Enzymes and their uses

Abstract

The present disclosure relates generally to polypeptides having esterase activity that can convert a terephthalic acid monoester to terephthalic acid and an alcohol; a terephthalic acid diester to terephthalic acid monoester and an alcohol; or a terephthalic acid diester to terephthalic acid and an alcohol, and methods of their use.

Description

[Technical Field]

[0001] This application claims priority to Australian Provisional Patent Application No. 2022902457, entitled "Enzymes and uses thereof," filed on August 26, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] The present invention relates to novel synthetic enzymes, and more particularly to recombinant enzymes that catalyze the hydrolysis of mono- and di-terephthalic acid esters, 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 plastic produces potentially harmful by-products that are released into the atmosphere; the rate at which plastic decomposes in landfills is typically very slow, posing a risk of toxic substances reaching groundwater; and mechanical degradation is relatively expensive and inefficient, often limiting the uses for the by-products.

[0006] More recently, biological (enzymatic) degradation of plastics has been considered as an alternative approach to reducing the accumulation of plastic waste. This approach involves the use of PETase, an enzyme of the esterase class that catalyzes the hydrolysis of PET to the monomer mono-2-hydroxyethyl terephthalate (MHET), which is further degraded by the action of MHETase to terephthalic acid and ethylene glycol. Terephthalic acid is a precursor to the polyester PET. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Yoshida et al. (2016) Science 351: 1196 pages [Non-patent document 2] Ion et al., 2021 Catalysis Today 366:177 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., 2016, Science, 352(6278): 1196 pages [Non-patent document 6] Belousov (1997) Nucleic Acids Res. 25:3440~3444 Summary of the Invention [Problem to be solved by the invention]

[0008] 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, in part due to its relative inefficiency, slow rate of enzymatic degradation, and low levels of enzyme expression in common industrial host organisms. Thus, there remains an urgent need for improved methods and reagents for the enzymatic degradation of plastics. [Means for solving the problem]

[0009] In one aspect disclosed herein, a polypeptide having esterase activity is provided, wherein the esterase activity is capable of converting a terephthalic acid monoester to terephthalic acid and an alcohol; a terephthalic acid diester to a terephthalic acid monoester and an alcohol; or a terephthalic acid diester to terephthalic acid and an alcohol; the polypeptide comprising an amino acid sequence selected from the group consisting of amino acids 5 to 261 of SEQ ID NO:2, or an amino acid sequence having at least 85% sequence identity thereto; amino acids 5 to 261 of SEQ ID NO:3, or an amino acid sequence having at least 77% sequence identity thereto; amino acids 5 to 261 of SEQ ID NO:4, or an amino acid sequence having at least 75% sequence identity thereto; amino acids 5 to 261 of SEQ ID NO:5, or an amino acid sequence having at least 95% sequence identity thereto; and amino acids 5 to 261 of SEQ ID NO:6, or an amino acid sequence having at least 96% sequence identity thereto; and the polypeptide is not SEQ ID NO:1 or SEQ ID NO:12.

[0010] In embodiments, the polypeptide comprises the amino acid sequence of amino acids 5 to 261 of SEQ ID NO: 6, or an amino acid sequence having at least 96% sequence identity thereto.

[0011] In embodiments, the polypeptide comprises the amino acid sequence of amino acids 5 to 261 of SEQ ID NO: 7, or an amino acid sequence having at least 97% identity thereto.

[0012] In embodiments, the polypeptide comprises the amino acid sequence of amino acids 5 to 261 of SEQ ID NO: 8, or an amino acid sequence having at least 96% identity thereto.

[0013] In embodiments, the polypeptide comprises the amino acid sequence of amino acids 5 to 261 of SEQ ID NO: 9, or an amino acid sequence having at least 97% identity thereto.

[0014] In embodiments, the polypeptide comprises the amino acid sequence of amino acids 5 to 261 of SEQ ID NO: 10, or an amino acid sequence having at least 98% identity thereto.

[0015] In embodiments, the polypeptide comprises the amino acid sequence of amino acids 5 to 261 of SEQ ID NO: 11, or an amino acid sequence having at least 98% identity thereto.

[0016] In another embodiment, the polypeptide comprises the amino acid sequence of SEQ ID NO:119.

[0017] In another aspect, a polypeptide having esterase activity is provided, wherein the esterase activity is capable of converting a terephthalic acid monoester to terephthalic acid and an alcohol; a terephthalic acid diester to terephthalic acid monoester and an alcohol; or a terephthalic acid diester to terephthalic acid and an alcohol, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:119.

[0018] In another aspect disclosed herein, a method is provided for the enzymatic hydrolysis of terephthalic acid monoesters and / or terephthalic acid diesters produced as by-products of the degradation of PET, wherein the terephthalic acid monoesters are optionally benzyl substituted mono-C1-C6 esters. 10 alkyl terephthalates, and the terephthalic acid monoester is not mono-(2-hydroxyethyl) terephthalate (MHET); and the terephthalic acid diester is di-C1-C optionally substituted by benzyl. 10 alkyl terephthalates; the method includes exposing a terephthalic acid monoester and / or a terephthalic acid diester to a polypeptide having esterase activity under conditions sufficient to enable the polypeptide to convert the terephthalic acid monoester to terephthalic acid and an alcohol; the terephthalic acid diester to a terephthalic acid monoester and an alcohol; or the terephthalic acid diester to terephthalic acid and an alcohol.

[0019] In embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having at least 70% sequence identity to SEQ ID NO:1.

[0020] In embodiments, the terephthalic acid monoester is selected from the group consisting of monobenzyl terephthalate (MBZT), monohexyl terephthalate, monoheptyl terephthalate (MHPT), and monooctyl terephthalate (MOCT). In preferred embodiments, the terephthalic acid monoester is MBZT or MOCT. In one embodiment, the polypeptide comprises the amino acid sequence of amino acids 5-261 of any one of SEQ ID NOs: 9-11, or an amino acid sequence having at least 70% sequence identity to any of the foregoing.

[0021] In embodiments, the terephthalic acid monoester is selected from the group consisting of dibenzyl terephthalate (DBZT), dihexyl terephthalate (DHXT), diheptyl terephthalate (DHPT), and dioctyl terephthalate (DOCT). In preferred embodiments, the terephthalic acid monoester is DBZT or DOCT. In one embodiment, the polypeptide comprises the amino acid sequence of amino acids 5-261 of any one of SEQ ID NOs: 6-8, or an amino acid sequence having at least 70% sequence identity to any of the foregoing.

[0022] In embodiments, the terephthalic acid monoesters and diesters are produced by the hydrolysis or degradation of polyethylene terephthalate (PET).

[0023] The present disclosure also extends to compositions comprising a polypeptide as described herein.

[0024] The present disclosure also extends to nucleic acid sequences encoding the polypeptides described herein.

[0025] The present disclosure also extends to expression vectors comprising the nucleic acid sequences described herein.

[0026] The present disclosure also extends to host cells comprising the nucleic acid sequences or expression vectors described herein.

[0027] In another aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: i) providing a polynucleotide described herein; ii) expressing the polynucleotide in a host cell under conditions sufficient to enable the host cell to produce the polypeptide; and iii) collecting the polypeptide produced by the host cell in ii). The present invention provides a method for producing a polypeptide having esterase activity, comprising:

[0028] The present disclosure also extends to a method of degrading a plastic article comprising a polyester, comprising contacting the plastic article with a polypeptide, composition, or host cell described herein under conditions sufficient to allow the polypeptide to degrade the plastic article. In an embodiment, the plastic article comprises the polyester polyethylene terephthalate (PET).

[0029] The present disclosure also extends to compositions comprising terephthalic acid and / or alcohol recovered by the methods disclosed herein.

[0030] In another aspect, there are provided host cells genetically engineered to express the polypeptides described herein.

[0031] In another aspect, there is provided a method for manufacturing a plastic article using the terephthalic acid and alcohol composition produced by the methods disclosed herein. [Brief explanation of the drawings]

[0032] [Figure 1] 1 shows a schematic representation of the enzymatic conversion of (i) a terephthalic acid monoester to terephthalic acid and alcohol, (ii) a terephthalic acid diester to terephthalic acid monoester and alcohol, and (iii) a terephthalic acid diester to terephthalic acid and alcohol, where X is C1-C10 and the reformed C1-C10 monoalcohol is represented by X-OH. [Figure 2]

[0023] Figure 1 demonstrates the DOCTase activity of polypeptides having the amino acid sequences of SEQ ID NOs: 69-118 (from left to right) disclosed herein as determined by the combined concentration (mg / mL) of monomer equivalent hydrolysis products MOCT and TPA as determined by U / HPLC (as represented on the Y-axis). SEQ ID NOs are provided on the X-axis. DETAILED DESCRIPTION OF THE INVENTION

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] While the enzymatic conversion of PET to the monomeric mono-2-hydroxyethyl terephthalate (MHET) and bis-(2-hydroxyethyl) terephthalic acid (BHET) by PETase enzymes and the enzymatic hydrolysis of MHET to terephthalate / TPA by MHETase are known (MHETase was originally discovered in conjunction with PETase in the bacterium Ideonella sakaiensis; the two enzymes enable the bacterium to survive on the plastic PET as a carbon source; Yoshida et al. (2016) Science 351:1196), enzymes capable of degrading or hydrolyzing other plastic or PET intermediates, including terephthalate esters (particularly terephthalate di- and monoesters other than MHET and BHET, see, e.g., Ion et al., 2021 Catalysis Today 366:177), have not yet been identified. This disclosure is based, at least in part, on the identification of enzymes capable of degrading or hydrolyzing other plastics or PET intermediates, including terephthalate esters (particularly terephthalate di- and monoesters other than MHET and BHET, see, e.g., Ion et al., 2021 Catalysis Today 366:177). 10 Alkyl terephthalates, especially mono- and di-C6-C 10 This is based on the inventors' identification of esterases that surprisingly have hydrolase / esterase activity towards the mono- and di-terephthalic acid esters of PET, such as alkyl terephthalates.

[0038] Accordingly, in aspects disclosed herein, there is provided a polypeptide having esterase activity, wherein the esterase activity is capable of converting a terephthalic acid monoester to terephthalic acid and an alcohol; a terephthalic acid diester to a terephthalic acid monoester and an alcohol; or a terephthalic acid diester to terephthalic acid and an alcohol; wherein the polypeptide comprises an amino acid sequence selected from the group consisting of amino acids 5 to 261 of SEQ ID NO:2, or an amino acid sequence having at least 85% sequence identity thereto; amino acids 5 to 261 of SEQ ID NO:3, or an amino acid sequence having at least 77% sequence identity thereto; amino acids 5 to 261 of SEQ ID NO:4, or an amino acid sequence having at least 75% sequence identity thereto; amino acids 5 to 261 of SEQ ID NO:5, or an amino acid sequence having at least 95% sequence identity thereto; and amino acids 5 to 261 of SEQ ID NO:6, or an amino acid sequence having at least 96% sequence identity thereto; and wherein the polypeptide is not SEQ ID NO:1 or SEQ ID NO:12.

[0039] In an embodiment, the terephthalic acid monoester is a mono-C1-C6 optionally substituted by benzyl. 10 alkyl terephthalate, the terephthalic acid diester being optionally benzyl substituted di-C1-C 10 In a preferred 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), and the terephthalic acid diester is selected from the group consisting of dibenzyl terephthalate (DBZT), dihexyl terephthalate (DHXT), diheptyl terephthalate (DHPT), and dioctyl terephthalate (DOCT).

[0040] References to terephthalic acid, as used herein, also extend to salts of terephthalic acid. Thus, in embodiments, the terephthalic acid is a salt of terephthalic acid. In embodiments, the salt of terephthalic acid is an alkali metal salt. In another embodiment, the terephthalic acid produced is disodium terephthalate.

[0041] By "at least 85%" it is meant that the polypeptide shares at least 85%, preferably at least 87%, preferably at least 88%, 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 at least 99% sequence identity to SEQ ID NO:2 or amino acids 5 to 261 of SEQ ID NO:2. Because the polypeptides described herein are synthetic polypeptides, it should be understood that in this context "at least 85%" can include 100% sequence identity across the entire sequence of SEQ ID NO:2 or across the entire amino acid sequence of amino acids 5 to 261 of SEQ ID NO:2.

[0042] By "at least 77%" it is meant that the polypeptide shares at least 77%, 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 at least 99% sequence identity to SEQ ID NO:3 or amino acids 5 to 261 of SEQ ID NO:3. Because the polypeptides described herein are synthetic polypeptides, it should be understood that in this context "at least 77%" can include 100% sequence identity across the entire sequence of SEQ ID NO:3 or across the entire sequence of amino acids 5 to 261 of SEQ ID NO:3.

[0043] By "at least 75%" is meant that the polypeptide shares 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 at least 99% sequence identity to SEQ ID NO: 4 or amino acids 5 to 261 of SEQ ID NO: 4. Because the polypeptides described herein are synthetic polypeptides, it should be understood that in this context "at least 85%" can include 100% sequence identity across the entire sequence of SEQ ID NO: 4 or across the entire sequence of amino acids 5 to 261 of SEQ ID NO: 4.

[0044] By "at least 95%" it is meant that the polypeptide shares at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, or more preferably at least 99% sequence identity to SEQ ID NO: 5 or amino acids 5 to 261 of SEQ ID NO: 5. Because the polypeptides described herein are synthetic polypeptides, it should be understood that in this context "at least 95%" can include 100% sequence identity across the entire sequence of SEQ ID NO: 5 or across amino acids 5 to 261 of SEQ ID NO: 5.

[0045] By "at least 96%" it is meant that the polypeptide shares at least 96%, preferably 97%, preferably at least 98%, or more preferably at least 99% sequence identity to SEQ ID NO: 6 or amino acids 5 to 261 of SEQ ID NO: 6. Because the polypeptides described herein are synthetic polypeptides, it should be understood that in this context "at least 95%" can include 100% sequence identity across the entire sequence of SEQ ID NO: 6 or across the entire sequence of amino acids 5 to 261 of SEQ ID NO: 6.

[0046] In one embodiment, the polypeptide comprises the amino acid sequence of amino acids 5 to 261 of SEQ ID NO: 2, or an amino acid sequence having at least 85% sequence identity thereto. In another embodiment, the polypeptide consists of the amino acid sequence of SEQ ID NO: 2.

[0047] In one embodiment, the polypeptide comprises the amino acid sequence of amino acids 5 to 261 of SEQ ID NO: 3, or an amino acid sequence having at least 77% sequence identity thereto. In another embodiment, the polypeptide consists of the amino acid sequence of SEQ ID NO: 3.

[0048] In one embodiment, the polypeptide comprises the amino acid sequence of amino acids 5 to 261 of SEQ ID NO: 4, or an amino acid sequence having at least 75% sequence identity thereto. In another embodiment, the polypeptide consists of the amino acid sequence of SEQ ID NO: 4.

[0049] In one embodiment, the polypeptide comprises the amino acid sequence of amino acids 5 to 261 of SEQ ID NO: 5, or an amino acid sequence having at least 95% sequence identity thereto. In another embodiment, the polypeptide consists of the amino acid sequence of SEQ ID NO: 5.

[0050] In embodiments, the polypeptide comprises the amino acid sequence of amino acids 5 to 261 of SEQ ID NO: 6, or an amino acid sequence having at least 96% sequence identity thereto.

[0051] In another embodiment, the polypeptide consists of the amino acid sequence of SEQ ID NO:6.

[0052] In one embodiment, the polypeptide comprises the amino acid sequence of amino acids 5 to 261 of SEQ ID NO: 7. In one embodiment, the polypeptide comprises the amino acid sequence of amino acids 5 to 261 of SEQ ID NO: 8. In one embodiment, the polypeptide comprises the amino acid sequence of amino acids 5 to 261 of SEQ ID NO: 9. In one embodiment, the polypeptide comprises the amino acid sequence of amino acids 5 to 261 of SEQ ID NO: 10. In one embodiment, the polypeptide comprises the amino acid sequence of amino acids 5 to 261 of SEQ ID NO: 11.

[0053] In another embodiment, the polypeptide consists of or consists essentially of the amino acid sequence of SEQ ID NO: 6. In one embodiment, the polypeptide consists of or consists essentially of the amino acid sequence of SEQ ID NO: 7. In one embodiment, the polypeptide consists of or consists essentially of SEQ ID NO: 8. In one embodiment, the polypeptide consists of or consists essentially of SEQ ID NO: 9. In one embodiment, the polypeptide consists of or consists essentially of SEQ ID NO: 10. In one embodiment, the polypeptide consists of or consists essentially of SEQ ID NO: 11.

[0054] In embodiments, the polypeptide comprises the amino acid sequence of amino acids 5 to 260 of any one of SEQ ID NOs: 1-118.

[0055] In embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 119. In embodiments, the amino acid residue at position X of SEQ ID NO: 119 is selected from the amino acid residue at the corresponding position of any one of SEQ ID NOs: 1-118.

[0056] In another embodiment, the polypeptide further comprises an N-terminal cellular export signal peptide or signal peptide selected from the amino acid sequences of SEQ ID NO: 120 (MAAN); SEQ ID NO: 121 (MAEN); SEQ ID NO: 122 (MQAN); SEQ ID NO: 123 (MADN); and SEQ ID NO: 124 (MQSN).

[0057] 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 10Formed 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.

[0058] Terephthalic acid diesters will be familiar to those skilled in the art. For example, as used herein, the term terephthalic acid diester refers to a 1,4-disubstituted benzene in which the substitution is an ester functional group. Terephthalic acid diesters include dialkyl terephthalates. In some embodiments, the terephthalic acid diester is a C1-C 10 Formed through the transesterification of PET with a monoalcohol. In certain embodiments, the terephthalic acid diester is a C6-C 10 Formed through the transesterification of PET with monoalcohols. In certain embodiments, terephthalic acid diesters are formed through the transesterification of PET with benzyl alcohol, hexanol, heptanol, or octanol.

[0059] The present disclosure also extends to compositions comprising a polypeptide as described herein.

[0060] The present disclosure also extends to nucleic acid sequences encoding the polypeptides described herein.

[0061] The present disclosure also extends to expression vectors comprising the nucleic acid sequences described herein.

[0062] The present disclosure also extends to host cells comprising the nucleic acid sequences or expression vectors described herein.

[0063] In another aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: i) providing a polynucleotide described herein; ii) expressing the polynucleotide in a host cell under conditions sufficient to enable the host cell to produce the polypeptide; and iii) harvesting the polypeptide produced by the host cell in (ii). The present invention provides a method for producing a polypeptide having esterase activity, comprising:

[0064] In yet another aspect, a method is provided for the enzymatic hydrolysis of terephthalic acid monoesters and / or terephthalic acid diesters produced as by-products of the degradation of PET, comprising: a. The terephthalic acid monoester is a mono-C1-C optionally benzyl substituted 10 alkyl terephthalate, and not terephthalic acid monoester mono-(2-hydroxyethyl) terephthalate (MHET); b. The terephthalic acid diester is a di-C1-C optionally benzyl substituted di-C 10 alkyl terephthalate; The method comprises: i. terephthalic acid monoester to terephthalic acid and alcohol; ii. terephthalic acid diester to terephthalic acid monoester and alcohol; or iii. exposing the terephthalic acid monoester and / or terephthalic acid diester to a polypeptide having esterase activity under conditions sufficient to permit conversion of the terephthalic acid diester to terephthalic acid and an alcohol.

[0065] For one embodiment, the polypeptide used in the hydrolysis of terephthalic acid monoesters and / or terephthalic acid diesters produced as by-products of PET degradation comprises the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having at least 70% sequence identity to SEQ ID NO:1.

[0066] In an embodiment, the terephthalic acid monoester is a mono-C-C optionally benzyl substituted terephthalic acid monoester. 10In one embodiment, the ester is a mono-C alkyl ester. In one embodiment, the ester is a mono-C alkyl ester. In one embodiment, the ester is a mono-C alkyl ester. In one embodiment, the ester is a mono-C alkyl ester. In another embodiment, the ester is a mono-C alkyl ester. In another embodiment, the ester is a mono-C alkyl ester. 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 or MOCT. In a preferred embodiment, the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 9-11 and an amino acid sequence having at least 70% sequence identity to any of the foregoing.

[0067] In an embodiment, the terephthalic acid monoester is a di-C-C optionally benzyl substituted terephthalic acid monoester. 10 In one embodiment, the ester is a di-C alkyl ester. In one embodiment, the ester is a di-C alkyl ester. In one embodiment, the ester is a di-C alkyl ester. In one embodiment, the ester is a di-C alkyl ester. In another embodiment, the ester is a di-C alkyl ester. In another embodiment, the ester is a di-C alkyl ester. 10 In an embodiment, the terephthalic acid diester is selected from the group consisting of dibenzyl terephthalate (DBZT), dihexyl terephthalate (DHXT), diheptyl terephthalate (DHPT), and dioctyl terephthalate (DOCT). In a preferred embodiment, the terephthalic acid monoester is DBZT or DOCT. In a preferred embodiment, the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 6 to 8 and an amino acid sequence having at least 70% sequence identity to any of the above.

[0068] In an embodiment, the terephthalic acid monoesters and terephthalic acid diesters are produced by hydrolysis or degradation of polyethylene terephthalate (PET). In another embodiment, the terephthalic acid monoesters and terephthalic acid diesters are produced by a process comprising exposing PET to sodium hydroxide and / or contacting PET with an esterase. In a preferred embodiment, the terephthalic acid monoesters and terephthalic acid diesters are C6-C 10 In a preferred embodiment, C6-C PET is 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. 10 The monoalcohol is 1-octanol.

[0069] The present disclosure also extends to a method of degrading a plastic article, including polyester, comprising contacting the plastic article with a polypeptide, composition, or host cell described herein under conditions sufficient to allow the polypeptide to degrade the plastic article.

[0070] 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).

[0071] The present disclosure also extends to compositions comprising terephthalic acid and / or ethylene glycol recovered by the methods disclosed herein.

[0072] In another aspect, there are provided host cells genetically engineered to express the polypeptides described herein.

[0073] In another aspect, there is provided a method for manufacturing a plastic article using the terephthalic acid and ethylene glycol composition produced by the methods disclosed herein.

[0074] The term "wild-type" is used herein to refer to a naturally occurring isoform of a polypeptide; i.e., a polypeptide as it occurs in nature. The term "extant" is used to refer to a naturally occurring isoform of a polypeptide that is found in organisms or species that still exist (i.e., have not become extinct).

[0075] Examples of existing cutinases will be familiar to those skilled in the art, including, but not limited to, leaf compost cutinase (LCC; registration number G9BY57), TfCut2 (cutinase from Thermobifida fusca; registration number E5BBQ3), E9LVH9 (Cut2 from Thermobifida cellulosilytica), and E5BBQ2 (Cut.1-KW3 cutinase from Thermobifida fusca).

[0076] 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).

[0077] The term "hydrolase," as used herein, typically refers to an enzyme that catalyzes the hydrolysis of chemical bonds, such as ester bonds, and belongs to the class of hydrolases classified as EC3 according to enzyme nomenclature.

[0078] The term "esterase," as used herein, typically refers to a hydrolase enzyme classified as EC 3.1 according to enzyme nomenclature, which catalyzes the hydrolysis of ester bonds to produce acids and alcohols. The term "cutinase" refers to a serine esterase enzyme classified as EC 3.1.1.74 according to enzyme nomenclature, which catalyzes the hydrolysis of cutin polymers (polyesters composed of hydroxy and hydroxyepoxy fatty acids) into cutin monomers.

[0079] The term "PETase," as used herein, typically refers to an esterase enzyme classified as EC 3.1.1.101 according to enzyme nomenclature, which catalyzes the hydrolysis of polyethylene terephthalate (PET) plastic to the monomer mono-2-hydroxyethyl terephthalate (MHET).

[0080] As used herein, the polypeptides of the invention are understood to have hydrolase or esterase activity, which has the ability to catalyze the hydrolysis of mono- and di-terephthalic acid esters.

[0081] 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 terephthalate and ethylene glycol (Palm et al. (2019, Nat. Comm. 10:1717), Sagong et al. (2020, ACS Catal. 10:4805), and Yoshida et al. (2016, Science 352(6278):1196)).

[0082] The terms "mutant" and "variant" are used interchangeably herein to refer to a polypeptide comprising an amino acid sequence derived from SEQ ID NO: 1 and further comprising modifications or changes (e.g., substitutions, insertions, and / or deletions) at one or more (e.g., several) positions when compared to an existing PETase or cutinase having PETase activity or the polypeptide of SEQ ID NO: 1, and having enhanced esterase activity in catalyzing the hydrolysis of mono- and di-terephthalic acid esters. 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.

[0083] 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).

[0084] Positions disclosed in the present application are numbered by reference to the amino acid sequence set forth in the specified SEQ ID NO. 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 the reference sequence. For example, the amino acid residue at position 5 of SEQ ID NO:6 (amino acid P / proline) would correspond to the amino acid residue at position 4 of SEQ ID NO:4 (amino acid P / proline) in SEQ ID NO:116. In another example, the amino acid residue at position 14 of SEQ ID NO:19 (amino acid N / asparagine) would correspond to the amino acid residue at position 13 of SEQ ID NO:113 (amino acid E / glutamic acid). In yet another example, in the context of SEQ ID NO:119, the amino acid residue at position 4 of SEQ ID NO:119 (amino acid P / proline) would correspond to the amino acid residue at position 12 of SEQ ID NO:44 (amino acid P / proline); and the amino acid residue at position 3 of SEQ ID NO:119 would correspond to the amino acid residue at position 11 of SEQ ID NO:44 (amino acid A / alanine). As a further example, in the context of SEQ ID NO: 119, the amino acid residue at position 3 of SEQ ID NO: 119 would correspond to the amino acid residue at position 10 of SEQ ID NO: 105 (amino acid D / aspartic acid).

[0085] 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.

[0086] The term "recombinant," as used herein, typically refers to a nucleic acid construct, vector, polypeptide, or cell that has been produced by genetic engineering.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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).

[0091] 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.

[0092] 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.

[0093] Suitable methods for determining or measuring the esterase / hydrolase activity of a polypeptide will be familiar to those of skill in the art, and illustrative examples are described elsewhere herein. Other illustrative examples are described in Palm et al. (2019, Nat. Comm., 10:1717), Sagong et al. (2020, ACS Catal. 10:4805), and Yoshida et al. (2016, Science, 352(6278):1196), the contents of which are incorporated herein by reference in their entireties. Another useful method for determining or measuring the esterase / hydrolase activity of a polypeptide is by measuring the amount of terephthalic acid or terephthalic acid monoester produced using analytical high-performance liquid chromatography (HPLC).

[0094] The esterase activity of novel engineered polypeptides and variants having esterase activity can be assigned absolute values ​​or values ​​relative to the esterase activity of a comparative standard. In embodiments, esterase activity is measured as the rate of monomer and / or oligomer (e.g., in mg or mol) released per hour and per mg or mol of enzyme under suitable conditions of temperature, pH, and buffer. In embodiments of the invention, the rate of monomer and / or oligomer released per hour is in the range of about 1 mol / h / mol enzyme to about 500 mol / h / mol enzyme. In another embodiment, the rate of monomer and / or oligomer released per hour (e.g., in mg) is in the range of about 50 mol / h / mol enzyme to about 400 mol / h / mol enzyme. In yet another embodiment, the rate of monomer and / or oligomer released per hour (e.g., in mg) is in the range of about 100 mol / h / mol enzyme to about 350 mol / h / mol enzyme.

[0095] Advantageously, the polypeptides described herein may be capable of catalyzing the hydrolysis of mono- and di-terephthalate esters within a temperature range of at least about 10° C. to about 80° C., about 20° C. to about 80° C., about 30° C. to about 80° C., about 30° C. to about 70° C., preferably about 40° C. to about 70° C., preferably about 50° C. to about 70° C., or even more preferably about 50° C. to about 60° C. In embodiments, the polypeptides described herein exhibit esterase activity at temperatures of about 10° C. to about 80° C., about 20° C. to about 80° C., about 30° C. to about 80° C., about 30° C. to about 70° C., preferably about 40° C. to about 70° C., preferably about 50° C. to about 70° C., or even more preferably about 50° C. to about 60° C. In embodiments, esterase activity can be measured at temperatures of about 10°C to about 70°C, about 20°C to about 80°C, about 30°C to about 80°C, about 30°C to about 70°C, preferably about 40°C to about 70°C, preferably about 50°C to about 70°C, or even more preferably about 50°C to about 60°C.

[0096] In embodiments, the polypeptide having esterase activity comprises hydrolase activity or catalyzes the hydrolysis of monoterephthalate esters at a temperature of about 10°C to about 80°C, about 20°C to about 80°C, about 30°C to about 80°C, about 30°C to about 70°C, preferably about 40°C to about 70°C, preferably about 50°C to about 70°C, or even more preferably about 50°C to about 60°C.

[0097] In another specific embodiment, the polypeptide having esterase activity should catalyze the hydrolysis of diterephthalate esters at temperatures between about 10°C and about 80°C, between about 20°C and about 80°C, between about 30°C and about 80°C, between about 30°C and about 70°C, preferably between about 40°C and about 70°C, preferably between about 50°C and about 70°C, or even more preferably between about 50°C and about 60°C.

[0098] In embodiments, the polypeptides described herein exhibit measurable hydrolase / esterase activity at least in the pH range of about 5 to about 11, preferably in the pH range of about 6 to about 10, preferably in the pH range of about 7 to about 10, and more preferably in the pH range of about 7.5 to about 9.5.

[0099] In embodiments, the polypeptides described herein exhibit measurable hydrolase / esterase activity in catalyzing the hydrolysis of monoterephthalic acid esters at least in a pH range of about 5 to about 11, preferably in a pH range of about 6 to about 10, preferably in a pH range of about 7 to about 10, and more preferably in a pH range of about 7.5 to about 9.5.

[0100] In embodiments, the polypeptides described herein exhibit measurable hydrolase / esterase activity in catalyzing the hydrolysis of diterephthalate esters at least in the pH range of about 5 to about 11, preferably in the pH range of about 6 to about 10, preferably in the pH range of about 7 to about 10, and more preferably in the pH range of about 7.5 to about 9.5.

[0101] In embodiments, the polypeptides described herein exhibit a melting temperature (Tm) of about 40°C to about 90°C, 50°C to about 90°C, preferably about 50°C to about 80°C.

[0102] 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, including, for example, modified linkages, modified purine or pyrimidine bases, or modified sugars. Nucleic acids of the invention can be in isolated or purified form and can 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 can also be synthesized in vitro by well-known chemical synthesis techniques, for example, as described in Belousov (1997) Nucleic Acids Res. 25:3440-3444.

[0103] 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).

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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.

[0110] 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.

[0111] 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 expression of the polypeptide expressed therein and / or the PETase activity of the host cell. For example, the polypeptides described herein can be used to complement a wild-type strain of a fungus or bacterium known to be capable of PETase activity in order to improve and / or increase the PETase activity of that strain.

[0112] The present disclosure also provides (a) providing a polynucleotide as described herein; (b) expressing the polynucleotide in a host cell to thereby produce a polypeptide; and (c) collecting the polypeptide produced in (b) from the host cell culture. The present invention also extends to a method for producing a polypeptide having esterase activity, comprising:

[0113] 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.

[0114] 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.

[0115] The host cells can be cultured in a nutrient medium suitable for production of the polypeptide using methods that will be known to those skilled in the art. Suitable examples include culturing the host cells in shake flask cultures or by small- or large-scale fermentation (including continuous, batch, fed-batch, or solid-state fermentation) in laboratory or industrial fermentors 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 catalogs of the American Type Culture Collection), or any other culture medium suitable for cell growth.

[0116] If the polypeptide is secreted into the nutrient medium, it can be recovered directly from the culture supernatant. Conversely, it can be recovered from cell lysates 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 polypeptide 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 a substantially pure polypeptide.

[0117] The polypeptides can be used in purified form, either alone or in combination with additional enzymes (e.g., PETase or MHETase, or carboxylesterase or cutinase with PETase activity), 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, for example, beads, columns, plates, etc.

[0118] It may be convenient to carry out the method of the invention with a polypeptide that is immobilized on a substrate. When carrying out the method of the invention in a semi-continuous or continuous mode, it may be advantageous to use an immobilized polypeptide.

[0119] In one embodiment, the polypeptides described herein are immobilized on a substrate.

[0120] 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.

[0121] In one embodiment, the polypeptide is immobilized on a resin. In one embodiment, the polypeptide is immobilized on 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. 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.

[0122] Suitable ion exchange resins for immobilizing polypeptides will generally comprise a polymer matrix or a polymer / ceramic hybrid matrix, examples of which include, but are not limited to, CM ceramic HyperD® ion exchange chromatography resins.

[0123] In one embodiment, the ion exchange resin is a cation exchange resin. For operation of the methods according to the invention, the polypeptide will typically be immobilized on a support resin and loaded onto a column.

[0124] The present disclosure also extends to compositions comprising the polypeptides, nucleic acids or host cells described herein.

[0125] 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.

[0126] 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.

[0127] The compositions described herein can further include additional polypeptides that exhibit broad PETase / MHETase activity as well as enzymatic activity that is not limited to PETase, esterase, carboxylesterase, MHETase, or cutinase.

[0128] In embodiments, the polypeptides described herein are 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.

[0129] In embodiments, the composition comprises at least one host cell expressing a polypeptide described herein, or an extract thereof. "Cell extract" refers to any fraction obtained from a cell, such as a cell supernatant, cell debris, cell wall, DNA extract, enzyme or enzyme preparation, or any preparation 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.

[0130] As described elsewhere herein, the inventors have surprisingly found that the polypeptides described herein (genetically engineered polypeptides and variants thereof) have greater esterase activity for hydrolyzing mono- and di-terephthalic acid esters when compared to existing PETases and cutinases with PETase activity. Accordingly, disclosed herein are methods for hydrolyzing mono- and di-terephthalic acid esters, comprising exposing the terephthalic acid ester 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; the terephthalic acid diester to terephthalic acid monoester and alcohol; or the terephthalic acid diester to terephthalic acid and alcohol. The present disclosure also extends to a method for degrading plastic articles comprising polyester, comprising exposing the plastic article to a polypeptide, composition, or host cell described herein.

[0131] The present disclosure extends to the use of the polypeptides, compositions, or host cells described herein in processes for degrading polyesters under aerobic or anaerobic conditions, and / or for regenerating polyester-containing materials, such as plastic products made from or containing polyesters, and / or for producing biodegradable plastic products containing polyesters. Such methods and uses are particularly useful for degrading plastic products, including PET.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] Advantageously, the degradation process is carried out at a temperature of about 10°C to about 80°C, preferably about 20°C to about 80°C, preferably about 30°C to about 80°C, preferably about 40°C to about 80°C, preferably about 50°C to about 80°C, more preferably about 60°C to about 80°C, even more preferably about 60°C to about 70°C, and even more preferably about 60°C. As one of skill in the art will appreciate, the temperature is typically maintained at an activation temperature, which corresponds to the temperature at which the polypeptide is active and / or the recombinant microorganism synthesizes, produces, or releases the polypeptides 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 80° C., preferably about 20° C. to about 80° C., preferably about 30° C. to about 80° C., preferably about 40° C. to about 80° C., preferably about 50° C. to about 80° C., more preferably about 60° C. to about 80° C., even more preferably about 60° C. to about 70° C., and even more preferably about 60° C. The process or method may be suitably carried out in a continuous manner, at a temperature that allows the polypeptide to be used and / or regenerated several times.

[0136] Advantageously, the degradation process or method is carried out at a pH in the range of about 5 to about 11, preferably in the range of about 6 to about 10, preferably in the range of about 6.5 to about 9, preferably in the range of about 7 to about 9, preferably in the range of about 7 to about 8, preferably at a pH of about 9.5 to about 11.

[0137] In embodiments, the polyester-containing material can be pretreated prior to contact with the polypeptide to physically or chemically alter its structure to improve access of the polyester and its intermediates to the enzyme.

[0138] 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.

[0139] 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.

[0140] 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.

[0141] The present disclosure also extends to a composition comprising a plastic compound and a polypeptide, and / or a host cell expressing said polypeptide or an extract thereof containing said polypeptide.

[0142] The present disclosure also extends to masterbatch compositions comprising polypeptides, compositions and / or host cells expressing said polypeptides or extracts thereof containing said polypeptides.

[0143] Advantageously, such plastic compounds or masterbatch compositions described herein can be used for the production of polyester-containing materials.

[0144] In an embodiment, the resulting plastic compound or masterbatch composition is a biodegradable plastic compound or masterbatch composition 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.

[0145] Advantageously, the degradation process of the polyester-containing material (i.e., plastic compound or masterbatch composition or plastic product) is carried out at a temperature of about 10°C to about 80°C, preferably about 20°C to about 80°C, preferably about 30°C to about 80°C, preferably about 40°C to about 80°C, preferably about 50°C to about 80°C, more preferably about 60°C to about 80°C, even more preferably about 60°C to about 70°C, and even more preferably about 60°C ± 5°C.

[0146] Alternatively, the degradation process of the polyester-containing material (ie, plastic compound or masterbatch composition or plastic article) is carried out at a temperature of about 50°C to about 70°C, more preferably at 60°C ± 5°C.

[0147] The engineered polypeptides with esterase activity disclosed herein are suitable for a range of applications, including 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 engineered polypeptides with esterase activity disclosed herein can be utilized in textile processing or textile manufacturing, where they can be used as exoesterases to suitably alter the properties of textile fibers.

[0148] 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]

[0149] Example 1: Ancestral sequence reconstruction All amino acid sequences belonging to the protein families PF12695, PF01738, and PF12740 were extracted from pfam. Redundancies were removed to 100% sequence identity using CD-HIT (Fu et al., 2012), and all-vs-all pBLAST was performed. The resulting table was visualized as a sequence similarity network in Cytoscape (Shannon et al., 2003). Edges connecting sequences with less than 40% sequence identity were deleted, and the network was then visualized as a prefuse-force directed network graph. All sequences belonging to the sequence cluster containing LCC, TfCut2, and IsPETase were extracted from uniprot and redundancies were removed to 95% sequence identity using CD-HIT. Signal peptides for Gram-positive and Gram-negative bacteria were identified using SignalP5.0 (Almagro Armenteros et al., 2019) and manually removed. A multiple sequence alignment of this dataset was generated using the GINSI protocol in MAFFT (Katoh and Standley 2013), which was manually edited according to the solved crystal structures. The final alignment had 397 aligned sequences.

[0150] One hundred independent iterations of maximum likelihood (ML) tree searches and phylogenetic reconstructions were performed in IQ-TREE (Minh et al., 2020) using default tree search parameters. The best-fitting sequence evolution model (WAG+F+R8) (Whelan and Goldman 2001) was identified using Akaike and Bayesian information criteria in ModelFinder (Kalyaanamoorthy et al., 2017). Branch support for each estimate was calculated using alternative likelihood ratio test statistics (Anisimova and Gascuel 2006) and the ultrafast bootstrap approximation (ufboot) (Hoang et al., 2018) for up to 1000 iterations. Because approximately unbiased (AU) (Shimodaira 2002) tests performed up to 10,000 iterations failed to reject any single topology as statistically less likely than others, 20 convergent topologies that represented most of the topological diversity within the full dataset were sampled for ASR. Ancestral sequences were reconstructed across the 20 topologies by ML in CodeML from the PAML (Yang 2007) suite using the sequence evolution model WAG+G4. Eighteen conserved insertions identified in extant sequences were treated as distinct binary properties (1 is the presence of an insertion, 0 is the absence of an insertion) and reconstructed by ML in the R package Ape (Paradis, Claude, and Strimmer 2004) using a two-way Jukes-Cantor-like equal-ratio model. Forty-eight reconstructed ancestral sequences were sampled from common nodes shared between LCC and TfCut2 across 20 topologies that had a minimum ufboot support of 0.9 and a minimum average posterior probability of 0.8.

[0151] Example 2: Protein expression and purification Plasmids were transformed into chemically competent E. cloni® cells (Lucigen) by heat shock, plated onto Lysogeny broth (LB) agar supplemented with 100 μg / L kanamycin, and incubated overnight at 37° C. A single colony was used to inoculate 1.5 mL of autoinduction medium supplemented with 100 μg / mL kanamycin in a 2.2 mL 96-well deep-well block and grown at 1050 rpm at 37° C. for 5 hours, followed by 16 hours at room temperature (RT; 25° C.).

[0152] Cells were harvested by centrifugation at 2000 x g for 15 min at RT and resuspended in lysis buffer (1 x BugBuster® Protein Extraction Reagent (Merck-Millipore), 20 mM Tris, 300 mM NaCl, 1 U / mL Turbonuclease (Sigma) pH 8). The cell suspension was incubated for 20 min at RT with gentle shaking. The lysate was separated from insoluble cell debris by centrifugation at 2250 x g for 1 h at RT. The cleared lysate was then diluted with 100 μL of equilibration buffer (20 mM Tris, 300 mM NaCl pH 8) and purified by nickel-loaded IMAC using a 96-well HisPur™ Ni-NTA spin plate (ThermoFisher Scientific) equilibrated in equilibration buffer. The sample was washed three times with 250 μL of wash buffer (20 mM Tris, 300 mM NaCl, 10 mM imidazole pH 8) and eluted with 250 μL of elution buffer (20 mM Tris, 300 mM NaCl, 150 mM imidazole pH 8). All centrifugation steps after the addition of wash or elution buffer were at RT for 1 min at 1000 × g. The eluate was stored at 4°C.

[0153] Example 3: HPLC activity assay to detect hydrolysis of MOCT and DOCT Assays for enzyme activity against monooctyl terephthalate (MOCT) or dioctyl terephthalate (DOCT) were performed using 1.5 mM substrate in reaction buffer (45 mM NaHPO, 90 mM NaCl, pH 7.5), 5% DMSO, and a 1:10 dilution of the eluate from the Ni-NTA purification.

[0154] Reactions were incubated at 50° C. for 64 minutes and then quenched by heating for at least 10 minutes at 95° C. Reactions were analyzed using high performance liquid chromatography (HPLC) and compared to control reactions containing no enzyme.

[0155] The concentrations of terephthalic acid and monooctyl terephthalate were determined by comparison with calibration curves generated using synthetic or commercially available standards.

[0156] The absorbance at 254 nm was used to compare the activity of all variants. The retention time of TPA for the HPLC method and the corresponding calibration curve were obtained using a TPA standard (Sigma-Aldrich). To characterize the MOCT and DOCT hydrolysis activity of selected variants, the peak corresponding to TPA was integrated and the peak area was obtained based on comparison with the standard sample. The selected variants exhibiting MOCT and DOCT hydrolysis activity are shown in Table 1.

[0157] Polypeptides having SEQ ID NOs: 32 and 34 appear to have higher activity toward DOCT than toward MOCT, while SEQ ID NOs: 44-54 appear to have significantly higher activity toward MOCT than toward DOCT. SEQ ID NOs: 6-8 demonstrated efficient hydrolysis of DOCT, and SEQ ID NOs: 9-11 demonstrated efficient hydrolysis of DOCT (see Tables 2-3).

[0158] Analysis of SEQ ID NOs: 1-118, which demonstrate activity in hydrolyzing MOCT and DOCT, showed that each of them falls within the consensus sequence—SEQ ID NO: 119.

[0159] Table 1A

[0160] Table 1B

[0161] Table 1C

[0162] Table 1D

[0163]

Table 1E

[0164]

Table 1F

[0165] Table 1G

[0166]

Table 1H

[0167]

Table 1I

[0168] Table 1J

[0169] Table 1K

[0170] Table 1L

[0171] Table 1M

[0172]

Table 1N

[0173]

Table 1O

[0174]

Table 1P

[0175]

Table 1Q

[0176]

Table 1R

[0177]

Table 1S

[0178]

Table 1T

[0179]

Table 1U

[0180] [Table 2]

[0181] [Table 3]

[0182] The disclosures of all patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety.

[0183] 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.

[0184] 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.

[0185] [Table 4A] [Table 4B] [Table 4C] [Table 4D] [Table 4E]

Claims

1. A polypeptide having esterase activity, the esterase activity being a. terephthalic acid monoester to terephthalic acid and alcohol; b. terephthalic acid diester to terephthalic acid monoester and alcohol; or c. Terephthalic acid diester to terephthalic acid and alcohol It is possible to convert The polypeptide is i. amino acids 5 to 261 of SEQ ID NO: 2, or an amino acid sequence having at least 85% sequence identity thereto; ii. amino acids 5 to 261 of SEQ ID NO: 3, or an amino acid sequence having at least 77% sequence identity thereto; iii. amino acids 5 to 261 of SEQ ID NO: 4, or an amino acid sequence having at least 75% sequence identity thereto; iv. amino acids 5 to 261 of SEQ ID NO: 5, or an amino acid sequence having at least 95% sequence identity thereto; and v. Amino acids 5 to 261 of SEQ ID NO: 6 or an amino acid sequence having at least 96% sequence identity thereto and comprising an amino acid sequence selected from the group consisting of: A polypeptide, wherein the polypeptide is not SEQ ID NO: 1 or SEQ ID NO:

12.

2. 2. The polypeptide of claim 1, comprising the amino acid sequence of amino acids 5 to 261 of SEQ ID NO: 6, or an amino acid sequence having at least 96% sequence identity thereto.

3. 3. The polypeptide of claim 2, comprising the amino acid sequence of amino acids 5 to 261 of SEQ ID NO:

6.

4. 2. The polypeptide of claim 1, comprising the amino acid sequence of amino acids 5 to 261 of SEQ ID NO: 7, or an amino acid sequence having at least 97% identity thereto.

5. 5. The polypeptide of claim 4, comprising the amino acid sequence of amino acids 5 to 261 of SEQ ID NO:

7.

6. 2. The polypeptide of claim 1, comprising the amino acid sequence of amino acids 5 to 261 of SEQ ID NO: 8, or an amino acid sequence having at least 96% identity thereto.

7. The polypeptide of claim 6, comprising the amino acid sequence of amino acids 5 to 261 of SEQ ID NO:

8.

8. 2. The polypeptide of claim 1, comprising the amino acid sequence of amino acids 5 to 261 of SEQ ID NO: 9, or an amino acid sequence having at least 97% identity thereto.

9. 9. The polypeptide of claim 8, comprising the amino acid sequence of amino acids 5 to 261 of SEQ ID NO:

9.

10. 2. The polypeptide of claim 1, comprising the amino acid sequence of amino acids 5 to 261 of SEQ ID NO: 10, or an amino acid sequence having at least 98% identity thereto.

11. 11. The polypeptide of claim 10, comprising the amino acid sequence of amino acids 5 to 261 of SEQ ID NO:

10.

12. 2. The polypeptide of claim 1, comprising the amino acid sequence of amino acids 5 to 261 of SEQ ID NO: 11, or an amino acid sequence having at least 98% identity thereto.

13. 13. The polypeptide of claim 12, comprising the amino acid sequence of amino acids 5 to 261 of SEQ ID NO:

11.

14. 14. The polypeptide of any one of claims 1 to 13, comprising the amino acid sequence of SEQ ID NO:

119.

15. 1. A process for the enzymatic hydrolysis of terephthalic acid monoesters and / or terephthalic acid diesters produced as by-products of the degradation of PET, comprising: a) The terephthalic acid monoester is optionally benzyl substituted mono-C 1 ~C 10 alkyl terephthalate, and not terephthalic acid monoester mono-(2-hydroxyethyl) terephthalate (MHET); b) The terephthalic acid diester is optionally benzyl substituted di-C 1 ~C 10 alkyl terephthalate; a terephthalic acid monoester and / or a terephthalic acid diester being reacted with a polypeptide having esterase activity, i. terephthalic acid monoester to terephthalic acid and alcohol; ii. terephthalic acid diester to terephthalic acid monoester and alcohol; or iii. Terephthalic acid diester to terephthalic acid and alcohol exposing under conditions sufficient to allow the transformation.

16. 16. The method of claim 15, wherein the polypeptide comprises the amino acid sequence of amino acids 5 to 261 of SEQ ID NO: 1 or an amino acid sequence having at least 70% sequence identity thereto.

17. Mono-C terephthalic acid monoester optionally substituted with benzyl 6 ~C 10 17. The method according to claim 15 or 16, wherein the alkyl terephthalate is an alkyl terephthalate.

18. 18. The method of claim 17, wherein the terephthalic acid monoester is selected from the group consisting of monobenzyl terephthalate (MBZT), monohexyl terephthalate, monoheptyl terephthalate (MHPT), and monooctyl terephthalate (MOCT).

19. 19. The method of claim 18, wherein the terephthalic acid monoester is MBZT or MOCT.

20. 20. The method of any one of claims 17 to 19, wherein the polypeptide comprises the amino acid sequence of amino acids 5 to 261 of any one of SEQ ID NOs: 9 to 11, or an amino acid sequence having at least 70% sequence identity to any of the foregoing.

21. 20. The method of any one of claims 17 to 19, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

119.

22. Di-C terephthalic acid diesters optionally substituted with benzyl 6 ~C 10 17. The method according to claim 15 or 16, wherein the alkyl terephthalate is an alkyl terephthalate.

23. 23. The method of claim 22, wherein the terephthalic acid diester is selected from the group consisting of dibenzyl terephthalate (DBZT), dihexyl terephthalate (DHXT), diheptyl terephthalate (DHPT), and dioctyl terephthalate (DOCT).

24. 24. The method of claim 23, wherein the terephthalic acid diester is DBZT or DOCT.

25. 25. The method of any one of claims 22 to 24, wherein the polypeptide comprises the amino acid sequence of amino acids 5 to 261 of any one of SEQ ID NOs: 6 to 8, or an amino acid sequence having at least 70% sequence identity to any of the foregoing.

26. The terephthalic acid monoester and / or the terephthalic acid diester are a. exposing the PET to sodium hydroxide; and / or b. contacting the esterase with the PET 26. The method of any one of claims 15 to 25, wherein the compound is produced by a method comprising:

27. The terephthalic acid monoester and / or the terephthalic acid diester are cC 1 ~C 10 subjecting the PET to a base-catalyzed transesterification with a monoalcohol; and / or d. contacting the esterase with the PET 27. The method of claim 26, wherein the compound is produced by a method comprising:

28. C 1 ~C 10 Monoalcohol is C 6 ~C 10 28. The method of claim 27, wherein the alcohol is a monoalcohol.

29. C 6 ~C 10 29. The method of claim 28, wherein the monoalcohol is benzyl alcohol, octanol, or heptanol.

30. C 6 ~C 10 30. The method of claim 29, wherein the monoalcohol is 1-octanol.

31. 31. The method of any one of claims 15 to 30, further comprising recovering terephthalic acid and / or alcohol.

32. 32. A composition comprising terephthalic acid and / or alcohol recovered by the method of claim 31.

33. A composition comprising a polypeptide according to any one of claims 1 to 14.

34. 15. A polynucleotide comprising a nucleic acid sequence encoding the polypeptide of any one of claims 1 to 14.

35. 35. An expression cassette or vector comprising the polynucleotide of claim 34.

36. a) a polypeptide according to any one of claims 1 to 14, b) a polynucleotide according to claim 34, or c) an expression cassette or vector according to claim 35 A host cell comprising:

37. 15. A host cell genetically modified to express a polypeptide according to any one of claims 1 to 14.

38. a) providing a polynucleotide according to claim 34; b) expressing the polynucleotide in the host cell under conditions sufficient to allow the host cell to produce the polypeptide; and c) harvesting the polypeptide produced by the host cell in (b). A method for producing a polypeptide having esterase activity, comprising:

39. a) decomposing PET by base-catalyzed transesterification to produce terephthalic acid monoesters and / or terephthalic acid diesters; and b) combining a terephthalic acid monoester and / or a terephthalic acid diester with a polypeptide according to any one of claims 1 to 13, a composition according to claim 33 or a host cell according to claim 36 or 37, wherein the polypeptide i. terephthalic acid monoester to terephthalic acid and alcohol; ii. terephthalic acid diester to terephthalic acid monoester and alcohol; or iii. Terephthalic acid diester to terephthalic acid and alcohol contacting under conditions sufficient to allow the conversion A method for recycling plastic products containing PET, including:

40. 40. The method of claim 39, wherein the step of degrading PET further comprises simultaneously or sequentially exposing PET to a PETase and / or a cutinase having PETase activity.

41. 35. The method of claim 33 or 34, wherein step b) further comprises simultaneously or sequentially exposing the terephthalic acid monoester and / or terephthalic acid diester to an MHETase or an enzyme having MHETase activity.

42. A polypeptide having esterase activity, the esterase activity being a. terephthalic acid monoester to terephthalic acid and alcohol; b. terephthalic acid diester to terephthalic acid monoester and alcohol; or c. Terephthalic acid diester to terephthalic acid and alcohol It is possible to convert; A polypeptide comprising the amino acid sequence of SEQ ID NO: 119.

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