Enzyme variants and uses thereof

Abstract

The disclosure generally relates to 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 to allow 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.

Description

[Technical Field]

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

[0002] The present invention relates to a novel enzyme, more particularly to a recombinant enzyme that hydrolyzes the ester bond of a monoester of terephthalic acid, and uses thereof. [Background technology]

[0003] All references cited in this specification, including any patents or patent applications, are incorporated herein by reference to enable a complete understanding of the present invention. Nevertheless, such references should not be read as constituting an admission that any of these documents form part of the common general knowledge in the art in Australia or any other country.

[0004] Global industrialization has had significant environmental impacts, particularly the increasing production of and reliance on plastics and plastic products. While widespread efforts are underway to find suitable and environmentally sustainable alternatives to plastics, including their production and disposal, such products remain a significant challenge and contribute to a large number of environmental pollutants. One of the major contributors to this problem is polyethylene terephthalate (PET) and its waste, millions of tons of which are generated globally each year. The environmental significance of this problem is due, at least in part, to the chemical properties of plastics, particularly PET-based products, as they do not readily decompose in nature.

[0005] Approaches to addressing the problem of plastic waste have typically included incineration, disposal in landfills, and mechanical degradation. However, these approaches also have significant environmental impacts. For example, incineration of plastics produces potentially harmful by-products that are released into the atmosphere; the rate at which plastics decompose in landfills is typically very slow, posing a risk of toxic substances reaching groundwater; and mechanical degradation is relatively expensive, often limiting the uses for the by-products.

[0006] More recently, chemical and biological (enzymatic) degradation of plastics have been considered as alternative approaches to reducing the accumulation of plastic waste. Chemical approaches have involved the cleavage of ester bonds in PET polymers by hydrolysis and transesterification, with the resulting oligomers or monomers being used in recycled plastic products. Widespread adoption of chemical recycling methods has been limited because they are energy- and resource-intensive and can be prohibitively expensive. Additionally, chemical recycling processes can produce oligomer or monomer products that cannot be efficiently recycled into other plastic products.

[0007] Enzymatic approaches include the use of PETase, an enzyme of the esterase class that catalyzes the hydrolysis of PET into the monomer mono-2-hydroxyethyl terephthalate (MHET) and some bis-(2-hydroxyethyl) terephthalic acid (BHET). MHETase is a class of esterase enzymes that hydrolyzes MHET into terephthalate / TPA (which can be recycled as a material suitable for the manufacture of new products, including plastics) and ethylene glycol. MHETase was originally discovered in conjunction with PETase in the bacterium Ideonella sakaiensis. The two enzymes allow the bacterium to survive by relying on the plastic PET as a carbon source (Yoshida et al. (2016) Science 351:1196).

[0008] As an esterase, MHETase does not appear to have broad substrate specificity; gallate esters, the substrates of its closest relatives in the tannase family, are not converted. p-Nitrophenyl esters of aliphatic monocarboxylic acids, such as p-nitrophenyl acetate, a widely used esterase substrate, are also not hydrolyzed. Native MHETase is also unable to hydrolyze BHET, mono(2-hydroxyethyl)-isophthalate (MHEI), or mono(2-hydroxyethyl)-furanoate (MHEF), which may be PETase degradation products resulting from the use of industrial chemicals and / or isophthalate comonomers (Knott et al. (2020) PNAS 117:25476). [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Yoshida et al. (2016) Science 351: 1196 pages [Non-patent document 2] Knott et al. (2020) PNAS 117:25476 pages [Non-patent document 3] Palm et al., 2019, Nat.Comms.10:1717 page [Non-patent document 4] Sagong et al., 2020, ACS Catal.10:4805 page [Non-Patent Document 5] Yoshida et al., 2020, Science, 352(6278): 1196 pages [Non-patent document 6] Belousov (1997) Nucleic Acids Res. 25:3440~3444 [Non-Patent Document 7] Raducanu et al., 2020, Journal of Biological Chemistry 295(34):12214~12223 Summary of the Invention [Problem to be solved by the invention]

[0010] Although the enzymatic degradation of plastics is an attractive alternative for reducing plastic waste and the environmental impact of their disposal, it has not yet seen widespread adoption due to, for example, its relative inefficiency, slow rate of enzymatic degradation, and low levels of enzyme expression in common industrial host strains. Thus, there remains an urgent need for improved methods and reagents for the enzymatic degradation of plastics. [Means for solving the problem]

[0011] In aspects disclosed herein, a method of hydrolyzing a terephthalic acid monoester is provided, comprising exposing the terephthalic acid monoester to a polypeptide having MHETase activity under conditions sufficient to allow 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 embodiments, the ester is a C1-C ester optionally substituted by benzyl. 10 In another embodiment, the ester is a C6-C alkyl ester optionally substituted with benzyl. 10 In one embodiment, the ester is a C6 alkyl ester. In one embodiment, the ester is a C7 alkyl ester. In one embodiment, the ester is a C8 alkyl ester. In another embodiment, the ester is a C9 alkyl ester. In another embodiment, the ester is a C 10 In an embodiment, the terephthalic acid monoester is selected from the group consisting of monobenzyl terephthalate (MBZT), monohexyl terephthalate, monoheptyl terephthalate (MHPT), and monooctyl terephthalate (MOCT). In a preferred embodiment, the terephthalic acid monoester is MBZT. In another preferred embodiment, the terephthalic acid monoester is MOCT.

[0012] In embodiments, the polypeptide comprises the amino acid sequence of amino acids 20 to 603 of SEQ ID NO: 1, or an amino acid sequence having at least 70% sequence identity thereto. In embodiments, the polypeptide comprises the amino acid sequence of amino acids 20 to 603 of SEQ ID NO: 1. In embodiments, the polypeptide has at least 70% sequence identity to amino acids 20 to 603 of SEQ ID NO: 1 and differs from amino acids 20 to 603 of SEQ ID NO: 1 by an amino acid substitution at one or more positions selected from the group consisting of: a position corresponding to amino acid position 156 of SEQ ID NO: 1; a position corresponding to amino acid position 159 of SEQ ID NO: 1; a position corresponding to amino acid position 192 of SEQ ID NO: 1; a position corresponding to amino acid position 196 of SEQ ID NO: 1; a position corresponding to amino acid position 197 of SEQ ID NO: 1; a position corresponding to amino acid position 252 of SEQ ID NO: 1; a position corresponding to amino acid position 260 of SEQ ID NO: 1; a position corresponding to amino acid position 264 of SEQ ID NO: 1; a position corresponding to amino acid position 267 of SEQ ID NO: 1; a position corresponding to amino acid position 286 of SEQ ID NO: 1; and a position corresponding to amino acid position 503 of SEQ ID NO: 1.

[0013] In embodiments, 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. In another embodiment, the amino acid substitutions are T159V, Y252F, and Y503W, or any of these conservative amino acid substitutions.

[0014] 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 any of these conservative amino acid substitutions. In a preferred embodiment, the polypeptide comprises the amino acid sequence of SEQ ID NO: 77.

[0015] In some embodiments, the terephthalic acid monoester is produced by hydrolysis or degradation of a terephthalic acid diester or polyethylene terephthalate (PET). In other embodiments, 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.

[0016] In another embodiment, the terephthalic acid monoester is a C-C 10 and / or exposing the terephthalic acid diester to an esterase. 10 The monoalcohol 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 explanation of the drawings]

[0017] [Figure 1] FIG. 1 shows the amino acid sequences of wild-type (WT) MHETase (SEQ ID NO: 1) and various consensus designs (SEQ ID NOs: 2-36, 73-78, 86). [Figure 2] FIG. 1 shows the nucleic acid sequences of wild-type (WT) MHETase (SEQ ID NO: 37) and various consensus designs (SEQ ID NOs: 38-72 and 79-85). [Figure 3] FIG. 1 shows the activity (dA465 / dt(min −1 )) of MHETase variants in whole cell suspensions against an analogue of MHET (1-naphthyl terephthalate). [Figure 4] Figure 1 shows the expression levels of wild-type MHETase and MHETase variants containing point mutations, including the MHETase variant N156G+T159V, in soluble cell lysates by SDS-PAGE gel electrophoresis and staining with NTA-Atto550 (Sigma). [Figure 5]Figure 1 shows the temperature 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 analysis at 222 nm (Y-axis) and temperatures ranging from 20 to 90°C (X-axis). [Figure 6] Figure 1 shows whole cell suspension FastBlue assay results for all tested MHETase variants from each mutagenesis round. Bar heights represent the average activity (dA465 / dt(min-1)) measured for each variant (n≧2, individual measurements shown), and error bars represent the standard error of the mean of the measurements. Highlighted bars represent variants used as parents in the next round of mutagenesis. [Figure 7] Shown is an SDS-PAGE gel of selected MHETase variants from each round stained with ATTO550 and imaged under UV transillumination. The expected size of the MHETase variants (approximately 64 kDa) is indicated. [Figure 8] FIG. 1 shows size exclusion chromatograms of selected MHETase variants. [Figure 9] Figure 1 shows Michaelis-Menten plots for selected MHETase variants obtained using the chromogenic assay described herein. Each point represents the average initial velocity of the reaction from three technical replicates, each incubated with 6 nM MHETase and 4 mM Fast Blue B Salt. Error bars represent the standard error of the mean. [Figure 10] Figure 1 shows the thermal stability of MHETase variants from triplicates as measured by circular dichroism analysis at 222 nm in sodium acetate pH 5.1. Data were fitted to a two-state unfolding model (linear), with error bars corresponding to the standard error of the mean. [Figure 11]FIG. 1 shows an HPLC assay comparing the activity of wild-type MHETase, round 5 Y252F (R5), and reversion of R5 to wild-type MHETase identity at positions 192, 156, 159, 252, and 503. [Figure 12] Figure 1 shows whole cell suspension FastBlue assay results for MHETase R5 reversion mutants. Mutations V159T, Y192M, F252Y, and W503Y were generated in the background of MHETase R5 (round 5 MHETase Y252F). Bar heights represent the average activity measured for each variant (n > 2), and error bars represent the standard error of the mean. [Figure 13] FIG. 1 shows the structures of mono-(2-hydroxyethyl) terephthalate (MHET) and other monoesters of terephthalic acid (TPA), 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. [Figure 14] Figure 1 shows an HPLC assay demonstrating the activity of MHETase round 5 Y252F (R5; SEQ ID NO: 77) on the substrates monooctyl terephthalate (MOCT) and monobenzyl terephthalate (MBZT). A) The increase in TPA concentration over time compared to the control (no enzyme) is shown when 200 nM R5 is incubated with 1.5 mM MOCT at 40°C. B) The corresponding decrease in MOCT concentration is shown. The data demonstrates that all of the MOCT is converted to TPA within 8 minutes. C) The increase in TPA concentration over time compared to the control (no enzyme) is shown when 200 nM R5 is incubated with 1.5 mM MBZT at 40°C. D) The corresponding decrease in MBZT concentration is shown. The data demonstrates that all of the MBZT is converted to TPA within 8 minutes. [Figure 15] Figure 1 shows the activity of engineered MHETase round 5 Y252F (R5; SEQ ID NO: 77) compared to S. scrofa esterase, T. lanuginosa lipase, and R. miehei lipase. Concentrations of A) MOCT and B) TPA over time are shown for all enzyme variants and a no-enzyme control. Concentrations were determined using high-performance liquid chromatography (HPLC). All data are substrate or product concentrations as % of the initial (0-minute) concentration. SEQ ID NO: 77 is shown to completely hydrolyze MOCT to TPA in less than 10 minutes, while S. scrofa esterase, T. lanuginosa lipase, and R. miehei lipase show no activity compared to the controls. DETAILED DESCRIPTION OF THE INVENTION

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For purposes of the present invention, the following terms are defined below.

[0019] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article, unless expressly stated otherwise. By way of example, "an element" means one element or more than one element.

[0020] As used herein, the term "about" means an amount, level, value, dimension, size, or quantity that varies by as much as 10% (e.g., 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%) relative to a reference amount, level, value, dimension, size, or amount.

[0021] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of the specified step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements.

[0022] The present disclosure is based, at least in part, on the inventors' unexpected discovery that polypeptides having MHETase activity can hydrolyze substrates other than MHET; i.e., terephthalic acid monoesters to terephthalic acid and alcohol. The inventors have also discovered that certain modifications can be made to the amino acid sequence of an MHETase to advantageously enhance its activity in hydrolyzing terephthalic acid monoesters to terephthalic acid and alcohol, where the terephthalic acid monoester is not mono-(2-hydroxyethyl)terephthalate. The inventors have also unexpectedly discovered that substitutions can be made to amino acid residues located outside the active site of a wild-type MHETase to enhance its activity in converting terephthalic acid monoesters to terephthalic acid and alcohol, where the terephthalic acid monoester is not mono-(2-hydroxyethyl)terephthalate.

[0023] Certain modifications also unexpectedly confer enhanced or improved activity on the modified MHETase in hydrolyzing terephthalic acid monoesters to terephthalic acid and alcohol, where the terephthalic acid monoester is not mono-(2-hydroxyethyl) terephthalate.

[0024] Accordingly, in aspects disclosed herein, there is provided a method of hydrolyzing a terephthalic acid monoester, the method comprising exposing the terephthalic acid monoester to a polypeptide having MHETase activity under conditions sufficient to allow 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.

[0025] Terephthalic acid monoesters will be familiar to those skilled in the art. For example, as used herein, the term terephthalic acid monoester refers to a 1,4-disubstituted benzene in which the substitutions are a carboxylic acid functional group and an ester functional group. Terephthalic acid monoesters include monoalkyl terephthalates. In some embodiments, the terephthalic acid monoester is a C1-C 10 Formed through the transesterification of PET with a monoalcohol. In certain embodiments, the terephthalic acid monoester is a C6-C 10 Formed through the transesterification of PET with a monoalcohol. In certain embodiments, terephthalic acid monoesters are formed through the transesterification of PET with benzyl alcohol, hexanol, heptanol, or octanol.

[0026] In embodiments, the polypeptide comprises the amino acid sequence of amino acids 20 to 603 of SEQ ID NO: 1, or an amino acid sequence having at least 70% sequence identity thereto. In embodiments, the polypeptide comprises the amino acid sequence of amino acids 20 to 603 of SEQ ID NO: 1. In embodiments, the polypeptide comprises an amino acid sequence that (i) has at least 70% sequence identity to amino acids 20 to 603 of SEQ ID NO: 1, and (ii) differs from amino acids 20 to 603 of SEQ ID NO: 1 by amino acid substitutions at one or more positions that do not otherwise contact the polyester substrate of the MHETase.

[0027] In an embodiment, the polypeptide comprises an amino acid sequence that (i) has at least 70% sequence identity to amino acids 20 to 603 of SEQ ID NO: 1, and (ii) differs from amino acids 20 to 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.

[0028] By "at least 70%" is meant that the polypeptide shares at least 70%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 92%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, or more preferably 99% sequence identity to SEQ ID NO: 1. Because the polypeptides described herein are variants of the naturally occurring (wild-type) MHETase of SEQ ID NO: 1, it should be understood that in this context, "at least 70%" does not include 100% sequence identity across the entire sequence of SEQ ID NO: 1 (residues 1-603 or residues 18-603). In some embodiments, the polypeptides, as described herein, can include amino acid insertions and / or deletions, such as at the N- and / or C-terminus.

[0029] In another embodiment, the polypeptide has (a) at least 70% sequence identity to amino acids 20-603 of SEQ ID NO:1; and (b) (i) a position corresponding to amino acid position 156 of SEQ ID NO: 1; (ii) a position corresponding to amino acid position 159 of SEQ ID NO: 1; (iii) a position corresponding to amino acid position 192 of SEQ ID NO: 1; and (iv) a position corresponding to amino acid position 503 of SEQ ID NO: 1 and wherein the amino acid sequence is different from amino acids 20 to 603 of SEQ ID NO: 1 due to amino acid substitutions at one or more positions selected from the group consisting of:

[0030] In embodiments, the amino acid sequence of the polypeptide differs from amino acids 20-603 of SEQ ID NO:1 by an amino acid substitution at a position corresponding to amino acid position 156 of SEQ ID NO:1.

[0031] In embodiments, 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.

[0032] In embodiments, the amino acid sequence of the polypeptide differs from amino acids 20-603 of SEQ ID NO:1 by an amino acid substitution at a position corresponding to amino acid position 159 of SEQ ID NO:1.

[0033] In embodiments, 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.

[0034] In embodiments, the amino acid sequence of the polypeptide differs from amino acids 20-603 of SEQ ID NO:1 by an amino acid substitution at a position corresponding to amino acid position 252 of SEQ ID NO:1.

[0035] In embodiments, 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.

[0036] In embodiments, the amino acid sequence of the polypeptide differs from amino acids 20-603 of SEQ ID NO:1 by an amino acid substitution at a position corresponding to amino acid position 503 of SEQ ID NO:1.

[0037] In embodiments, 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.

[0038] 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, 252, and 503 of SEQ ID NO: 1. In preferred embodiments, the amino acid substitutions are T159V, Y252F, and Y503W, or any of these conservative amino acid substitutions.

[0039] 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 any of these conservative amino acid substitutions.

[0040] 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 any of these conservative amino acid substitutions.

[0041] 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 any of these conservative amino acid substitutions.

[0042] 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 any of these conservative amino acid substitutions.

[0043] In another embodiment, the polypeptide has (a) at least 70% sequence identity to amino acids 20-603 of SEQ ID NO:1; and (b) (i) a position corresponding to amino acid position 156 of SEQ ID NO: 1; (ii) a position corresponding to amino acid position 159 of SEQ ID NO: 1; (iii) a position corresponding to amino acid position 196 of SEQ ID NO: 1; (iv) a position corresponding to amino acid position 197 of SEQ ID NO: 1; (v) a position corresponding to amino acid position 260 of SEQ ID NO: 1; (vi) a position corresponding to amino acid position 264 of SEQ ID NO: 1; (vii) a position corresponding to amino acid position 267 of SEQ ID NO: 1; (viii) a position corresponding to amino acid position 286 of SEQ ID NO: 1; and (ix) a position corresponding to amino acid position 503 of SEQ ID NO: 1 and wherein the amino acid sequence is different from amino acids 20 to 603 of SEQ ID NO: 1 due to amino acid substitutions at one or more positions selected from the group consisting of:

[0044] In embodiments, the amino acid sequence of the polypeptide differs from amino acids 20-603 of SEQ ID NO:1 by an amino acid substitution at a position corresponding to amino acid position 156 of SEQ ID NO:1.

[0045] In embodiments, 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.

[0046] In embodiments, the amino acid sequence of the polypeptide differs from amino acids 20-603 of SEQ ID NO:1 by an amino acid substitution at a position corresponding to amino acid position 159 of SEQ ID NO:1.

[0047] In embodiments, 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.

[0048] In embodiments, the amino acid sequence of the polypeptide differs from amino acids 20-603 of SEQ ID NO:1 by an amino acid substitution at a position corresponding to amino acid position 196 of SEQ ID NO:1.

[0049] In embodiments, 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.

[0050] In embodiments, the amino acid sequence of the polypeptide differs from amino acids 20-603 of SEQ ID NO:1 by an amino acid substitution at a position corresponding to amino acid position 197 of SEQ ID NO:1.

[0051] In embodiments, 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.

[0052] In embodiments, the amino acid sequence of the polypeptide differs from amino acids 20-603 of SEQ ID NO:1 by an amino acid substitution at a position corresponding to amino acid position 260 of SEQ ID NO:1.

[0053] In embodiments, 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.

[0054] In embodiments, the amino acid sequence of the polypeptide differs from amino acids 20-603 of SEQ ID NO:1 by an amino acid substitution at a position corresponding to amino acid position 264 of SEQ ID NO:1.

[0055] In embodiments, 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.

[0056] In embodiments, the amino acid sequence of the polypeptide differs from amino acids 20-603 of SEQ ID NO:1 by an amino acid substitution at a position corresponding to amino acid position 267 of SEQ ID NO:1.

[0057] In embodiments, 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.

[0058] In embodiments, the amino acid sequence of the polypeptide differs from amino acids 20-603 of SEQ ID NO:1 by an amino acid substitution at a position corresponding to amino acid position 286 of SEQ ID NO:1.

[0059] In embodiments, 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.

[0060] In embodiments, the amino acid sequence of the polypeptide differs from amino acids 20-603 of SEQ ID NO:1 by an amino acid substitution at a position corresponding to amino acid position 503 of SEQ ID NO:1.

[0061] In embodiments, 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.

[0062] 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 embodiments, a polypeptide includes a combination of amino acid substitutions at at least two, preferably at least three, preferably at least four, preferably at least five, preferably at least six, preferably at least seven, preferably at least eight, preferably at least nine, or more preferably at least ten positions corresponding to positions in SEQ ID NO: 1 as described herein.

[0063] In 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 197 of SEQ ID NO:1.

[0064] In embodiments, the amino acid substitutions are N156G, T159V and Y197V, or any of these conservative amino acid substitutions.

[0065] In one embodiment, the polypeptide is not a boar derived esterase or a T. lanuginosa or R. miehei derived lipase.

[0066] The polypeptides can be used in purified form, alone or in combination with other enzymes (e.g., PETase or MHETase, or carboxylesterases or cutinases with PETase or MHETase or esterase activity), to catalyze enzymatic reactions involved in the degradation and / or regeneration of materials containing polyesters or mono / diesters of TPA, such as plastic articles containing polyesters or mono / diesters of TPA. The polypeptides described herein can be in soluble form or can be immobilized on a substrate. Suitable substrates will be familiar to those skilled in the art, and illustrative examples include cell membranes, lipid vesicles, glass, plastic, polymers, filters, membranes, beads, columns, and plates.

[0067] Performing the methods of the invention with polypeptides immobilized on a substrate can be convenient, for example, when performing the methods of the invention in a semi-continuous or continuous fashion.

[0068] In embodiments, the polypeptides described herein are immobilized on a substrate.

[0069] The polypeptides can be immobilized on any suitable substrate using techniques known to those skilled in the art, for example, the polypeptides can be immobilized on a support resin by ion exchange, adsorption (e.g., hydrophobic adsorption), or covalent coupling.

[0070] In embodiments, the substrate is a resin. Suitable resins will be known to those skilled in the art, and an illustrative example is an ion exchange resin. 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.

[0071] Thus, in embodiments, the substrate is an ion exchange resin. Those skilled in the art are familiar with the general principles of enzymatic immobilization techniques, which principles can be advantageously applied in the context of immobilizing polypeptides on substrates in accordance with the present invention.

[0072] Suitable ion exchange resins will generally comprise a polymer matrix or a polymer / ceramic hybrid matrix. Illustrative examples of such resins include, but are not limited to, CM Ceramic HyperD® ion exchange chromatography resins.

[0073] In embodiments, the ion exchange resin is a cation exchange resin. For operation of the methods described herein, the polypeptide will typically be immobilized on a support resin and loaded onto a column.

[0074] The present disclosure also extends to compositions comprising a polypeptide as described herein.

[0075] The present disclosure also extends to nucleic acid sequences encoding the polypeptides described herein.

[0076] The present disclosure also extends to expression vectors comprising the nucleic acid sequences described herein.

[0077] The present disclosure also extends to host cells comprising the nucleic acid sequences or expression vectors described herein.

[0078] In embodiments, the terephthalic acid monoester is produced as a by-product of the decomposition, hydrolysis, or recycling of polyethylene terephthalate (PET). In embodiments, the terephthalic acid monoester is produced as by the decomposition or hydrolysis of a terephthalic acid diester. In another embodiment, the terephthalic acid monoester is produced by a process comprising exposing the terephthalic acid diester to sodium hydroxide and / or contacting the terephthalic acid diester with an esterase.

[0079] In another embodiment, the terephthalic acid monoester is a C-C 10 In a preferred embodiment, C6-C olefins are produced by a process comprising the steps of: subjecting PET to base-catalyzed transesterification with a monoalcohol; and / or contacting PET with an esterase. 10 The monoalcohol is benzyl alcohol, octanol or heptanol.

[0080] The present disclosure also extends to compositions comprising terephthalic acid and / or alcohol recovered by the methods described herein.

[0081] The present disclosure also extends to a method of degrading a plastic article comprising polyester, the method comprising exposing the plastic article to a polypeptide, composition or host cell described herein.

[0082] In embodiments, the polyester is selected from the group consisting of polylactic acid (PLA), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polyethylene terephthalate (PET), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), polycaprolactone (PCL), polyethylene adipate (PEA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), and any combination thereof. In embodiments, the polyester is polyethylene terephthalate (PET).

[0083] In another embodiment, the methods disclosed herein comprise: 10 Terephthalic acid monoesters C6-C by subjecting PET to base-catalyzed transesterification with monoalcohols 10 A step of producing a monoalcohol derivative, and a step of producing a C6-C terephthalic acid monoester of the polypeptide. 10 terephthalic acid monoester C6-C6 under conditions sufficient to permit conversion of the monoalcohol derivative to terephthalic acid and alcohol. 10 The method comprises contacting a monoalcohol derivative with a polypeptide.

[0084] In one embodiment, C to C 10 The monoalcohol is selected from hexanol, pentanol, octanol, nonanol, decanol and benzyl alcohol. 10 The monoalcohols are hexanol, pentanol and octanol.

[0085] The transesterification carried out in accordance with the process of the present invention is base-catalyzed. There are no particular limitations on the type of base catalyst that can be used.

[0086] In one embodiment, the transesterification is catalyzed using an alkali metal base. 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, the transesterification is catalyzed using sodium hydroxide or potassium hydroxide.

[0087] The present disclosure also extends to compositions comprising terephthalic acid and / or alcohol recovered by the methods disclosed herein.

[0088] In another aspect, there are provided host cells genetically engineered to express the polypeptides described herein.

[0089] As used herein, the terms "peptide," "polypeptide," "protein," and "enzyme" should be understood to mean 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 one-letter or three-letter code 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).

[0090] The term "hydrolase" refers to an enzyme belonging to the class of hydrolases classified as EC3 according to enzyme nomenclature, which catalyzes the hydrolysis of peptide bonds in peptides or proteins to produce shorter peptides. The term "esterase," as used herein, typically refers to an enzyme belonging to the class of hydrolases (enzyme class EC3.1) that hydrolyzes esters to acids and alcohols. The term "MHETase," as used herein, typically refers to a carboxylesterase enzyme (enzyme class EC3.1.1.102) that hydrolyzes 2-hydroxyethyl terephthalate to terephthalic acid and alcohol.

[0091] The terms "wild-type" or "parent" are used interchangeably herein to refer to a naturally occurring isoform of a polypeptide; i.e., the polypeptide as it occurs in nature. In the present disclosure, wild-type polypeptide refers to a mono-(2-hydroxyethyl)terephthalate hydrolase having the amino acid sequence as set forth in SEQ ID NO: 1 (EC 3.1.1.102; UniProt Accession No. A0A0K8P8E7) or comprising amino acids 20 to 603 of SEQ ID NO: 1.

[0092] As noted by Palm et al. (2019, Nat. Comms. 10:1717), two recently discovered bacterial enzymes that specifically degrade polyethylene terephthalate (PET) represent promising solutions for otherwise environmentally burdensome polyester-containing products. First, Ideonella sakaiensis PETase, a structurally well-characterized α / β-hydrolase-fold enzyme, converts PET to mono-(2-hydroxyethyl) terephthalate (MHET). The second key enzyme, MHETase, hydrolyzes MHET to 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)).

[0093] The amino acid and nucleic acid sequences of wild-type MHETases will be well known to those of skill in the art, and an illustrative example thereof is SEQ ID NO:1.

[0094] The terms "mutant" and "variant" can be used interchangeably herein to refer to a polypeptide comprising an amino acid sequence derived from SEQ ID NO: 1 and which further comprises a modification or change (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. The terms "modification," "alteration," "substitution," and the like, when used herein in reference to an amino acid residue or position, typically mean that the amino acid at a particular position is altered compared to the amino acid in the wild-type or parent polypeptide.

[0095] Suitable substitutions include the replacement of an amino acid residue with another residue selected from the 20 standard naturally occurring amino acid residues, rare naturally occurring amino acid residues (e.g., hydroxyproline, hydroxylysine, allohydroxylysine, 6-N-methyllysine, N-ethylglycine, N-methylglycine, N-ethylasparagine, alloisoleucine, N-methylisoleucine, N-methylvaline, pyroglutamine, aminobutyric acid, ornithine, norleucine, norvaline), and often synthetically produced non-naturally occurring amino acid residues (e.g., cyclohexylalanine). Preferably, the substitution involves the replacement of an amino acid residue with another 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 changes can be identified herein using the following terms: Y197V indicates that the amino acid residue tyrosine (Y) at position 197 of the parent polypeptide sequence is replaced with valine (V). Y197V / I / M indicates that the amino acid residue tyrosine (Y) at position 197 of the parent sequence can be replaced with one of the following amino acids valine (V), isoleucine (I), or methionine (M). The substitution can be a conservative or non-conservative substitution. Examples of conservative substitutions will be well known to those of skill in the art, and illustrative examples include substitutions within the group of basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine, asparagine, and threonine), hydrophobic amino acids (methionine, leucine, isoleucine, cysteine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, and serine).

[0096] Unless otherwise specified, positions disclosed in this application are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1. In this context, the term "corresponding to," when used in reference to an amino acid position, is intended to mean 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.

[0097] As used herein, the term "sequence identity" or "identity" refers to the number of matches (or proportion expressed as a percentage %) between two polypeptide sequences (identical amino acid residues). In a preferred embodiment, sequence identity is determined by comparing sequences when aligned to maximize overlap and identity while minimizing sequence gaps. Depending on the length of the two sequences, sequence identity can be determined using any of a number of mathematical global or local alignment algorithms known to those skilled in the art. Sequences of similar length can be aligned using a global alignment algorithm (e.g., the Needleman and Wunsch algorithm; Needleman and Wunsch, 1970) that optimally aligns sequences across their entire length, while sequences of substantially 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 purposes of determining percent amino acid sequence identity can be accomplished by any means available to those of skill in the art, illustrative examples of which include publicly available computer programs such as those 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 appropriate parameters for measuring alignment, including, for example, any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.As used herein, % sequence identity typically refers to the value generated using a pairwise sequence alignment (e.g., using the Needleman-Wunsch algorithm) to create an optimal global alignment of two sequences, with all search parameters set to default values, e.g., scoring matrix=BLOSUM62, gap open=10, gap extension=0.5, end gap penalty=false, end gap open=10, and end gap extension=0.5.

[0098] The term "recombinant," as used herein, typically refers to a nucleic acid construct, vector, polypeptide, or cell that has been produced by genetic engineering.

[0099] The term "expression," as used herein, typically means any step involved in producing a polypeptide, such as by transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0100] The term "expression cassette" refers to a nucleic acid construct that comprises a coding region and, preferably, a regulatory region to which the coding region is operably linked.

[0101] The term "expression vector" typically refers to a DNA or RNA molecule that contains an expression cassette. Expression vectors can be linear or circular double-stranded DNA molecules.

[0102] The term "polymer," as used herein, typically refers to a compound or mixture of compounds whose structure is composed of multiple monomers (repeating units) linked by covalent chemical bonds. Within the context of the present invention, the term polymer includes natural or synthetic polymers composed of a single type of repeating unit (i.e., homopolymers) or a mixture of different repeating units (i.e., copolymers or heteropolymers).

[0103] As used herein, the terms "polyester-containing material," "polyester-containing product," and the like should be understood to mean products, such as plastic products, that contain at least one polyester in crystalline, semi-crystalline, or completely amorphous form. A polyester-containing material can refer to any article made from at least one plastic material, such as plastic sheets, tubes, rods, profiles, shapes, films, blocks, fibers, fabrics, and the like, containing at least one polyester and, optionally, other substances or additives, such as plasticizers, mineral or organic fillers. In an embodiment, the polyester-containing material is a fabric or textile comprising at least one polyester-containing fiber. In another embodiment, the polyester-containing material is a plastic compound or plastic agent, in a molten or solid state, suitable for producing a plastic product.

[0104] Suitable polyesters will be well known to those skilled in the art, and illustrative examples include polylactic acid (PLA), polyethylene terephthalate (PET), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polyhydroxyalkanoates (PHAs), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), polycaprolactone (PCL), and poly(ethylene adipate) (PEA). Thus, in embodiments, the polyester is selected from the group consisting of polylactic acid (PLA), polyethylene terephthalate (PET), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), polycaprolactone (PCL), poly(ethylene adipate) (PEA), and any combination thereof.

[0105] As described elsewhere herein, the present inventors have unexpectedly discovered that naturally occurring MHETases and functional variants thereof are capable of converting terephthalic acid monoesters to terephthalic acid and alcohols.

[0106] This newly identified activity of MHETase makes it particularly suitable for use in the degradation of plastic products, especially those containing PET. Furthermore, the inventors have surprisingly discovered that amino acid residues in the protein structure that are not otherwise intended to contact polyester substrates can be advantageously modified to enhance the activity of MHETase in converting terephthalic acid monoesters to terephthalic acid and alcohol, where the terephthalic acid monoester is not mono-(2-hydroxyethyl) terephthalate.

[0107] In some embodiments, a polypeptide is provided that comprises: (i) an amino acid sequence having at least 70% sequence identity to amino acids 20-603 of SEQ ID NO:1; and (ii) an amino acid sequence that differs from amino acids 20-603 of SEQ ID NO:1 by an amino acid substitution at one or more positions that do not contact a polyester substrate of an MHETase, wherein the polypeptide is capable of converting a terephthalic acid monoester to terephthalic acid and an alcohol, and the terephthalic acid monoester is not mono-(2-hydroxyethyl) terephthalate. In some embodiments, the ester is a C1-C ester optionally substituted with benzyl. 10 In another embodiment, the ester is a C6-C alkyl ester optionally substituted with benzyl. 10 In one embodiment, the ester is a C6 alkyl ester. In one embodiment, the ester is a C7 alkyl ester. In one embodiment, the ester is a C8 alkyl ester. In another embodiment, the ester is a C9 alkyl ester. In another embodiment, the ester is a C 10The terephthalic acid monoester is an alkyl ester. In an embodiment, the terephthalic acid monoester is selected from the group consisting of monobenzyl terephthalate (MBZT), monohexyl terephthalate (MHXT), monoheptyl terephthalate (MHPT), and monooctyl terephthalate (MOCT). In a preferred embodiment, the terephthalic acid monoester is MBZT. In another preferred embodiment, the terephthalic acid monoester is MOCT. The term "contact" in this context typically refers to direct contact between the MHETase of SEQ ID NO: 1 and the polyester substrate via the amino acid residues of SEQ ID NO: 1. The amino acid residues of SEQ ID NO: 1 that contact the polyester substrate are expected to be familiar to those skilled in the art. 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 differs from amino acids 20-603 of SEQ ID NO: 1 by amino acid substitutions at one or more positions that are outside the active site of the MHETase of SEQ ID NO: 1. The term "active site" typically refers to the region of SEQ ID NO: 1 that is capable of contacting and hydrolyzing the polyester substrate (i.e., MHET). Amino acid positions of SEQ ID NO: 1 that are located outside the active site of the MHETase of SEQ ID NO: 1 are expected to be familiar to those of skill in the art.

[0108] In the context of this disclosure, reference to increased or enhanced activity means the ability to convert a terephthalic acid monoester to terephthalic acid and alcohol; where the terephthalic acid monoester is not mono-(2-hydroxyethyl) terephthalate.

[0109] In embodiments, a polypeptide as disclosed herein is capable of converting monobenzyl terephthalate (MBZT) to terephthalic acid and alcohol, wherein the terephthalic acid monoester is not mono-(2-hydroxyethyl) terephthalate. In embodiments, a polypeptide as disclosed herein is capable of converting monobenzyl terephthalate (MBZT) to terephthalic acid and benzyl alcohol.

[0110] In embodiments, a polypeptide as disclosed herein is capable of converting monohexyl terephthalate (MHXT) to terephthalic acid and an alcohol, wherein the terephthalic acid monoester is not mono-(2-hydroxyethyl) terephthalate. In embodiments, a polypeptide as disclosed herein is capable of converting monohexyl terephthalate (MHXT) to terephthalic acid and a heptanol.

[0111] In embodiments, a polypeptide as disclosed herein is capable of converting monoheptyl terephthalate (MHPT) to terephthalic acid and an alcohol, wherein the terephthalic acid monoester is not mono-(2-hydroxyethyl) terephthalate. In embodiments, a polypeptide as disclosed herein is capable of converting monoheptyl terephthalate (MHPT) to terephthalic acid and a heptanol.

[0112] In embodiments, a polypeptide as disclosed herein is capable of converting monooctyl terephthalate (MOCT) to terephthalic acid and an alcohol, wherein the terephthalic acid monoester is not mono-(2-hydroxyethyl) terephthalate. In embodiments, a polypeptide as disclosed herein is capable of converting monooctyl terephthalate (MOCT) to terephthalic acid and an octanol.

[0113] In embodiments, the activity of a polypeptide described herein in converting a terephthalic acid monoester to terephthalic acid and an alcohol, where the terephthalic acid monoester is not mono-(2-hydroxyethyl) terephthalate, is similar to the activity of the MHETase of SEQ ID NO: 1. In embodiments, the activity of a polypeptide described herein to convert a terephthalic acid monoester to terephthalic acid and alcohol, where 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, compared to the MHETase of SEQ ID NO: 1. Suitable methods for determining or measuring a specific activity of a polypeptide will be familiar to those of skill in the art, and illustrative examples thereof are provided elsewhere herein. For example, the activity of a polypeptide in converting a terephthalic acid monoester to 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 entireties.In embodiments, the activity in converting a terephthalic acid monoester to terephthalic acid and alcohol, where 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, compared to the MHETase of SEQ ID NO: 1, when determined by an assay using a terephthalic acid monoester as a substrate.

[0114] The activity to convert a terephthalic acid monoester to terephthalic acid and alcohol, where the terephthalic acid monoester is not mono-(2-hydroxyethyl)terephthalate, can be assigned an absolute value or a value relative to the activity of a comparative standard (e.g., the MHETase of SEQ ID NO: 1). In embodiments, the activity to convert a terephthalic acid monoester to terephthalic acid and alcohol, where the terephthalic acid monoester is not mono-(2-hydroxyethyl)terephthalate, is measured as the rate of monomer and / or oligomer (e.g., in mg) released per hour and per mg of enzyme under suitable conditions of temperature, pH, and buffer.

[0115] The activity of converting a terephthalic acid monoester to terephthalic acid and an alcohol, where the terephthalic acid monoester is not mono-(2-hydroxyethyl) terephthalate, can be measured or assayed using purified enzyme. Alternatively, the activity of converting a terephthalic acid monoester to terephthalic acid and an alcohol, where the terephthalic acid monoester is not mono-(2-hydroxyethyl) terephthalate, can be measured as a function of the activity of the enzyme when recombinantly expressed in a host cell system (also referred to herein as cellular catalytic activity or whole cell activity).

[0116] Advantageously, the polypeptides described herein exhibit activity to convert a terephthalic acid monoester to terephthalic acid and alcohol, wherein the terephthalic acid monoester is not mono-(2-hydroxyethyl)terephthalate, at a temperature range of at least 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 about 45°C. In embodiments, the polypeptides described herein exhibit activity at temperatures of about 10°C to about 60°C, preferably about 20°C to about 60°C, preferably about 30°C to about 60°C, more preferably about 40°C to about 60°C, even more preferably about 40°C to about 50°C, or even more preferably about 45°C. In embodiments, the activity is measurable at temperatures of about 40°C to about 60°C, preferably about 40°C to about 50°C, or even more preferably about 45°C. In another particular embodiment, polyester degrading activity is still measurable at temperatures of about 10°C to about 30°C, preferably about 15°C to about 28°C, which corresponds to the average temperature in the natural environment (room temperature).

[0117] In embodiments, the polypeptide has activity to convert a terephthalic acid monoester of SEQ ID NO: 1 to terephthalic acid and alcohol, where the terephthalic acid monoester is not mono-(2-hydroxyethyl) terephthalate, at a temperature of about 10°C to about 60°C, preferably about 20°C to about 60°C, preferably about 30°C to about 60°C, more preferably about 40°C to about 60°C, even more preferably about 40°C to about 50°C, or even more preferably about 45°C, compared to the activity at the same temperature. %, 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 of the activity converting the terephthalic acid monoester to terephthalic acid and alcohol, where the terephthalic acid monoester is not mono-(2-hydroxyethyl) terephthalate.

[0118] In another specific embodiment, the polypeptides described herein have increased activity to convert a terephthalic acid monoester to terephthalic acid and alcohol, where the terephthalic acid monoester is not mono-(2-hydroxyethyl) terephthalate, compared to the polypeptide of SEQ ID NO: 1 at a temperature of about 10°C to about 60°C, preferably about 20°C to about 60°C, preferably about 30°C to about 60°C, preferably about 40°C to about 60°C, preferably about 40°C to about 50°C, or more preferably about 45°C. In embodiments, the polypeptides described herein have 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 activity to convert a terephthalic acid monoester of SEQ ID NO:1 to terephthalic acid and alcohol, where the terephthalic acid monoester is not mono-(2-hydroxyethyl) terephthalate, between about 20° C. and about 60° C., compared to the activity at the same temperature to convert the terephthalic acid monoester of SEQ ID NO:1 to terephthalic acid and alcohol, where the terephthalic acid monoester is not mono-(2-hydroxyethyl) terephthalate.

[0119] In another embodiment, the polypeptides described herein have increased activity for converting a terephthalic acid monoester to terephthalic acid and alcohol, where the terephthalic acid monoester is not mono-(2-hydroxyethyl) terephthalate, compared to the polypeptide of SEQ ID NO: 1 at a temperature of about 10°C to about 30°C, preferably about 15°C to about 30°C, even more preferably about 20°C to about 30°C, or even more preferably at about 28°C. In embodiments, the polypeptides described herein have 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 activity to convert a terephthalic acid monoester of SEQ ID NO:1 to terephthalic acid and alcohol, where the terephthalic acid monoester is not mono-(2-hydroxyethyl) terephthalate, at a temperature of about 10° C. to about 30° C., compared to the activity at the same temperature to convert the terephthalic acid monoester of SEQ ID NO:1 to terephthalic acid and alcohol, where the terephthalic acid monoester is not mono-(2-hydroxyethyl) terephthalate.

[0120] In embodiments, the polypeptides described herein exhibit measurable activity in converting a terephthalic acid monoester to terephthalic acid and alcohol, where the terephthalic acid monoester is not mono-(2-hydroxyethyl) terephthalate, at least in the pH range of 5 to 11, preferably in the pH range of 6 to 10, more preferably in the pH range of 6.5 to 9, and even more preferably in the pH range of 7 to 8.

[0121] The present disclosure also extends to polynucleotides comprising a nucleic acid sequence encoding the MHETase polypeptides described herein. In an aspect disclosed herein, a polynucleotide comprising a nucleic acid sequence encoding a polypeptide described herein is provided. 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 sequence," "polynucleotide," "oligonucleotide," and "nucleotide sequence" are used interchangeably and refer to a sequence of deoxyribonucleotides and / or ribonucleotides. A nucleic acid can be DNA (cDNA or gDNA), RNA, or a mixture of the two. It can be in single-stranded or double-stranded form or a mixture of the two. It can be of recombinant, artificial, and / or synthetic origin and can contain modified nucleotides, for example, containing modified linkages, modified purine or pyrimidine bases, or modified sugars. The nucleic acids of the invention may be in isolated or purified form and may be produced, isolated, and / or manipulated by techniques known per se in the art, such as cloning and expression of cDNA libraries, amplification, enzymatic synthesis, or recombinant techniques. Nucleic acids may also be synthesized in vitro by well-known chemical synthesis techniques, for example, as described in Belousov (1997) Nucleic Acids Res. 25:3440-3444.

[0122] The nucleic acid sequences disclosed herein can be suitably codon-optimized. Suitable methods for codon optimization will be familiar to those skilled in the art, and illustrative examples thereof are described in the reference manual Sambrook et al. (Sambrook et al., 2001).

[0123] The nucleic acid sequences described herein can be suitably deduced from the amino acid sequences of the polypeptides described herein, and the codon usage can be adapted depending on the host cell in which the nucleic acid is expected to be transcribed.

[0124] In some embodiments, the nucleic acid sequences described herein may suitably include additional nucleotide sequences such as regulatory regions, i.e., promoters, enhancers, silencers, terminators, signal peptides, etc., that can be used to cause or regulate expression of the polypeptide in a selected host cell or system. Alternatively, or in addition, the nucleic acid sequences described herein may further include additional nucleotide sequences encoding fusion proteins, such as maltose binding protein (MBP) or glutathione S-transferase (GST), that can be used to favor polypeptide expression and / or solubility.

[0125] As described elsewhere herein, the present disclosure also extends to expression vectors and expression cassettes comprising the nucleic acid sequences described herein, optionally operably linked to one or more control sequences that direct expression of the nucleic acid sequence in a suitable host cell. Typically, the expression vector or cassette contains the nucleic acid sequence described herein operably linked to a control sequence, such as a transcription promoter and / or a transcription terminator. Control sequences include promoters recognized by a host cell or in vitro expression system for expression of a nucleic acid encoding a polypeptide described herein. The promoter will typically include a transcription control sequence that mediates expression of the polypeptide. The promoter can be any polynucleotide that exhibits transcriptional activity in a host cell, including mutant, truncated, and hybrid promoters, and can be suitably obtained from a gene encoding an extracellular or intracellular polypeptide, either homologous or heterologous to the host cell. The control sequence can also be a transcription terminator recognized by the host cell to terminate transcription. The terminator is typically operably linked to the 3' end of the nucleic acid encoding the polypeptide. Any terminator that is functional in the host cell can be used in this context. Typically, the expression vector or cassette contains a nucleic acid sequence described herein operably linked to a transcription promoter and a transcription terminator.

[0126] The term "vector" typically refers to a DNA molecule used as a vehicle for transferring recombinant genetic material into a host cell. Suitable vectors include plasmids, bacteriophages, viruses, fosmids, cosmids, and artificial chromosomes. A vector is typically a DNA sequence that contains an insert (heterologous nucleic acid sequence, transgene) and a larger sequence that serves as the "backbone" of the vector. The purpose of a vector to transfer genetic information into a host is typically to isolate, amplify, or express the insert in a target cell. Expression vectors (also called expression constructs) are specifically adapted for expression of a heterologous sequence in a target cell and generally have a promoter sequence that drives the expression of the heterologous sequence encoding a polypeptide.

[0127] Generally, regulatory elements used in expression vectors include a transcription promoter, a ribosome binding site, a terminator, and optionally an operator. Expression vectors can further include an origin of replication for autonomous replication in host cells, a selectable marker, a limited number of useful restriction enzyme sites, and the potential for high copy number. Suitable expression vectors will be well known to those skilled in the art, and illustrative examples include cloning vectors, modified cloning vectors, plasmids, and viruses. Expression vectors capable of providing suitable levels of polypeptide expression in different hosts are also well known in the art. The choice of vector typically depends on the compatibility of the vector with the host cell into which the vector is to be introduced. In one embodiment, the vector is the bacterial expression vector pET-28a(+) (SEQ ID NO: 85).

[0128] The present disclosure also extends to host cells containing the nucleic acid sequences described herein. Host cells can be transformed, transfected, or transduced in a transient or stable manner. The nucleic acid, expression cassette, or vector can be introduced into the host cell so that the nucleic acid, cassette, or vector is maintained as a chromosomal integrant or as a self-replicating extrachromosomal vector. The term "host cell" encompasses any progeny of a parent host cell that is not identical to the parent host cell due to mutations that occur during replication. Host cells can be any cell useful in producing the variants of the present invention, for example, a prokaryotic or eukaryotic cell. Prokaryotic host cells can be any gram-positive or gram-negative bacteria. Host cells can also be eukaryotic cells, such as yeast, fungi, mammalian, insect, or plant cells. In certain embodiments, the host cell is selected from the group of Escherichia coli, Pseudomonas, Bacillus, Streptomyces, Trichoderma, Aspergillus, Saccharomyces, Pichia, Thermus, or Yarrowia.

[0129] The nucleic acids, expression cassettes or expression vectors according to the invention can be introduced into host cells 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.

[0130] In embodiments, the host cell is a genetically modified host cell or microorganism. In this context, the host cell or microorganism can be genetically modified to enhance the expression and / or activity of the polypeptide expressed therein. For example, the polypeptides described herein can be used to complement a wild-type strain of a fungus or bacterium already known to be capable of MHETase activity and / or converting terephthalic acid monoester to terephthalic acid and alcohol, where the terephthalic acid monoester is not mono-(2-hydroxyethyl) terephthalate, in order to improve and / or increase the activity of the strain to convert terephthalic acid monoester to terephthalic acid and alcohol, where the terephthalic acid monoester is not mono-(2-hydroxyethyl) terephthalate.

[0131] The present disclosure also provides a) providing a nucleic acid sequence as described herein; b) expressing the nucleic acid sequence in a host cell culture, thereby producing a polypeptide; and c) recovering the polypeptide produced in (b) from the host cell culture. The present invention also extends to a method for producing a polypeptide having an activity for converting a terephthalic acid monoester into terephthalic acid and an alcohol, the activity comprising:

[0132] The present disclosure also extends to in vitro methods of producing the polypeptides described herein, comprising the steps of: (a) contacting a nucleic acid, cassette, or vector of the invention with an in vitro expression system; and (b) recovering the produced polypeptide. In vitro expression systems are well known to those of skill in the art and are commercially available.

[0133] Suitable host cells will be well known to those skilled in the art, and illustrative examples include recombinant Bacillus, E. coli, Pseudomonas, Aspergillus, Trichoderma, Streptomyces, Saccharomyces, Pichia, Thermus, or Yarrowia. In an embodiment, the host cell is E. coli. In another embodiment, the host cell is Bacillus.

[0134] The host cells can be cultured in a nutrient medium suitable for the production of the polypeptide using methods known to those skilled in the art. Suitable examples include culturing the host cells in shake flask cultures or small- or large-scale fermentation (including continuous, batch, fed-batch, or solid-state fermentation) in laboratory or industrial fermentors, conducted in a suitable medium and under conditions that allow the enzyme to be expressed and / or isolated. Culturing will typically be carried out in a suitable nutrient medium prepared from a commercial supplier or according to published compositions (e.g., in the catalogs of the American Type Culture Collection), or any other culture medium suitable for cell growth. If the polypeptide is expressed and / or secreted in 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. Alternatively, the polypeptide can be recovered directly from the culture supernatant. Conversely, the polypeptide can be recovered from a cell lysate or after permeabilization of the host membrane. The polypeptide can be recovered using any suitable method known to those skilled in the art, illustrative examples of which include harvesting, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. Optionally, the polypeptides can be partially or completely 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 substantially pure polypeptides.

[0135] The polypeptides can be used in purified form, either alone or in combination with additional enzymes (e.g., PETase), to catalyze enzymatic reactions involved in the degradation and / or regeneration 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 polypeptides can be bound to cell membranes or lipid vesicles, or to synthetic supports such as glass, plastic, polymers, filters, membranes, etc., in the form of beads, columns, plates, etc.

[0136] The present disclosure also extends to compositions comprising the polypeptides, nucleic acids or host cells described herein.

[0137] The composition may be in liquid or dry form, e.g., powder form. In some embodiments, the composition is a lyophilized material. For example, the composition may include a polypeptide, a nucleic acid, and / or a host cell, and optionally, an excipient and / or a reagent. Suitable excipients include buffers commonly used in biochemistry, agents for adjusting pH, preservatives such as sodium benzoate, sodium sorbate, or sodium ascorbate, conservatives, protectants or stabilizers such as starch, dextrin, gum arabic, salts, sugars, e.g., sorbitol, trehalose, or lactose, glycerol, polyethylene glycol, polyethylene glycol (polyethene glycol), polypropylene glycol, propylene glycol, divalent ions such as calcium, sequestrants 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, and the like.

[0138] In an embodiment, the composition comprises a polypeptide described herein (the polypeptide can be present in the composition in isolated or at least partially purified form). In an embodiment, the composition comprises a polypeptide described herein in an amount of about 0.1% to about 99.9% by weight, preferably about 0.1% to about 50% by weight, preferably about 0.1% to about 30% by weight, and preferably 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 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 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 suitably adapted by one skilled in the art, depending, for example, on the nature and / or amount of the polyester-containing material to be degraded (hydrolyzed) and / or the presence or absence of any additional enzymes / polypeptides in the composition.

[0139] The compositions described herein can further include additional polypeptides that exhibit enzymatic activity, including but not limited to MHETase.

[0140] In embodiments, the polypeptides described herein can be solubilized in an aqueous medium with one or more excipients, such as excipients that can suitably stabilize or protect the polypeptide from degradation. For example, the polypeptides described herein can be solubilized in water and then mixed with excipients such as glycerol, sorbitol, dextrin, starch, glycols such as propanediol, salts, etc. The resulting mixture can then be dried to obtain a powder. Methods for drying such mixtures are well known to those skilled in the art and include, but are not limited to, lyophilization, freeze-drying, spray drying, supercritical drying, downdraft evaporation, thin-layer evaporation, centrifugal evaporation, conveyor drying, fluidized-bed drying, drum drying, or any combination thereof.

[0141] In embodiments, the composition comprises at least one host cell expressing a polypeptide described herein, or an extract thereof. By "cell extract" is meant any fraction obtained from a cell, such as a cell supernatant, cell debris, cell wall, DNA extract, enzyme or enzyme preparation, or any preparation derived from cells by chemical, physical, and / or enzymatic treatment that is essentially free of viable cells. A preferred extract is an enzymatically active extract. The composition can comprise one or more host cells or extracts thereof containing a polypeptide described herein, and optionally one or more additional cells.

[0142] As described elsewhere herein, the inventors have surprisingly discovered that the polypeptides described herein are capable of converting a terephthalic acid monoester to terephthalic acid and alcohol, wherein the terephthalic acid monoester is not mono-(2-hydroxyethyl)terephthalate. Accordingly, disclosed herein is a method for converting a terephthalic acid monoester to terephthalic acid and alcohol, the method comprising exposing the terephthalic acid monoester to a polypeptide, composition, or host cell described herein under conditions sufficient to allow the polypeptide to convert the terephthalic acid monoester to terephthalic acid and alcohol, wherein the terephthalic acid monoester is not mono-(2-hydroxyethyl)terephthalate. The present disclosure also extends to a method for degrading a plastic article comprising polyester, the method comprising exposing the plastic article to a polypeptide, composition, or host cell described herein.

[0143] The present disclosure extends to the use of the polypeptides, compositions, or host cells described herein to degrade polyesters under aerobic or anaerobic conditions, and / or to regenerate polyester-containing materials, such as plastic products made from or containing polyesters, and / or to produce biodegradable plastic products containing polyesters. Such methods and uses are particularly useful for degrading plastic products, including PET.

[0144] Advantageously, the polyesters of the polyester-containing material are depolymerized to monomers and / or oligomers. In embodiments, at least one polyester is degraded to produce repolymerizable monomers and / or oligomers, which are advantageously removed or recovered for further use.

[0145] In embodiments, the polyester of the polyester-containing material is completely degraded.

[0146] As described elsewhere herein, the plastic product can include at least one polyester selected from the group consisting of polylactic acid (PLA), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polyethylene terephthalate (PET), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), polycaprolactone (PCL), poly(ethylene adipate) (PEA), and any combination thereof. The plastic product can include at least one polymer selected from the group consisting of polypropylene, polystyrene, polyvinyl chloride, synthetic rubber, phenol formaldehyde resin (or Bakelite), neoprene, nylon, polyacrylonitrile, PVB, and silicone.

[0147] The time required to degrade a polyester-containing material may vary depending on the polyester-containing material itself (i.e., the nature and origin of the plastic product, its composition, shape, etc.), the type and amount of polypeptide used, and various process parameters (i.e., temperature, pH, additional agents, etc.) A person skilled in the art can easily adapt the process parameters to the polyester-containing material.

[0148] Advantageously, the degradation process is carried out at a temperature of about 10°C to about 60°C, preferably about 20°C to about 60°C, preferably about 30°C to about 60°C, more preferably about 40°C to about 60°C, even more preferably about 40°C to about 50°C, or even more preferably about 45°C. The temperature is typically maintained below the inactivation temperature, corresponding to the temperature at which the polypeptide is inactive and / or the recombinant microorganism does not synthesize, produce, or release the polypeptide described herein. In embodiments, the temperature is maintained below the glass transition temperature (Tg) of the polyester in the polyester-containing material. In embodiments, the degradation process or method is carried out at a temperature of about 10°C to about 60°C, preferably about 20°C to about 60°C, preferably about 30°C to about 60°C, more preferably about 40°C to about 60°C, even more preferably about 40°C to about 50°C, or even more preferably about 45°C. The process or method can be suitably carried out in a continuous manner, at a temperature that allows the polypeptide to be used and / or regenerated several times.

[0149] Advantageously, the degradation process or method is carried out at a pH comprised between 5 and 11, preferably between 6 and 10, more preferably between 6.5 and 9, and even more preferably between 7 and 8.

[0150] In embodiments, the polyester-containing material can be pretreated prior to contact with the polypeptide to physically alter its structure so as to increase the contact surface between the polyester and the enzyme.

[0151] Monomers resulting from the depolymerization or decomposition process or method can be suitably recovered sequentially or continuously. A single type of monomer or several different types of monomers can be recovered depending on the starting polyester-containing material.

[0152] The recovered monomer can be further purified and prepared in a repolymerizable form using any suitable purification method, including, combined or not, stripping processes, separation with aqueous solutions, selective steam condensation, filtration and concentration of post-bioprocessing medium, separation, distillation, vacuum evaporation, extraction, electrodialysis, adsorption, ion exchange, precipitation, crystallization, concentration and acid dehydration and precipitation, nanofiltration, acid catalyzed treatment, semi-continuous or continuous mode distillation, solvent extraction, evaporative concentration, evaporative crystallization, liquid / liquid extraction, hydrogenation, azeotropic distillation processes, adsorption, column chromatography, simple vacuum distillation, and microfiltration.

[0153] The repolymerizable monomers can be used to synthesize new polyesters. Advantageously, polyesters of the same nature are repolymerized. However, it is possible to mix the recovered monomers with other monomers, for example, to synthesize new copolymers. Alternatively, the recovered monomers can be used as chemical intermediates to produce new compounds of interest.

[0154] The present disclosure also extends to plastic compounds comprising the polypeptides, compositions and / or host cells expressing said polypeptides or extracts thereof containing said polypeptides.

[0155] The present disclosure also extends to masterbatch compositions comprising polypeptides, compositions and / or host cells expressing said polypeptides or extracts thereof containing said polypeptides.

[0156] Advantageously, such plastic compounds or masterbatch compositions described herein can be used for the production of polyester-containing materials and / or plastic articles that are expected to include the polypeptides described herein.

[0157] In embodiments, the resulting plastic compound, masterbatch composition or plastic article is a biodegradable plastic compound, masterbatch composition or plastic article that complies with at least one of the relevant standards and / or labels known to those skilled in the art, such as EN 13432 standard, ASTM D6400 standard, OK Biodegradation Soil (Vincotte label), OK Biodegradation Water (Vincotte label), OK Compost (Vincotte label), OK Home Compost (Vincotte label), etc.

[0158] Advantageously, the degradation process of the polyester-containing material (i.e. the plastic compound, masterbatch composition or plastic product) is carried out at a temperature comprised between 10°C and 50°C, preferably between 15°C and 40°C, more preferably between 20°C and 30°C, more preferably at 28°C, ±2°C.

[0159] Alternatively, the degradation process of the polyester-containing material (i.e., plastic compound, masterbatch composition or plastic article) is carried out at a temperature comprised between 50°C and 60°C, more preferably 55°C, ±2°C.

[0160] The MHETase polypeptides disclosed herein are suitable for a range of applications, e.g., industrial applications, illustrative examples of which include surfactants, feed compositions (including for animal feed), textile manufacturing, and additives in electronics and biomedical applications. For example, the polypeptides disclosed herein can be utilized in textile processing or textile manufacturing, where they can be used as exoesterases to favorably alter the properties of textile fibers.

[0161] The present invention will now be described with reference to the following examples which illustrate certain preferred aspects of the invention. It should be understood, however, that the details of the following description of the invention should not supersede the generality of the preceding description of the invention. [Example]

[0162] Materials and Methods Construction, expression and purification of recombinant MHETase A. Consensus Design and Construction Using wild-type I. sakaiensis MHETase (SEQ ID NO: 1) as the seed sequence, 5,000 sequences were collected by BLAST+, using an E-value threshold of 10-5. SEQ ID NO: 1 (UniProt accession number A0A0K8P8E7)

[0163] [ka]

[0164] 315 nonredundant sequences showing high similarity to MHETase were retrieved from the UniProt database. Peptide export signals were identified and deleted using SignalP 4.0. All sequences were aligned using a sequence alignment algorithm based on the PROMALS3D library and available MHETase structures (6QGB), and the final curated alignment was then manually refined. A consensus of the alignment at each amino acid position was constructed using several different thresholds (95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, and 50%). A truncated, codon-optimized version of the MHETase gene (GenBank accession number A0A0K8P8E7) lacking the first 19 amino acids at the N'-terminus, as well as 10 consensus designs, were commercially synthesized and cloned into pET-28a(+) (Twist Bioscience).

[0165] B. Protein Expression and Purification Plasmids were transformed by electroporation into electrocompetent E. coli SHuffle T7 Express cells (New England Biolabs), plated onto Lysogeny broth (LB) agar supplemented with 100 μg / mL ampicillin, and incubated overnight at 37°C. A single colony was used to inoculate 10 mL of LB supplemented with 100 μg / mL ampicillin (LBA) and incubated overnight at 30°C. This culture was added to 1 L of LBA and incubated at 30°C until an OD600 of 1.0 was reached. Isopropyl β-D-1-thiogalactopyranoside was added to a final concentration of 1 mM, and the culture was transferred to an 18°C ​​incubation for 16 hours.

[0166] Cells were harvested by centrifugation at 5,000 × 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 a 5-minute pulse time, 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 subsequently purified by nickel-loaded IMAC using a 5 mL HisTrap HP (GE Healthcare Life Sciences) equilibrated in lysis buffer and eluted with lysis buffer (500 mM NaCl, 500 mM imidazole, 0.5 mM dithiothreitol (DTT), and 25 mM HEPES pH 7.5). The elution with MHETase was collected, concentrated, and filtered through a 0.2 μm filter. The filtered product was further purified using a HiLoad26 / 600 Superdex200 (GE Healthcare Life Sciences) equilibrated in SEC buffer (150 mM NaCl, 25 mM HEPES pH 7.5).

[0167] C. Chromogenic Assay 50 μL of cell suspension of expressed protein was diluted in 150 μL of reaction buffer (90 mM NaCl, 45 mM sodium phosphate pH 7.5). The reaction was initiated by the addition of 50 μL of 10 mM 1-naphthyl terephthalate (1NT) and 5 mM Fast Blue B Salt dye in 100% (v / v) DMSO. Absorbance at 465 nm was monitored for 30 min using an Epoch microplate spectrophotometer (BioTek).

[0168] For kinetic determinations, homogenous MHETase at a final concentration of 7.5 nM was used instead of soluble cell lysate, with the concentration of 1NT varying from 1 mM to 7.8 μM. The concentration of Fast Blue B Salt remained constant. Absorbance was converted to product concentration using a calibration curve of 1 mM to 7.8 μM 1-naphthol.

[0169] D. SDS-PAGE Atto550 The method was adapted from Raducanu et al. (2020, Journal of Biological Chemistry 295(34):12214-12223). One milliliter of cell pellet was resuspended in 1x BugBuster (Merck-Millipore) diluted in SEC buffer and incubated at room temperature for 10 minutes. The mixture was centrifuged at 15,000 x g for 10 minutes, and 5 μL of the soluble cell lysate was run on an SDS-PAGE gel at 140 V for 60 minutes. The gel was microwaved twice for 30 seconds in Milli-Q water (MQ), followed by 2 minutes in fixative solution (40% (v / v) methanol, 10% (v / v) acetic acid in MQ). The now fixed protein gel was again microwaved in MQ for 10 minutes and incubated in a 1:3000 dilution of NTA-Atto550 dye in PBS buffer for 1 hour on a shaker in a dark environment. The gel was then transferred to a warm MQ container for an additional 30 minutes of shaking. The gel was then imaged using a ChemiDoc MP imaging system (BIO-RAD) with the DyLight 550 fluorophore option.

[0170] E. Circular Dichroism Analysis and Temperature Stability Circular dichroism spectra for MHETase were measured in 1 mm quartz cuvettes on an Applied Photophysics Chirascan spectrometer. The homogeneous enzyme was diluted to 0.2 mg / mL in 25 mM sodium acetate, pH 7.5. CD spectra from 200 nm to 260 nm were measured at 20 °C using a 1 nm bandwidth and a 0.5 s scan rate with adaptive sampling enabled. Spectra were measured in triplicate, and a buffer blank was subtracted from the results. For thermal melting assessment, CD spectra at 222 nm were recorded while the solution temperature was increased from 20 °C to 90 °C at 1 °C / min. A standard sigmoidal curve was fitted to the thermal melting data to determine the Tm.

[0171] High-Performance Liquid Chromatography (HPLC) Activity Assessment of F. MHETase The HPLC assay was adapted from Palm et al. (2019). Homogeneous MHETase was diluted in reaction buffer (80 μL) to a final concentration of 7.5 nM. The reaction was initiated by adding 20 μL of 1 mM MHET dissolved in 100% DMSO. The reaction was quenched after set time points (0, 10, 30, and 60 min) by adding 100 μL quench buffer (160 mM sodium phosphate pH 2) and heating to 80°C for 10 min. A 10 μL volume of the reaction mix was loaded onto an Agilent ZORBAX SB-C18, 3.5 μm, 4.6 x 150 mm column. TPA and MHET were separated using a flow rate of 1 mL / min at 30°C, equilibrated in 50% phosphate buffer (20 mM sodium phosphate pH 2.0) and 50% acetonitrile, over a 7-minute run time. TPA and MHET were detected at 240 nm and quantified against a calibration curve.

[0172] G. Monoesterase HPLC Activity Assay Assays of enzyme activity toward terephthalic acid monoester substrates (e.g., MBZT, MHXT, MHPT, and MOCT substrates) were performed using 1.5 mM substrate, 5% DMSO, and 200 nM enzyme. Reactions were incubated at 40°C for 64 minutes and then quenched at various time points by heating to 95°C for at least 10 minutes. Reactions were analyzed using high-performance liquid chromatography (HPLC) and compared to control reactions containing no enzyme. Product concentrations (terephthalic acid or terephthalic acid monoester substrates) were determined by comparison to calibration curves generated using synthetic or commercially available standards.

[0173] Example 1 Variant MHETase A consensus-based design was performed using aligned sequences of MHETase and its closely related enzymes. From this design, a number of different combinatorial MHETase sequences were constructed using different consensus thresholds: 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, and 50%. For example, a consensus design at a 95% threshold represents all of the differences observed in 95% of the aligned sequences but not in WT MHETase. The amino acid sequences of WT MHETase (SEQ ID NO: 1) and various consensus designs (SEQ ID NOs: 2-36, 73-78, and 86) are shown in Figure 1. The nucleic acid sequences of WT MHETase and various consensus designs (SEQ ID NOs: 37-72 and 79-85) are shown in Figure 2. The various consensus designs resulting from thresholds of 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55% and 50% are referred to herein as Round 1 Consensus A, B, C, D, E, F, G, H, I and J, respectively.

[0174] MHETase activity / expression was measured using whole cell suspensions using a chromogenic assay as described above.

[0175] As shown in Figure 3, the variant "Round 1 Consensus A" (SEQ ID NO: 73) exhibited higher whole-cell activity than either WT MHETase or other consensus designs. Round 1 Consensus A contained two amino acid substitutions compared to WT; namely, N156G and T159V. As an initial study, point mutations were added to this variant based on other consensus residues identified through multiple sequence alignment to identify mutations that further stabilize / improve activity. The remaining variants (mutants; SEQ ID NOs: 3-36) contained one of the following point mutations compared to the Round 1 Consensus A sequence (designations refer to amino acid positions in wild-type MHETase, SEQ ID NO: 1): T68V, A78P, E110A, M117L, E128Q, S131G, N134D, A161G, G156N, D191L, M192Y, S196A, Y197V, G204A, A207L, A216P, E226N, L234A, S235A, P255V, G258A, S260A, T264L, T265L, N284L, L295V, S296A, T355R, A377P, S463L, A493P, Y503W, E594A and N496S

[0176] As also shown in Figure 3, when made against the round 1 consensus A sequence, the point mutants S196A, Y197V, S235A, P255V, S260A, S286A, Y503W showed much higher whole cell activity when expressed in E. coli.

[0177] To determine the enzyme expression rate, soluble cell lysates were run through SDS-PAGE and stained with NTA-Atto550. MHETAse migration at 64 kDa on the gel is shown in Figure 4. This gel shows that the majority of MHETAse variants (including the control) are not visible even under more specific staining methods. This result is consistent with low soluble protein expression. Variants containing point mutations S196A, Y197V, S235A, P255V, S260A, S286A, or Y503W relative to the round 1 consensus A and variant B backbone appeared to exhibit increased heterologous expression in E. coli relative to the wild type (WT).

[0178] The stability of selected generated variants was also tested using purified protein variants. As shown in Figure 5, the thermal melting (T m ) were all around 59°C, while the introduction of the Y197V point mutation into round 1 consensus A resulted in T m The temperature was reduced to 52°C.

[0179] One of the key limitations of the use of wild-type MHETase in industrial applications is its low expression in industrial strains. The increase in whole cell activity (combined catalytic efficiency and expression yield of active protein) as demonstrated by the variants disclosed herein advantageously reduces production and use costs.

[0180] Example 2 Engineering MHETase with improved activity Because Round 1 consensus A was shown to have improved whole-cell MHETase activity with little change to temperature stability (Figure 3), Round 1 consensus A (containing the N156G and T159V point mutations) was selected as the basis for further engineering to generate variants with improved temperature stability (Figure 5). In this novel engineering process, point mutations were added to Round 1 consensus A to generate a library of mutants (Round 2) and screened for whole-cell activity. The process involves selecting the most promising variants and iteratively introducing single point mutations over several design rounds.

[0181] In round 2, the following point mutations were introduced into the round 1 consensus A backbone (designations refer to amino acid positions in wild-type MHETase, SEQ ID NO: 1): T593D, P543A, Y503W, S463L, P449A, T355R, G301A, S296A, H293Q, S286A, N284L, S267A, T265L, T264L, S26 0A, P255V, S235A, K218R, A216P, A207L, Y197B, S196A, D191L, L190I, E110A, Y107Q, A99N, A81P, A78P, T68V

[0182] The best performing variant selected from round 2 is "Round 2 Y503W" (containing N156G, T159V and Y503W point mutations; SEQ ID NO: 74).

[0183] In round 3, the following point mutations were introduced into "round 2 Y503W": E594A, A493P, A377P, S286A, S267A, T264L, S260A, S196A, M192Y

[0184] The best performing variant selected from round 3 is "Round 3 M192Y" (containing N156G, T159V, M192Y and Y503W point mutations; SEQ ID NO: 75).

[0185] In round 4, the following point mutations were introduced into round 3 M192Y, where "+" indicates the insertion of an amino acid at a particular position: +N564, S561A, G534A, L486V, A469P, +P467, H467M, W398K, A377P, M361F, I357L, S288T, L282D, S286A, S267A, S260A , Y252F, M233V, G231A, E230H, V208I, Q202P, V200L, T162S, A161S, G156N, V159T, L137V, G130N, R114E, L112G, A79G

[0186] The best performing variant selected from round 4 is "Round 4 G156N" (containing T159V, M192Y and Y503W point mutations; SEQ ID NO: 76).

[0187] In round 5, the following point mutations were introduced into round 4 G156N, where "+" indicates the insertion of an amino acid at a particular position: N592E, I582V, G562R, R537Q, A494V, W466F, Q461E, M361F, I357L, N316D, I283L, Y252F, V246L, Y242F, G231A, V200L, G164A, I104V, E90A, E71T

[0188] The best performing variant selected from round 5 was "Round 5 Y252F" (comprising T159V, M192Y, Y252F and Y503W; SEQ ID NO: 77), which showed a 16-fold increase in whole-cell activity compared to the wild-type (Figure 6).

[0189] Expression of the best variants from each round was quantitatively measured using SDS-PAGE stained with the His-tag-specific fluorescent label ATTO550. Expression of WT MHETase was too low to be conclusively detected relative to background endogenous E. coli protein expression. However, round 2 of genetic engineering improved the recombinant expression level of the variant MHETase (round 2 Y503W) to a level that was visually detectable using the ATTO550 fluorescent dye (Figure 7).

[0190] Using the chromogenic assay described herein with purified proteins (Figure 8), the kinetic parameters of these enzymes were determined. The catalytic efficiency (kcat / KM) of all variants remained relatively constant relative to that of wild-type MHETase. Figure 9 shows a Michaelis-Menten plot of the rates of enzyme function at various substrate concentrations. From these data, two constants, kcat and KM, were obtained, as shown in Table 1.

[0191] [Table 1]

[0192] To validate the data observed using the chromogenic assay described herein, the activity of wild-type MHETase and the best variant from Round 5 (Round 5 Y252F; SEQ ID NO: 77) against the natural substrate MHET was determined by high performance liquid chromatography (HPLC). HPLC was used to measure the concentrations of terephthalic acid (TPA) and MHET in enzymatic reactions containing wild-type MHETase or Round 5 Y252F over time. The data corroborate the kDa activity observed using the chromogenic assay. cat and K.M This is consistent with the reduction in , confirming that round 5 Y252F hydrolyzes MHET to TPA (see Figure 11).

[0193] Stability studies for the consensus design were also performed using purified protein variants. Mutations at positions 192 and 252 improve the temperature stability (Tm) compared to WT MHETase (Figure 10).

[0194] Overall, these stabilizing properties contribute to the improved expression and activity of round 5 Y252F compared to wild-type MHETase. However, despite these improvements, the specific activity of these variants remains poor due to their relatively low k cat is reduced compared to the wild type, as demonstrated by (Table 1).

[0195] Further genetic engineering was performed to revert some of the individual mutations in round 5 Y252F to their wild-type residues. In the case of position 156, the mutation N156G was reintroduced into the MHETase R5 background to investigate the impact of reverting this residue to its wild-type identity in round 4. Whole cell activity, as determined by FastBlue assay, is shown in Figure 12.

[0196] When the mutation M192Y in round 5 Y252F was reverted to the wild-type residue (M) ("R5-Y192M"; SEQ ID NO: 78), an approximately three-fold increase in specific activity was observed compared to round 5 Y252F (Figure 11), restoring specific activity to a level more comparable to that of the wild-type MHETase of SEQ ID NO: 1. Reversion of other point mutations in round 5 Y252F either reduced or did not significantly change enzyme activity. The temperature stability of the MHETase variants is shown in Table 2.

[0197] [Table 2]

[0198] Example 3 Engineered MHETase efficiently hydrolyzes the monoester of TPA The native substrate of MHETase is mono-(2-hydroxyethyl) terephthalate (MHET), a monoester of terephthalic acid (TPA). However, the inventors have demonstrated that the enzyme disclosed herein can also convert C6 to C8 between PETs. 10 It was surprisingly found that other monoesters of TPA formed by base-catalyzed transesterification with monoalcohols (see Figure 13) can also be hydrolyzed to TPA. As shown in Figure 14, the MHETase R5 polypeptide (SEQ ID NO: 77) was able to hydrolyze monobenzyl terephthalate and monooctyl terephthalate to TPA (see Figure 14).

[0199] In comparison, known and commercially available esterases known to hydrolyze ester bonds, such as boar esterase and two lipases from T. lanuginosa and R. miehei (all from Sigma-Aldrich), do not demonstrate hydrolysis of the major monoester, MOCT, when tested in parallel with MHETase R5 (see Figure 15).

[0200] The disclosures of all patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety.

[0201] The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.

[0202] Throughout this specification, the objective has been to describe preferred embodiments of the present invention without limiting the invention to any one embodiment or particular collection of features. Accordingly, those skilled in the art will appreciate that, in light of this disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All such modifications and changes are intended to be included within the scope of the appended claims.

Claims

1. 1. A method for hydrolyzing a terephthalic acid monoester, comprising exposing the terephthalic acid monoester to a polypeptide having MHETase activity under conditions sufficient to allow 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.

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

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

1.

4. 3. The method of claim 2, wherein the polypeptide comprises an amino acid sequence having at least 70% sequence identity to amino acids 20 to 603 of SEQ ID NO:1 and differing from amino acids 20 to 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. C wherein the ester is optionally substituted by benzyl 1 ~C 10 5. The method of claim 1, wherein the hydroxyl group is an alkyl ester.

6. C wherein the ester is optionally substituted by benzyl 6 ~C 10 6. The method of claim 1, wherein the hydroxyl group is an alkyl ester.

7. 7. The method of claim 1, wherein the terephthalic acid monoester is selected from the group consisting of monobenzyl terephthalate (MBZT), monohexyl terephthalate, monoheptyl terephthalate (MHPT), and monooctyl terephthalate (MOCT).

8. 8. The method of claim 1, wherein the terephthalic acid monoester is MBZT.

9. 8. The method according to claim 1, wherein the terephthalic acid monoester is MOCT.

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

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

1.

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

1.

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

1.

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

1.

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

1.

20. 20. The method of any one of claims 10 to 19, wherein the amino acid sequence of the polypeptide differs from amino acids 20 to 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.

21. 21. The method of any one of claims 10 to 20, wherein the amino acid sequence of the polypeptide differs from amino acids 20 to 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. 19. The method of any one of claims 10 to 18, wherein the amino acid sequence of the polypeptide differs from amino acids 20 to 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. 23. The method of any one of claims 10 to 18 or 22, wherein the amino acid sequence of the polypeptide differs from amino acids 20 to 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. 19. The method of any one of claims 10 to 18, wherein the amino acid sequence of the polypeptide differs from amino acids 20 to 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. 20. The method of any one of claims 10 to 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. 21. The method of claim 20, wherein the amino acid substitution is N156G, T159V, and Y503W, or any of these conservative amino acid substitutions.

27. 22. The method of claim 21, wherein the amino acid substitution is N156G, T159V, M192Y, or Y503W, or any of these conservative amino acid substitutions.

28. 23. The method of claim 22, wherein the amino acid substitution is T159V, Y252F, or Y503W, or any of these conservative amino acid substitutions.

29. 24. The method of claim 23, wherein the amino acid substitution is T159V, M192Y, Y252F, or Y503W, or any conservative amino acid substitution therein.

30. 25. The method of claim 24, wherein the amino acid substitution is T159V, M192Y, or Y503W, or any of these conservative amino acid substitutions.

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

1.

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

1.

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

1.

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

1.

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

1.

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

1.

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

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

45. Terephthalic acid monoester is C 6 ~C 10 44. The method of claim 43, produced by a process comprising subjecting PET to base-catalyzed transesterification with a monoalcohol.

46. Terephthalic acid diester is C 6 ~C 10 44. The method of claim 43, produced by a process comprising subjecting PET to base-catalyzed transesterification with a monoalcohol.

47. C 6 ~C 10 47. The method of claim 45 or 46, wherein the monoalcohol is benzyl alcohol, octanol or heptanol.

48. C 6 ~C 10 48. The method of claim 47, wherein the monoalcohol is 1-octanol.

49. 45. The method of claim 44, wherein the esterase is PETase.

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

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

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