host cell is Escherichia coli. In another embodiment, the host cell is Bacillus.

[0167] The host cells can be cultured in a nutrient medium suitable for producing the polypeptide using methods known to those skilled in the art. Suitable examples include culturing the host cells by shake flask culture, or small-scale or large-scale fermentation (including continuous, batch, fed-batch or solid-state fermentation) in a suitable medium and under conditions that permit enzyme expression and / or isolation in a laboratory or industrial fermenter. The culture is generally carried out in a suitable nutrient medium from a commercial supplier or prepared according to published compositions (e.g., in the catalog of the American Type Culture Collection) or any other medium suitable for cell growth. When the polypeptide is expressed and / or secreted into the nutrient medium, the polypeptide can be used in the form of a cell / supernatant mixture or in the form of a crude cell lysate. Optionally, the polypeptide can be recovered directly from the culture supernatant. Alternatively, the polypeptide can be recovered from the 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. Optionally, the polypeptide can be partially or fully purified by various procedures known in the art, including but not limited to heat 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.

[0168] The polypeptide can be used alone in purified form or in combination with another enzyme (e.g., PETase) to catalyze enzymatic reactions involving 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, the polypeptide can be bound to a cell membrane or lipid vesicle, or bound to a synthetic support such as glass, plastic, polymer, filter, membrane, e.g., in the form of beads, columns, plates, etc.

[0169] The present disclosure also extends to compositions comprising the polypeptides, nucleic acids or host cells described herein.

[0170] The compositions can be liquid or dry, for example in the form of a powder. In some embodiments, the composition is a lyophilizate. For example, the composition can comprise a polypeptide, nucleic acid, and / or host cell and optionally excipients and / or reagents, etc. Suitable excipients can include buffers commonly used in biochemistry, agents for adjusting pH, preservatives such as sodium benzoate, sodium sorbate or sodium ascorbate, conservatives, protecting agents or stabilizers such as starch, dextrin, gum arabic, salts, sugars (e.g., sorbitol, trehalose or lactose), glycerol, polyethylene glycol, polyethene glycol, polypropylene glycol, propylene glycol, divalent ions such as calcium, chelating agents such as EDTA, reducing agents (e.g., β-mercaptoethanol, dithiothreitol, ascorbic acid, tris(2-carboxyethyl)phosphine), amino acids, carriers such as solvents or aqueous solutions, etc.

[0171] In embodiments, the composition comprises a polypeptide described herein (the polypeptide can be present in the composition in isolated or at least partially purified form). In embodiments, the composition comprises a polypeptide described herein in an amount of from about 0.1% to about 99.9%, preferably from about 0.1% to about 50%, preferably from about 0.1% to about 30%, preferably from about 0.1% to about 5% by weight of the total weight of the composition. In a preferred embodiment, the composition comprises a polypeptide described herein in an amount of from about 0.1% to about 5% by weight of the total weight of the composition. In another embodiment, the composition comprises a polypeptide described herein in an amount of from about 0.1% to about 0.2% by weight of the total weight of the composition. The amount of polypeptide in the composition can be appropriately adjusted by those skilled in the art, depending on, for example, the nature and / or amount of the polyester-containing material to be degraded (hydrolyzed), and / or the presence or absence of any additional enzyme / polypeptide in the composition.

[0172] The compositions described herein can also comprise additional polypeptides that exhibit enzymatic activity, not limited to MHETase.

[0173] In embodiments, the polypeptides described herein are dissolved in an aqueous medium together with one or more excipients, such as excipients that can suitably stabilize or protect the polypeptides from degradation. For example, the polypeptides described herein can be dissolved in water and then mixed with excipients such as glycerol, sorbitol, dextrin, starch, diols such as propylene glycol, salts, etc. The resulting mixture can then be dried to obtain a powder. Methods for drying such mixtures are well known to those skilled in the art and include, but are not limited to, lyophilization, freeze-drying, spray drying, supercritical drying, down-draft evaporation, thin-film evaporation, centrifugal evaporation, conveyor drying, fluidized-bed drying, drum drying, or any combination thereof.

[0174] In embodiments, the composition comprises at least one host cell expressing the polypeptides described herein or an extract thereof. "Cell extract" means any fraction obtained from cells by chemical, physical, and / or enzymatic treatment, such as cell supernatant, cell debris, cell wall, DNA extract, enzyme or enzyme preparation, or any preparation derived from cells, which is substantially free of living cells. Preferred extracts are enzymatically active extracts. The composition can comprise one or several host cells expressing the polypeptides described herein or an extract thereof, and optionally one or several additional cells.

[0175] As described elsewhere herein, the inventors have surprisingly found that the polypeptides described herein are capable of converting a terephthalic acid monoester into terephthalic acid and an alcohol, wherein the terephthalic acid monoester is not terephthalic acid mono(2-hydroxyethyl) ester. Accordingly, there is disclosed a method for converting a terephthalic acid monoester into terephthalic acid and an alcohol, wherein the terephthalic acid monoester is not terephthalic acid mono(2-hydroxyethyl) ester, the method comprising exposing the terephthalic acid monoester to the polypeptides, compositions, or host cells described herein under conditions sufficient to enable the polypeptides described herein to convert the terephthalic acid monoester into terephthalic acid and an alcohol. The present disclosure also extends to a method for degrading a plastic product comprising a polyester, the method comprising exposing the plastic product to the polypeptides, compositions, or host cells described herein.

[0176] The present disclosure extends to the use of the polypeptides, compositions, or host cells described herein for degrading polyesters and / or recycling polyester-containing materials (such as plastic products made of or containing polyesters) and / or producing biodegradable plastic products containing polyesters under aerobic or anaerobic conditions. Such methods and uses are particularly useful for degrading plastic products comprising PET.

[0177] Advantageously, the polyester in the polyester-containing material is depolymerized into monomers and / or oligomers. In embodiments, at least one polyester is degraded to produce polymerizable monomers and / or oligomers, which are advantageously retrieved or recycled for further use.

[0178] In an embodiment, the polyester in the polyester-containing material is completely degraded.

[0179] As described elsewhere herein, the plastic product 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), polyhydroxyalkanoates (PHA), polybutylene succinate (PBS), polybutylene adipate succinate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furandicarboxylate (PEF), polycaprolactone (PCL), polyethylene adipate (PEA), and combinations of any of the foregoing. The plastic product may comprise at least one polymer selected from the group consisting of: polypropylene, polystyrene, polyvinyl chloride, synthetic rubber, phenolic resin (or Bakelite), neoprene, nylon, polyacrylonitrile, PVB, and silicone.

[0180] The time required to degrade the polyester-containing material may vary depending on the polyester-containing material itself (i.e., the nature and source of the plastic product, its composition, shape, etc.), the type and amount of the 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.

[0181] Advantageously, the degradation process is carried out at a temperature of from about 10 °C to about 60 °C, preferably from about 20 °C to about 60 °C, preferably from about 30 °C to about 60 °C, more preferably from about 40 °C to about 60 °C, even more preferably from about 40 °C to about 50 °C, or even more preferably at about 45 °C. The temperature is generally maintained 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 polypeptides 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 from about 10 °C to about 60 °C, preferably from about 20 °C to about 60 °C, preferably from about 30 °C to about 60 °C, more preferably from about 40 °C to about 60 °C, even more preferably from about 40 °C to about 50 °C, or even more preferably at about 45 °C. The process or method may suitably be carried out in a continuous manner at a temperature at which the polypeptide can be used and / or recycled multiple times.

[0182] Advantageously, the degradation process or method is carried out at a pH between 5 and 11, preferably between 6 and 10, more preferably between 6.5 and 9, even more preferably between 7 and 8.

[0183] In embodiments, the polyester-containing material can be pretreated prior to contact with the polypeptide in order to physically alter its structure, thereby increasing the contact surface between the polyester and the enzyme.

[0184] Monomers produced by a depolymerization or degradation process or method can be suitably recovered sequentially or continuously. Depending on the starting polyester-containing material, a single type of monomer or several different types of monomers can be recovered.

[0185] The recovered monomers can be further purified using any suitable purification method and conditioned in a form suitable for repolymerization. Illustrative examples of suitable purification methods include stripping processes, aqueous solution separation, vapor selective condensation, filtration and concentration of the medium after a biological process, separation, distillation, vacuum evaporation, extraction, electrodialysis, adsorption, ion exchange, precipitation, crystallization, concentration and acid dehydration and precipitation, nanofiltration, acid catalyst treatment, semi-continuous mode distillation or continuous mode distillation, solvent extraction, evaporation concentration, evaporation crystallization, liquid / liquid extraction, hydrogenation, azeotropic distillation process, adsorption, column chromatography, simple vacuum distillation, and microfiltration, with or without combination.

[0186] The repolymerizable monomers can be used to synthesize new polyesters. Advantageously, polyesters of the same nature are repolymerized. However, the recovered monomers can be mixed with other monomers, for example, to synthesize new copolymers. Optionally, the recovered monomers can be used as chemical intermediates to produce new chemical compounds of interest.

[0187] The present disclosure also extends to plastic compounds comprising a polypeptide, a composition, and / or a host cell expressing the polypeptide or an extract thereof containing the polypeptide.

[0188] The present disclosure also extends to masterbatch compositions comprising a polypeptide, a composition, and / or a host cell expressing the polypeptide or an extract thereof containing the polypeptide.

[0189] Advantageously, such plastic compounds or masterbatch compositions described herein can be used to produce polyester-containing materials and / or plastic articles comprising the polypeptides described herein.

[0190] In embodiments, the resulting plastic compound, masterbatch composition, or plastic article is a biodegradable plastic compound, masterbatch composition, or plastic article that meets at least one of the relevant standards and / or labels known to those skilled in the art, such as Standard EN 13432, Standard ASTM D6400, OK BiodegradationSoil (Vincotte label), OK Biodegradation Water (Vincotte label), OK Compost (Vincotte label), OK Home Compost (Vincotte label).

[0191] Advantageously, the degradation process of the polyester-containing material (i.e., plastic compound, masterbatch composition or plastic product) is carried out at a temperature between 10 °C and 50 °C, preferably between 15 °C and 40 °C, more preferably between 20 °C and 30 °C, and even more preferably at 28 °C ± 2 °C.

[0192] Optionally, the degradation process of the polyester-containing material (i.e., plastic compound, masterbatch composition or plastic product) is carried out at a temperature including between 50 °C and 60 °C, more preferably at 55 °C ± 2 °C.

[0193] The MHETase polypeptides disclosed herein are suitable for a range of applications, including industrial applications, illustrative examples of which include as additives in detergents, feed compositions (including for animal feed), textile production, electronics and biomedical applications. For example, the polypeptides disclosed herein can be applied in textile processing or textile production, where they can be used as exoesterases to suitably modify the properties of textile fibers.

[0194] The present invention will now be described with reference to the following examples, which illustrate some preferred aspects of the invention. However, it should be understood that the particularity of the following description of the invention does not supersede the generality of the previous description of the invention. Examples

[0195] Materials and Methods

[0196] Construction, Expression and Purification of Recombinant MHETase

[0197] A. Construction of Consensus Sequence

[0198] Using the wild-type MHETase from Sakai, Osaka (SEQ ID NO:1) as a seed sequence, 5,000 sequences were collected by BLAST+, using an E-value threshold of 10 -5 of.

[0199] SEQ ID NO:1 (UniProt accession number A0A0K8P8E7)

[0200] MQTTVTTMLLASVALAACAGGGSTPLPLPQQQPPQQEPPPPPVPLASRAACEALKDGNGD

[0201] MVWPNAATVVEVAAWRDAAPATASAAALPEHCEVSGAIAKRTGIDGYPYEIKFRLRMPAE

[0202] WNGRFFMEGGSGTNGSLSAATGSIGGGQIASALSRNFATIATDGGHDNAVNDNPDALGTVA

[0203] FGLDPQARLDMGYNSYDQVTQAGKAAVARFYGRAADKSYFIGCSEGGREGMMLSQRFPS

[0204] HYDGIVAGAPGYQLPKAGISGAWTTQSLAPAAVGLDAQGVPLINKSFSDADLHLLSQAILGT

[0205] CDALDGLADGIVDNYRACQAAFDPATAANPANGQALQCVGAKTADCLSPVQVTAIKRAM

[0206] AGPVNSAGTPLYNRWAWDAGMSGLSGTTYNQGWRSWWLGSFNSSANNAQRVSGFSARS

[0207] WLVDFATPPEPMPMTQVAARMMKFDFDIDPLKIWATSGQFTQSSMDWHGATSTDLAAFRD

[0208] RGGKMILYHGMSDAAFSALDTADYYERLGAAMPGAAGFARLFLVPGMNHCSGGPGTDRF

[0209] DMLTPLVAWVERGEAPDQISAWSGTPGYFGVAARTRPLCPYPQIARYKGSGDINTEANFAC

[0210] AAPP

[0211] Three hundred and fifteen non-redundant sequences highly similar to MHETase were retrieved from the UniProt database. The peptide transport signals were identified using SignalP 4.0 and removed. All sequences were aligned using a sequence alignment algorithm based on the PROMALS3D library and the available MHETase structure (6QGB), and then the final curated alignment was manually refined. Consensus sequences for each amino acid position alignment were constructed using many different thresholds (95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55% and 50%). A truncated codon-optimized version of the commercially synthesized MHETase gene (GenBank accession number A0A0K8P8E7) (lacking the first 19 amino acids at the N'-terminus) and 10 consensus sequences were designed and cloned into pET-28a(+) (Twist Bioscience).

[0212] B. Protein Expression and Purification

[0213] Plasmids were transformed into electrocompetent Escherichia coli SHuffle T7 Express cells (New England Biolab) by electroporation and plated on 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 at a final concentration of 1 mM was added and the culture was transferred to 18 °C for 16 h incubation.

[0214] Cells were harvested by centrifugation at 5000 × g for 15 min at 4 °C and resuspended in lysis buffer (500 mM NaCl, 30 mM imidazole, 0.5 mg / mL lysozyme, 1% (v / v) Triton X-100, 1 U / ml Turbonuclease (Sigma), 0.5 mM dithiothreitol (DTT), and 25 mM HEPES pH 7.5). The cell suspension was lysed by two rounds of sonication at 50% power and 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-based IMAC using a 5 mL 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 related to MHETase was collected, concentrated and filtered through a 0.2 μm filter. The filtered 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).

[0215] C. Colorimetric Assay

[0216] Dilute 50 μL of the cell suspension expressing the protein in 150 μL of reaction buffer (90 mM NaCl, 45 mM sodium phosphate pH 7.5). Initiate the reaction by adding 50 μL of 10 mM 1NT (1-naphthyl terephthalate) in 100% (v / v) DMSO and 5 mM Fast Blue B salt dye. Monitor the absorbance at 465 nm for 30 minutes using an Epoch microplate spectrophotometer (BioTek).

[0217] For kinetic determination, use homogeneous MHETase at a final concentration of 7.5 nM instead of the soluble cell lysate, and different concentrations of 1NT from 1 mM to 7.8 μM. The concentration of Fast Blue B salt remains constant. Convert the absorbance to product concentration using a calibration curve of 1-naphthol from 1 mM to 7.8 μM.

[0218] D. SDS-PAGE Atto550

[0219] The method was adapted from Raducanu et al. (2020, Journal of Biological Chemistry 295(34):12214-12223). Resuspend 1 mL of cell pellet in 1x BugBuster (Merck-Millipore) diluted in SEC buffer and incubate at room temperature for 10 min. Centrifuge the mixture at 15,000×g for 10 min and run 5 μL of the soluble cell lysate on an SDS-PAGE gel at 140 V for 60 min. Microwave the gel in Milli-Q water (MQ) twice for 30 sec and then in a fixing solution (40% (v / v) methanol, 10% (v / v) acetic acid in MQ) for 2 min. The now-fixed protein gel is microwaved again in MQ for 10 min and incubated in the dark on an orbital shaker in NTA-Atto550 dye diluted 1:3000 in PBS buffer for 1 hour. Then transfer the gel to a container with warm MQ and shake for an additional 30 min. Then image the gel using a ChemiDoc MP imaging system (BIO-RAD) with the DyLight 550 fluorophore option.

[0220] E. Circular Dichroism and Thermal Stability

[0221] Measurements of the circular dichroism of MHETase were performed in a 1 mm quartz cuvette 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 at 20 °C between 200 nm and 260 nm, using a bandwidth of 1 nm and a scan rate of 0.5 s, with adaptive sampling enabled. Spectra were measured in triplicate, and the buffer blank was subtracted from the results. For thermal melt evaluation, the CD at 222 nm was recorded as the solution temperature was increased from 20 °C to 90 °C at 1 °C / min. A standard sigmoidal curve was fit to the thermal melt data to determine the Tm.

[0222] F. HPLC activity assessment of MHETase

[0223] The HPLC assay was adapted from Palm et al. (2019). The homogeneous MHETase was diluted to a final concentration of 7.5 nM (80 μL) in reaction buffer. The reaction was initiated by adding 20 μL of 1 mM MHET dissolved in 100% DMSO. After set time points (0, 10 min, 30 min, and 60 min), the reaction was quenched by adding 100 μL of quenching buffer (160 mM sodium phosphate pH 2) and heated to 80 °C for 10 min. A 10 μL volume of the reaction mixture was loaded onto an Agilent ZORBAX SB-C18, 3.5 um, 4.6×150 mm column. TPA and MHET were separated at 30 °C using a flow rate of 1 mL / min in 50% phosphate buffer (20 mM sodium phosphate pH 2.0) and 50% acetonitrile, with a 7-minute run time. TPA and MHET were detected at 240 nm and quantified against a calibration curve.

[0224] G. Monoesterase HPLC activity assay

[0225] Enzyme activity against terephthalic acid monoester substrates (e.g., MBZT, MHXT, MHPT, and MOCT substrates) was assayed with 1.5 mM substrate, 5% DMSO, and 200 nM enzyme. The reaction was incubated at 40 °C for 64 minutes and then quenched at different time points by heating at 95 °C for at least 10 minutes. The reaction was analyzed using high-performance liquid chromatography (HPLC) and compared to a control reaction without enzyme. The concentration of the product (terephthalic acid or terephthalic acid monoester substrate) was determined by comparison to a calibration curve generated using synthetic or commercial standards.

[0226] Example 1: Variant MHETase

[0227] Consensus sequence-based designs were performed using the alignment sequences of MHETase and its closest relatives. Thus, a number of different combinatorial MHETase sequences were constructed using the following different consensus sequence thresholds: 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55% and 50%. For example, the consensus sequence design at the 95% threshold represents all the differences observed in 95% of the alignment sequences but not in WT MHETase. The amino acid sequences of WT MHETase (SEQ ID NO:1) and different consensus sequence designs (SEQ ID NO:2-36, 73-78 and 86) are shown in Figure 1 The nucleic acid sequences of WT MHETase and different consensus sequence designs (SEQ ID NO:37-72 and 79-85) are shown in Figure 2 The different consensus sequence designs generated by the thresholds of 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55% and 50% are herein referred to as Round 1 Consensus Sequences A, B, C, D, E, F, G, H, I and J, respectively.

[0228] The activity / expression of MHETase was measured using whole cell suspensions using the colorimetric assay described above.

[0229] As shown in Figure 3 the variant "Round 1 Consensus Sequence A" (SEQ ID NO:73) showed more whole cell activity than WT MHETase or other consensus sequence designs. When compared to WT, Round 1 Consensus Sequence A contains 2 amino acid substitutions: namely N156G and T159V. As a preliminary study, point mutations were added to this variant based on other consensus sequence residues identified by multiple sequence alignment to identify mutations that further stabilize / improve activity. When compared to the Round 1 Consensus Sequence A sequence, the remaining variants (mutants: SEQ ID NO:3-36) contain one of the following point mutations (this nomenclature refers to the amino acid positions of 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.

[0230] It was also in Figure 3As shown, when point mutations S196A, Y197V, S235A, P255V, S260A, S286A, and Y503W are introduced into the consensus sequence A of the first round, much higher whole-cell activity is shown when expressed in Escherichia coli.

[0231] The soluble cell lysates were run via SDS-PAGE and stained with NTA-Atto550 to identify the expression rate of the enzyme. Figure 4 As shown, MHETase runs at 64 kDa on the gel. The gel shows that even with this more specific staining method, most MHETase mutants (including the control) are invisible. This result is consistent with the low soluble protein expression. The consensus sequence A of the first round and variants containing point mutations S196A, Y197V, S235A, P255V, S260A, S286A, or Y503W in the backbone of variant B appear to show increased heterologous expression in Escherichia coli compared to the wild type (WT).

[0232] The stability of the selected generated variants was also tested using purified protein variants. As shown in Figure 5 the thermal melts (T m ) of WTMHETase, the consensus sequence A of the first round, and the consensus sequence A + Y503W of the first round are all approximately 59 °C, while introducing the Y197V point mutation into the consensus sequence A of the first round reduces T m to 52 °C.

[0233] One of the key limitations in using wild-type MHETase in industrial applications is the low expression of wild-type MHETase in industrial strains. As shown by the variants disclosed herein, the increase in whole-cell activity (a combination of catalytic efficiency and active protein expression yield) advantageously reduces the production and use costs.

[0234] Example 2: Engineered MHETase with Enhanced Activity

[0235] To generate variants with improved thermal stability, the consensus sequence A of the first round (containing the point mutations N156G and T159V) was selected as the basis for further engineering because the consensus sequence A of the first round shows increased whole-cell MHETase activity ( Figure 3 ), while the change in thermal stability is small ( Figure 5 ). In this new engineering process, point mutations were added to the consensus sequence A of the first round to generate a mutant library (the second round), and whole-cell activity was screened. This process requires selecting the most promising variants and iteratively introducing single point mutations in several rounds of design.

[0236] In the second round, the following point mutations (this nomenclature refers to the amino acid positions of wild-type MHETase SEQ ID NO:1) were introduced into the consensus sequence A backbone of the first round: T593D, P543A, Y503W, S463L, P449A, T355R, G301A, S296A, H293Q, S286A, N284L, S267A, T265L, T264L, S260A, P255V, S235A, K218R, A216P, A207L, Y197B, S196A, D191L, L190I, E110A, Y107Q, A99N, A81P, A78P, T68V.

[0237] The best-performing variant selected from the second round was "Round 2 Y503W" (containing the N156G, T159V, and Y503W point mutations; SEQ ID NO:74).

[0238] In the third round, the following point mutations were introduced into "Round 2 Y503W": E594A, A493P, A377P, S286A, S267A, T264L, S260A, S196A, M192Y.

[0239] The best-performing variant selected from the third round was "Round 3 M192Y" (containing the N156G, T159V, M192Y, and Y503W point mutations; SEQ ID NO:75).

[0240] In the fourth round, the following point mutations were introduced into Round 3 M192Y (where '+' indicates an amino acid insertion at a specific position):

[0241] +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.

[0242] The best-performing variant selected from the fourth round was "Round 4 G156N" (containing the T159V, M192Y, and Y503W point mutations; SEQ ID NO:76).

[0243] In the fifth round, the following point mutations were introduced into Round 4 G156N (where '+' indicates an amino acid insertion at a specific position):

[0244] N592E, I582V, G562R, R537Q, A494V, W466F, Q461E, M361F, I357L, N316D, I283L, Y252F, V246L, Y242F, G231A, V200L, G164A, I104V, E90A, E71T.

[0245] The best-performing variant selected from round 5 was "Round 5 Y252F" (comprising T159V, M192Y, Y252F, and Y503W; SEQ ID NO:77), which exhibited a 16-fold increase in whole-cell activity relative to the wild type ( Figure 6 ).

[0246] The expression of the best variant of each round was qualitatively measured by SDS-PAGE stained with His-tag-specific fluorescent label ATTO550. The expression of WT MHETase was too low to be conclusively detected above the background of endogenous E. coli protein expression. However, through engineering in round 2, the recombinant expression level of the variant MHETase (Round 2 Y503W) was increased to a level that was clearly detectable using the ATTO550 fluorescent dye ( Figure 7 ).

[0247] The colorimetric assay described herein ( Figure 8 ) was used on the purified proteins to determine the kinetic parameters of these enzymes. The catalytic efficiency (kcat / KM) of all variants remained relatively constant compared to wild-type MHETase. Figure 9 Michaelis plots of the enzymatic function rate at various substrate concentrations are shown. From these data, two constants - kcat and KM - were obtained, as shown in Table 1.

[0248] Table 1. Michaelis kinetic parameters of selected MHETase variants

[0249]

[0250] To verify the data observed using the colorimetric assay described herein, the activities of wild-type MHETase and the best variant from round 5 (Round 5 Y252F; SEQ ID NO:77) against the native substrate MHET were determined by high-performance liquid chromatography (HPLC). The concentrations of terephthalic acid (TPA) and MHET over time in the enzyme reactions containing wild-type MHETase or Round 5 Y252F were measured using HPLC. This data was consistent with the decrease in k cat and K M observed using the colorimetric assay and confirmed that Round 5 Y252F hydrolyzes MHET to TPA. (See Figure 11 ).

[0251] Purified protein variants were also used to test the stability of the consensus sequence design. Mutations at positions 192 and 252 increased the thermal stability (Tm) relative to WT MHETase ( Figure 10 ).

[0252] Overall, these stabilizing features contributed to increased expression and activity of Round 5 Y252F relative to wild-type MHETase. However, despite these improvements, the specific activity of this variant was reduced compared to the wild type, as shown by its lower k cat (Table 1).

[0253] Further engineering was performed to revert some of the single mutations in Round 5 Y252F to their wild-type residues. In the case of position 156, the mutation N156G was reintroduced into the background of MHETase R5 to study the effect of reverting this residue to wild-type identity in Round 4. The whole-cell activity determined by the Fast Blue assay is shown in Figure 12 .

[0254] 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 relative to Round 5 Y252F ( Figure 11 ), and the specific activity was restored to a level more comparable to the wild-type MHETase of SEQ ID NO:1. Reversion of other point mutations in Round 5 Y252F decreased enzyme activity or did not significantly alter enzyme activity. The thermal stability of the MHETase variants is shown in Table 2.

[0255] Table 2: Thermal stability of wild-type (WT) MHETase and selected MHETase variants

[0256]

[0257] Example 3: Engineered MHETase Efficiently Hydrolyzes Monoester of TPA

[0258] The natural substrate of MHETase is mono(2-hydroxyethyl) terephthalate (MHET), a monoester of terephthalic acid (TPA). However, the inventors surprisingly found that the enzymes disclosed herein are also capable of hydrolyzing other monoesters of TPA formed by base-catalyzed transesterification between PET and C 6 -C 10 monoalcohols (see Figure 13 ). As shown in Figure 14 , the MHETase R5 polypeptide (SEQ ID NO:77) can hydrolyze monobenzyl terephthalate and monooctyl terephthalate to TPA (see Figure 14 ).

[0259] In contrast, when tested in parallel with MHETase R5, known and commercially available esterases, including a wild boar esterase and two lipases from T. lanuginosa and Rhizomucor miehei (both from Sigma Aldrich) (which are known to hydrolyze ester bonds), do not show hydrolysis of the primary monoester MOCT (see Figure 15 ).

[0260] The disclosure of each patent, patent application, and publication cited herein is hereby incorporated by reference in its entirety.

[0261] The citation of any reference herein shall not be construed as an admission that such reference is available as "prior art" to the present application.

[0262] Throughout the specification, the aim is to describe preferred embodiments of the invention without restricting the invention to any one embodiment or specific set of features. Accordingly, those skilled in the art will understand that various modifications and changes can be made to the specific embodiments illustrated in accordance with the present disclosure without departing from the scope of the invention. All such modifications and changes are intended to be included within the scope of the appended claims.

Claims

1. A method for hydrolyzing a terephthalic acid monoester, the method comprising exposing the terephthalic acid monoester to a polypeptide having MHETase activity under conditions sufficient for the polypeptide to convert the terephthalic acid monoester to terephthalic acid and an alcohol, wherein the terephthalic acid monoester is not terephthalic acid mono(2-hydroxyethyl) ester.

2. The method according to claim 1, wherein the polypeptide comprises the amino acid sequence of amino acids 20 - 603 of SEQ ID NO:1 or an amino acid sequence having at least 70% sequence identity therewith.

3. The method according to claim 2, wherein the polypeptide comprises the amino acid sequence of amino acids 20 - 603 of SEQ ID NO:

1.

4. The method according to claim 2, wherein the polypeptide comprises an amino acid sequence having at least 70% sequence identity with amino acids 20 - 603 of SEQ ID NO:1 and is different from amino acids 20 - 603 of SEQ ID NO:1 by amino acid substitutions at one or more positions selected from the group consisting of positions corresponding to amino acid positions 156 to 396, 398 to 410, and 425 to 603 of SEQ ID NO:

1.

5. The method according to any one of claims 1 - 4, wherein the ester is a C 1 -C 10 hydrocarbyl ester optionally substituted by benzyl.

6. The method according to any one of claims 1-5, wherein the ester is a C 6 -C 10 hydrocarbyl ester optionally substituted by benzyl.

7. The method according to any one of claims 1 - 6, wherein the terephthalic acid monoester is selected from the group consisting of benzyl terephthalate (MBZT), hexyl terephthalate, heptyl terephthalate (MHPT), and octyl terephthalate (MOCT).

8. The method according to any one of claims 1 - 7, wherein the terephthalic acid monoester is MBZT.

9. The method according to any one of claims 1 - 7, wherein the terephthalic acid monoester is MOCT.

10. The method according to any one of claims 1 - 9, wherein the polypeptide comprises an amino acid sequence having at least 70% sequence identity with amino acids 20 - 603 of SEQ ID NO:1 and is different from amino acids 20 - 603 of SEQ ID NO:1 by amino acid substitutions at one or more positions selected from the group consisting of: i. the position corresponding to amino acid position 156 of SEQ ID NO:1; ii. the position corresponding to amino acid position 159 of SEQ ID NO:1; iii. the position corresponding to amino acid position 192 of SEQ ID NO:1; iv. the position corresponding to amino acid position 196 of SEQ ID NO:1; v. the position corresponding to amino acid position 197 of SEQ ID NO:1; vi. the position corresponding to amino acid position 252 of SEQ ID NO:1; vii. the position corresponding to amino acid position 260 of SEQ ID NO:1; viii. the position corresponding to amino acid position 264 of SEQ ID NO:1; ix. the position corresponding to amino acid position 267 of SEQ ID NO:1; x. The position corresponding to amino acid position 286 of SEQ ID NO:1; and xi. The position corresponding to amino acid position 503 of SEQ ID NO:

1.

11. The method according to claim 10, wherein the polypeptide comprises an amino acid sequence different from amino acids 20-603 of SEQ ID NO:1 by an amino acid substitution at the position corresponding to amino acid position 156 of SEQ ID NO:

1.

12. The method according to claim 11, 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.

13. The method according to any one of claims 10-12, wherein the polypeptide comprises an amino acid sequence different from amino acids 20-603 of SEQ ID NO:1 by an amino acid substitution at the position corresponding to amino acid position 159 of SEQ ID NO:

1.

14. The method according to claim 13, 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.

15. The method according to any one of claims 10-14, wherein the amino acid sequence of the polypeptide is different from amino acids 20-603 of SEQ ID NO:1 by an amino acid substitution at the position corresponding to amino acid position 252 of SEQ ID NO:

1.

16. The method according to claim 15, 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.

17. The method according to any one of claims 10-16, wherein the amino acid sequence of the polypeptide is different from amino acids 20-603 of SEQ ID NO:1 by an amino acid substitution at the position corresponding to amino acid position 503 of SEQ ID NO:

1.

18. The method according to claim 17, 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.

19. The method according to any one of claims 10-18, wherein the amino acid sequence of the polypeptide is different from amino acids 20-603 of SEQ ID NO:1 by amino acid substitutions at the positions corresponding to amino acid positions 156 and 159 of SEQ ID NO:

1.

20. The method according to any one of claims 10-19, wherein the amino acid sequence of the polypeptide is different from amino acids 20-603 of SEQ ID NO:1 by amino acid substitutions at the positions corresponding to amino acid positions 156, 159 and 503 of SEQ ID NO:

1.

21. The method according to any one of claims 10 - 20, wherein the amino acid sequence of the polypeptide differs from amino acids 20 - 603 of SEQ ID NO:1 by amino acid substitutions at positions corresponding to amino acid positions 156, 159, 192 and 503 of SEQ ID NO:

1.

22. The method according to any one of claims 10 - 18, wherein the amino acid sequence of the polypeptide differs from amino acids 20 - 603 of SEQ ID NO:1 by amino acid substitutions at positions corresponding to amino acid positions 159, 252 and 503 of SEQ ID NO:

1.

23. The method according to any one of claims 10 - 18 or 22, wherein the amino acid sequence of the polypeptide differs from amino acids 20 - 603 of SEQ ID NO:1 by amino acid substitutions at positions corresponding to amino acid positions 159, 192, 252 and 503 of SEQ ID NO:

1.

24. The method according to any one of claims 10 - 18, wherein the amino acid sequence of the polypeptide differs from amino acids 20 - 603 of SEQ ID NO:1 by amino acid substitutions at positions corresponding to amino acid positions 159, 192 and 503 of SEQ ID NO:

1.

25. The method according to any one of claims 10 - 19, wherein the amino acid substitution at the position corresponding to amino acid position 156 is N156G or a conservative amino acid substitution thereof, and 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.

26. The method according to claim 20, wherein the amino acid substitutions are N156G, T159V and Y503W or conservative amino acid substitutions of any of the foregoing.

27. The method according to claim 21, wherein the amino acid substitutions are N156G, T159V, M192Y and Y503W, or conservative amino acid substitutions of any of the foregoing.

28. The method according to claim 22, wherein the amino acid substitutions are T159V, Y252F and Y503W, or conservative amino acid substitutions of any of the foregoing.

29. The method according to claim 23, wherein the amino acid substitutions are T159V, M192Y, Y252F and Y503W, or conservative amino acid substitutions of any of the foregoing.

30. The method according to claim 24, wherein the amino acid substitutions are T159V, M192Y and Y503W, or conservative amino acid substitutions of any of the foregoing.

31. The method according to any one of claims 10 - 30, wherein the amino acid sequence of the polypeptide differs from amino acids 20 - 603 of SEQ ID NO:1 by an amino acid substitution at the position corresponding to amino acid position 196 of SEQ ID NO:

1.

32. The method according to claim 31, wherein the amino acid substitution at the position corresponding to amino acid position 196 of SEQ ID NO:1 is S196A, or a conservative amino acid substitution thereof.

33. The method according to any one of claims 10 - 32, wherein the amino acid sequence of the polypeptide differs from amino acids 20 - 603 of SEQ ID NO:1 by an amino acid substitution at the position corresponding to amino acid position 197 of SEQ ID NO:

1.

34. The method according to claim 33, wherein the amino acid substitution at the position corresponding to amino acid position 197 of SEQ ID NO:1 is Y197V, or a conservative amino acid substitution thereof.

35. The method according to any one of claims 10 - 34, wherein the amino acid sequence of the polypeptide differs from amino acids 20 - 603 of SEQ ID NO:1 by an amino acid substitution at the position corresponding to amino acid position 260 of SEQ ID NO:

1.

36. The method according to claim 35, wherein the amino acid substitution at the position corresponding to amino acid position 260 of SEQ ID NO:1 is S260A, or a conservative amino acid substitution thereof.

37. The method according to any one of claims 10 - 36, wherein the amino acid sequence of the polypeptide differs from amino acids 20 - 603 of SEQ ID NO:1 by an amino acid substitution at the position corresponding to amino acid position 264 of SEQ ID NO:

1.

38. The method according to claim 37, wherein the amino acid substitution at the position corresponding to amino acid position 264 of SEQ ID NO:1 is S264L, or a conservative amino acid substitution thereof.

39. The method according to any one of claims 10 - 38, wherein the amino acid sequence of the polypeptide differs from amino acids 20 - 603 of SEQ ID NO:1 by an amino acid substitution at the position corresponding to amino acid position 267 of SEQ ID NO:

1.

40. The method according to claim 39, wherein the amino acid substitution at the position corresponding to amino acid position 267 of SEQ ID NO:1 is S267A, or a conservative amino acid substitution thereof.

41. The method according to any one of claims 10 - 40, wherein the amino acid sequence of the polypeptide differs from amino acids 20 - 603 of SEQ ID NO:1 by an amino acid substitution at the position corresponding to amino acid position 286 of SEQ ID NO:

1.

42. The method according to claim 41, wherein the amino acid substitution at the position corresponding to amino acid position 286 of SEQ ID NO:1 is S286A, or a conservative amino acid substitution thereof.

43. The method according to any one of claims 1 - 42, wherein the terephthalic acid monoester is produced by hydrolysis or degradation of terephthalic acid diester or polyethylene terephthalate (PET).

44. The method according to claim 43, wherein the terephthalic acid monoester is produced by a process comprising: a. exposing the terephthalic acid diester to sodium hydroxide, and / or b. exposing the terephthalic acid diester to an esterase.

45. The method according to claim 43, wherein the terephthalic acid monoester is produced by a process comprising subjecting the PET to a base-catalyzed transesterification reaction with a C 6 -C 10 monohydric alcohol.

46. The method according to claim 43, wherein the terephthalic acid diester is produced by a process comprising subjecting the PET to a base-catalyzed transesterification reaction with a C 6 -C 10 monohydric alcohol.

47. The method according to claim 45 or claim 46, wherein the C 6 -C 10 monohydric alcohol is benzyl alcohol, octanol or heptanol.

48. The method according to claim 47, wherein said C 6 -C 10 monohydric alcohol is 1-octanol.

49. The method according to claim 44, wherein the esterase is PETase.

50. The method according to any one of claims 1-49, further comprising recovering the terephthalic acid and / or the alcohol.

51. A composition comprising the terephthalic acid and / or alcohol recovered by the method according to claim 50.

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