Methods for depolymerising and repolymerising

Abstract

The present invention relates to a method for the formation of a mono or di-ester of terephthalic acid from polyethylene terephthalate (PET) using a hydrolase enzyme in the presence of at least one C1-C6 alkyl alcohol, and further relates to a method for re-polymerisation to form PET.

Description

[0001] METHODS FOR DEPOLYMERISING AND REPOLYMERISING

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the field of polymer recycling, particularly to the field of polyethylene terephthalate (PET) recycling.

[0004] BACKGROUND TO THE INVENTION

[0005] The recycling of polymers, and the recovery of components that make up such polymers (e.g. monomers), has become of increasing importance and complexity.

[0006] PET is the most common thermoplastic polymer resin of the polyester family and is used in fibres for clothing, containers for liquids and foods, and in thermoforming processes such as moulding 3D printing, as well as in combination with glass fibre for engineering resins. Recycling of PET to recover the components that make it up ( / .e. oligomers or monomers), generates considerable amounts of waste and results in poor yield of said component. The recycling of PET products also consumes considerable amounts of energy due to the high activation energy associated with breaking and reforming each ester bond comprised therein.

[0007] It is an aim of the present invention to provide a method for the depolymerisation of PET, as well as a method for re-polymerising PET using the components recovered during said depolymerisation method, wherein each method is energy efficient, rapid, safe, cheap, and of improved yield. It is a particular aim of the present invention to provide a method for the depolymerisation of PET which allows energy efficient, rapid and safe separation of unreacted starting materials from the components recovered during said depolymerisation method. It is another particular aim of the present invention to provide a method for re-polymerising PET using the components recovered during said depolymerisation method which allows energy efficient, rapid and safe separation of said re-polymerised PET from any by-products.

[0008] BRIEF DESCRIPTION OF THE INVENTION

[0009] The present invention relates to a method for formation of a diester of terephthalic acid from polyethylene terephthalate (PET) using a hydrolase enzyme in the presence of at least one alcohol selected from the group consisting of: a Ci-Ce alkyl alcohol and a Ci-Ce alkylene diol.

[0010] The present invention further relates to a method for formation of a diester of terephthalic acid from polyethylene terephthalate (PET) using a hydrolase enzyme in the presence of at least one alcohol selected from the group consisting of Ci-Ce alkyl alcohols; particularly ethanol and methanol. The use of a Ci-Ce alkyl alcohol as the alcohol starting material in the method for formation of a diester of terephthalic acid from PET renders it less energy intensive, faster and safer to separate the diester and / or monoester from any unreacted alcohol than when the alcohol is a Ci-Ce alkylene diol. As a result, when an excess of said alcohol is employed, it is less energy intensive to use a Ci- Ce alkyl alcohol as opposed to a Ci-Ce alkylene diol.

[0011] The present invention also relates to a method for re-polymerisation of PET, wherein the method comprises the following steps:

[0012] (i) forming a diester of terephthalic acid from said PET using a hydrolase enzyme in the presence of at least one alcohol selected from the group consisting of: a Ci-Ce alkyl alcohol and a Ci-Ce alkylene diol;

[0013] (ii) isolating the diester resulting from step (i); and

[0014] (iii) polymerising the diester with ethylene glycol or bis(2-hydroxyethyl)terephthalate to form new PET.

[0015] The present invention also relates to a method for re-polymerisation of PET, wherein the method comprises the following steps:

[0016] (i) forming a diester of terephthalic acid from said PET using a hydrolase enzyme in the presence of at least one alcohol selected from the group consisting of a Ci-Ce alkyl alcohols; preferably methanol or ethanol;

[0017] (ii) isolating the diester resulting from step (i); and

[0018] (iii) polymerising the diester with ethylene glycol or bis(2-hydroxyethyl)terephthalate to form PET.

[0019] In addition, the present invention also relates to a method for formation of a monoester of terephthalic acid from PET using a hydrolase enzyme in the presence of at least one alcohol selected from the group consisting of: a Ci-Ce alkyl alcohol and a Ci-Ce alkylene diol, preferably methanol, ethanol or ethylene glycol;

[0020] The present invention also relates to a method for formation of a monoester of terephthalic acid from PET using a hydrolase enzyme in the presence of at least one alcohol selected from the group consisting of Ci-Ce alkyl alcohols; preferably methanol or ethanol.

[0021] The present invention also relates to a method for formation of a diester of terephthalic acid from PET using a hydrolase enzyme in the presence of at least one Ci-Ce alkyl alcohol, preferably methanol or ethanol; wherein said hydrolase enzyme is an enzyme selected from the group consisting of: a carboxylesterase, a lipase such as triacylglycerol lipase, a cutinase, an acetylxylan esterase and a carboxymethylenebutenolidase. The present invention also relates to a method for formation of a diester of terephthalic acid from PET using a hydrolase enzyme selected from the group consisting of: a carboxylesterase (EC 3.1 .1 .1); a triacylglycerol lipase (EC 3.1 .1 .3); an acetylxylan esterase (EC 3.1 .1 .72); a cutinase (EC 3.1 .1 .74); and a carboxymethylenebutenolidase (EC 3.1 .1 .45), in the presence of at least one alcohol selected from the group consisting of: methanol, ethanol and ethylene glycol, preferably wherein said PET from which a diester of terephthalic acid is formed is a treated PET obtained by subjecting a first PET to at least one of a thermomechanical treatment and a chemical treatment, wherein said treated PET has lower crystallinity and greater surface area than said first PET.

[0022] The present invention also relates to a method for formation of a diester of terephthalic acid from PET using a hydrolase enzyme selected from the group consisting of: a carboxylesterase (EC 3.1.1.1); a lipase (EC 3.1.1 .3) such as triacylglycerol lipase; an acetylxylan esterase (EC 3.1.1.72); a cutinase (EC 3.1.1.74); and a carboxymethylenebutenolidase (EC 3.1.1 .45), in the presence of at least one alcohol selected from methanol and ethanol, preferably wherein the PET from which a diester of terephthalic acid is formed is a treated PET obtained by subjecting a first PET to at least one of a mechanical treatment, thermomechanical treatment and chemical treatment; and wherein the treated PET has lower crystallinity and greater surface area than the first PET.

[0023] Furthermore, the present invention also relates to a method for re-polymerisation of PET, wherein the method comprises the following steps:

[0024] (i) forming a diester of terephthalic acid from said PET using a hydrolase enzyme selected from the group consisting of: a carboxylesterase (EC 3.1.1.1); a triacylglycerol lipase (EC 3.1.1.3); an acetylxylan esterase (EC 3.1.1.72); a cutinase (EC 3.1.1.74); and a carboxymethylenebutenolidase (EC 3.1.1.45), in the presence of at least one alcohol selected from the group consisting of: a Ci-Ce alkyl alcohol and a Ci-Ce alkylene diol, preferably methanol, ethanol or ethylene glycol;

[0025] (ii) isolating the diester resulting from step (i); and

[0026] (iii) polymerising the diester with ethylene glycol or bis(2-hydroxyethyl)terephthalate to form new PET, preferably wherein said PET from which a diester of terephthalic acid is formed in step (i) is a treated PET obtained by subjecting a first PET to at least one of a thermomechanical treatment and a chemical treatment, wherein said treated PET has lower crystallinity and greater surface area than said first PET.

[0027] Furthermore, the present invention also relates to a method for re-polymerisation of PET, wherein the method comprises the following steps:

[0028] (i) forming a diester of terephthalic acid from said PET using a hydrolase enzyme selected from the group consisting of: a carboxylesterase (EC 3.1 .1.1); a lipase (EC 3.1.1.3) such as a triacylglycerol lipase; an acetylxylan esterase (EC 3.1.1.72); a cutinase (EC 3.1.1.74); and a carboxymethylenebutenolidase (EC 3.1.1.45), in the presence of at least one alcohol selected from the group consisting of: a Ci-Ce alkyl alcohols, preferably methanol or ethanol; (ii) isolating the diester resulting from step (i); and

[0029] (iii) polymerising the diester with ethylene glycol or bis(2-hydroxyethyl)terephthalate to form PET, preferably wherein said PET from which a diester of terephthalic acid is formed in step (i) is a treated PET obtained by subjecting a first PET to at least one of a thermomechanical treatment and a chemical treatment, wherein said treated PET has lower crystallinity and greater surface area than said first PET.

[0030] DETAILED DESCRIPTION OF THE INVENTION

[0031] The present invention relates to a method for formation of a diester of terephthalic acid (TPA) from PET, as well as to a method for re-polymerisation of PET. The method for re-polymerisation of PET includes the method for formation of a diester of TPA from PET as step (i).

[0032] Each method involves formation of a diester of terephthalic acid from said PET. However, also described herein is a method for formation of a monoester of terephthalic acid from PET using a hydrolase enzyme in the presence of at least one alcohol selected from the group consisting of: a Ci-Ce alkyl alcohol and a Ci-Ce alkylene diol. Each method also involves formation of a diester of terephthalic acid from said PET. However, also described herein is a method for formation of a monoester of terephthalic acid from PET using a hydrolase enzyme in the presence of at least one alcohol selected from the group consisting of a Ci-Ce alkyl alcohols.

[0033] Thus, the methods described herein may result in formation of said diester and / or said monoester and therefore may be described as involving transesterification of PET. In a preferred embodiment of each method of the present invention, said method of formation of a diester of terephthalic acid additionally forms a monoester of terephthalic acid. In a more preferred embodiment of each method of the present invention, said method forms less monoester than diester.

[0034] PET [also known as polyethylene terephthalate or polyethylene terephthalate)] comprises a polymer chain represented by the generic chemical formula (I): wherein the average value for n in the bulk polymer is >50, preferably >70, more preferably >100. Said polymer chain is a polyester ( / .e. said PET is a condensation product formed by polycondensation or condensative chain polymerization).

[0035] Said PET may be considered as comprised in a material. In non-limiting examples, said material may comprise at least one of a powder, pellets, block, rod, sheet, film, composite, fabric, textile, yarn, thread, fibre, filament or particle, or any combination thereof.

[0036] Said material may be a material composed of one substance or more than one substance.

[0037] When said material consists of one substance, it consists of said PET ( / .e substantially pure PET wherein >99% of the material is PET matrix). By way of example and without limitation, substantially pure PET carbonated drink bottles and substantially pure PET fabrics are each exemplary materials which comprise only said PET.

[0038] When said material comprises more than one substance, it comprises said PET in combination with at least one other substance. Each other substance may be independently selected from another polyester (e.g. a polyester comprised in a polyurethane block copolymer); a polymer other than a polyester (e.g. a nylon), a metal or alloy (e.g. steel; brass; or aluminium such as in aluminised biaxially oriented PET), a natural fibre (e.g. cellulose such as cotton; silk; mohair; or wool), a treated natural fibre (e.g. dyed cotton; or viscose), a composite (e.g. glass; or ceramic), and an adhesive (e.g. an epoxy polymer; a polyurethane; or an acrylic polymer).

[0039] When said PET is in combination with at least one other substance in said material, each other substance may be either associated chemically to said PET or associated physically with said PET.

[0040] When at least one other substance is associated chemically with said PET, it is either bonded directly to said PET (via a covalent bond to the PET chain) or bonded indirectly to said PET (via a covalent bond to a spacer moiety bound to the PET chain). By way of example and without limitation, copolymers such as a PET-PBT copolymer, glycol-modified PET (PETG), and a PET-polycarbonate copolymer are each exemplary materials which comprise PET in combination (chemical association) with at least one other substance.

[0041] When another substance is associated physically with said PET, it is either in physical contact with said PET or spatially separated from said PET. By way of example and without limitation, PET- polypropylene (PP) blends ( / .e. miscible blends of PET and PP); PET-polyamide laminated bottles (such as Nylon-MXD6 / PET laminated bottles); PET carpets; PET-GoreTex two-layer sandwich fabric; and blended PET-cotton yarn are each exemplary materials which comprise PET in combination (physical association) with at least one other substance. Said material may also be subjected to a treatment which increases its surface area : volume ratio prior to subjecting said PET to formation of a diester of TPA ( / .e. prior to subjecting said PET to a hydrolase enzyme). Such a treatment is preferably a mechanical treatment. Said mechanical treatment may, in some embodiments, involve cutting, shearing, ripping, tearing, abrasion, deformation, heating, or a combination thereof. Preferably, said treatment is achieved by shredding or granulation. In a preferred embodiment of the methods of the present invention, and, hence the products of the present invention which are formed at steps thereof, each method comprises an additional step prior to subjecting said PET to formation of a diester of TPA ( / .e. prior to subjecting said PET to a hydrolase enzyme or, where relevant, to an initial base-catalysed transesterification, as described herein) of shredding said material.

[0042] The PET which is used to form said diester and / or monoester of terephthalic acid may be treated PET.

[0043] Treated PET may be obtained by subjecting a first PET (such as that described herein) to at least one of a mechanical treatment, thermomechanical treatment and a chemical treatment, resulting in a PET which has lower crystallinity and / or greater surface area : volume ratio than said first PET. The treated PET can then be used in formation of a diester instead of first (untreated) PET. In a preferred embodiment of the present invention, the PET which is used to form said diester of terephthalic acid ( / .e. the PET from which a diester of terephthalic acid is formed) is a treated PET obtained by subjecting a first PET to at least one of a thermomechanical treatment and a chemical treatment, wherein said treated PET has lower crystallinity and greater surface area : volume ratio than said first PET. Thermomechanical treatment may be selected from, but is not limited to, the following: hot melt extrusion, foaming extrusion, or cryomilling. Chemical treatment may be selected from, but is not limited to, the following: detergent destabilization, swelling with solvent / ionic liquid, or dissolution with or without the addition of chemicals to disrupt crystallinity.

[0044] PET which has a lower crystallinity and / or greater surface area : volume ratio is more susceptible to attack by said hydrolase enzyme, thereby resulting in faster and higher-yielding formation of said diester and / or monoester. Thus, in some embodiments, the PET which is used to form said diester and / or monoester of terephthalic acid may be amorphous or semi-crystalline ( / .e. having a degree of crystallinity of < 40 %). In some embodiments, said PET is not amorphous ( / .e. has a degree of crystallinity greater than 0). Preferably, said PET has a degree of crystallinity of between 0.1 and 30 %. Even more preferably, said PET has a degree of crystallinity of between 1 and 25 %, still more preferably between 5 and 20 %. A degree of crystallinity, as defined herein, is measured according to differential scanning calorimetry using a TA Instruments Q2000 DSC configured with a Refrigerated Cooling System (RCS90) under a nitrogen atmosphere (50 mL I minute) at a heating rate of 10 °C per minute from -50 °C to 300 °C, with samples of 5 to 10 mg in a crimped aluminium pan. Treated PET may also be a PET which has been subjected to an initial transesterification reaction with a C1-C10 mono-alcohol. In other words, said treated PET may be an initial transesterification product which is subsequently used to form the aforementioned diester and / or monoester of terephthalic acid by transesterification using said hydrolase enzyme. Said initial transesterification may be an acid or base-catalysed transesterification using said C1-C10 alcohol. In an embodiment of the methods of the present invention, and hence the products of the present invention which are formed at steps thereof, the formation of a diester of terephthalic acid from said PET comprises: subjecting said PET to base-catalysed transesterification ( / .e. initial transesterification) with a C1-C10 alcohol to produce a composition comprising a corresponding initial transesterification product; and treating said initial transesterification product with said hydrolase enzyme in the presence of said at least one alcohol selected from the group consisting of: a Ci-Ce alkyl alcohol and a Ci-Ce alkylene diol, preferably the group consisting of Ci-Ce alkyl alcohols, more preferably methanol or ethanol; to form said diester of terephthalic acid.

[0045] The initial transesterification has the effect of further improving the yield of the diester of terephthalic acid that is formed using a hydrolase enzyme.

[0046] The initial transesterification product comprised in the composition produced by said base-catalysed transesterification may be an ester of terephthalic acid in which one carboxylic acid moiety thereof has esterified with a molecule of said C1-C10 alcohol, or an ester of terephthalic acid in which both carboxylic acid moieties thereof have each esterified with a molecule of said C1-C10 alcohol. The composition may comprise either or both of said initial transesterification products. Herein the ester(s) present in the composition produced by said base-catalysed initial transesterification are referred to as initial transesterification product(s) so as to distinguish them from the diester or monoester which are produced in the enzyme catalysed reaction.

[0047] The formation of the diester of terephthalic acid, and the formation of the monoester of terephthalic acid, is conducted in the presence of at least one alcohol. Said alcohol can be a Ci-Ce alkyl alcohol or a Ci-Ce alkylene diol, such as a Ci, C2, C3, C4, C5 or Ce alkyl alcohol or a Ci, C2, C3, C4, C5 or Ce alkylene diol, respectively; particularly a Ci, C2, C3, C4, C5 or Ce alkyl alcohol

[0048] Herein a Ci-Ce alkyl alcohol is defined as a monovalent saturated aliphatic alcohol having from 1 to 6 carbons. In some embodiments of the method of the present invention a Ci-Ce alkyl alcohol is a linear, branched and / or cyclic alkyl alcohol. Each Ci-Ce alkyl alcohol may be selected from the group consisting of: a Ci alkyl alcohol (methanol), a C2 alkyl alcohol (ethanol), a C3 alkyl alcohol (n-propyl, isopropyl or cyclopropyl alcohol), a C4 alkyl alcohol (n-butyl, sec-butyl, isobutyl, te / Y-butyl, cyclobutyl, cyclopropylmethyl, 2-methylcyclopropyl or 1 -methylcyclopropyl alcohol), a C5 alkyl alcohol (n-pentyl, 2-methylbutan-2-yl, 2,2-dimethylpropyl, 3-methylbutyl, pentan-2-yl, pentan-3-yl, 3-methylbutan-2-yl, 2-methylbutyl, cyclopentyl, cyclobutylmethyl, 1 -methylcyclobutyl, 2-methylcyclobutyl, cyclopropylethyl, 1 ,2-dimethylcyclopropyl, 2,2-dimethylcyclopropyl or 2,3-dimethylcyclopropyl alcohol), and a Ce alkyl alcohol [n-hexyl, hexan-2-yl, hexan-3-yl, 2-methylpentyl, 3-methylpentyl, 4- methylpentyl, 2-methylpentan-2-yl, 3-methylpentan-2-yl, 4-methylpentan-2-yl, 2-methylpentan-3-yl, 3-methylpentan-3-yl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 2,3-dimethylbutan-2-yl,

[0049] 3.3-dimethylbutan-2-yl, 2-ethylbutyl, cyclohexyl, cyclopentylmethyl, 1 -methylcyclopentyl, 2- methylcyclopentyl, 3-methylcyclopentyl, (2-cyclobutyl)ethyl, 1 -methylcyclobutylmethyl, 2- methylcyclobutylmethyl, 1 ,2-dimethylcyclobutyl, 1 ,3-dimethylcyclobutyl, 2,2-dimethylcyclobutyl, 2,3- dimethylcyclobutyl, 3,3-dimethylcyclobutyl, 1 -ethylcyclobutyl, 2-ethylcyclobutyl, 3-ethylcyclobutyl, 1- propylcyclopropyl, 1 -(1 -methylethyl)cyclopropyl, 2-propylcyclopropyl, 2-(1 -methylethyl)cyclopropyl,

[0050] 1-ethyl-2-methylcyclopropyl, 1-methyl-2-ethylcyclopropyl, 1-methyl-2,2-dimethylcyclopropyl, 1- methyl-2,3-dimethylcyclopropyl, (3-cyclopropyl)propyl, (2-cyclopropyl)propyl, (l-cyclopropyl)propan-

[0051] 2-yl, (2-cyclopropyl)propan-2-yl, (l-methylcyclopropyl)ethyl, (2-methylcyclopropyl)ethyl, 2-(1- methylcyclopropyl)ethyl 2-(2-methylcyclopropyl)ethyl, (l-ethylcyclopropyl)methyl, (2- ethylcyclopropyl)methyl, (1 ,2-dimethylcyclopropyl)methyl, (2,2-dimethylcyclopropyl)methyl or (2,3- dimethylcyclopropyl)methyl alcohol], and any L or R isomers thereof. Preferably each Ci-Ce alkyl alcohol is independently selected from the group consisting of: a Ci alkyl alcohol (methanol), a C2 alkyl alcohol (ethanol), a C3 alkyl alcohol (n-propyl, isopropyl alcohol), a C4 alkyl alcohol (n-butyl, secbutyl, isobutyl or fe / Y-butyl alcohol), a C5 alkyl alcohol (n-pentyl, 2-methylbutan-2-yl, 2,2- dimethylpropyl, 3-methylbutyl, pentan-2-yl, pentan-3-yl, 3-methylbutan-2-yl or 2-methylbutyl alcohol) and a Ce alkyl alcohol (n-hexyl, hexan-2-yl, hexan-3-yl, 2-methylpentyl, 3-methylpentyl, 4- methylpentyl, 2-methylpentan-2-yl, 3-methylpentan-2-yl, 4-methylpentan-2-yl, 2-methylpentan-3-yl,

[0052] 3-methylpentan-3-yl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 2,3-dimethylbutan-2-yl,

[0053] 3.3-dimethylbutan-2-yl or 2-ethylbutyl alcohol). More preferably, each Ci-Ce alkyl alcohol is independently selected from the group consisting of C1-4 alkyl alcohols. More preferably, each Ci-Ce alkyl alcohol is independently selected from the group consisting of: a Ci alkyl alcohol (methanol), a C2 alkyl alcohol (ethanol), a C3 alkyl alcohol (n-propyl or isopropyl alcohol) and a C4 alkyl alcohol (n- butyl, sec-butyl, isobutyl or fe / Y-butyl alcohol). Even more preferably, each Ci-Ce alkyl alcohol is a C1-C3 alkyl alcohol, wherein each C1-C3 alkyl alcohol is independently selected from the group consisting of: a Ci alkyl alcohol (methanol), a C2 alkyl alcohol (ethanol) and a C3 alkyl alcohol (n- propyl or isopropyl alcohol). In preferred embodiments of the present invention, each Ci-Ce alkyl and C1-C3 alkyl is independently selected from methanol or ethanol.

[0054] Herein a Ci-Ce alkylene diol is defined as a bivalent saturated aliphatic di-alcohol having from 1 to 6 carbons. In some embodiments of the method of the present invention a Ci-Ce alkylene diol is a linear, branched and / or cyclic diol. Thus, each Ci-Ce alkylene diol may be selected from the group consisting of: a Ci alkylene diol (methylene glycol), a C2 alkylene diol (ethylene glycol or ethane-1 ,1- diol), a C3 alkylene diol [propane-1 ,3-diol, propylene glycol (propane-1 ,2-diol), propane-2, 2-diol, propane-1 ,1-diol, cyclopropan-1 ,1-diol or cyclopropan-1 ,2-diol], a C4 alkylene diol (butane-1 ,4-diol, butane-1 ,3-diol, butane-1 ,2-diol, butane-1 ,1-diol, butane-2,3-diol, butane-2, 2-diol, 2-methyl-propan-

[0055] 1 .3-diol, 2-methyl-propan-1 ,2-diol, 2-methyl-propan-1 ,1-diol, cyclobutane-1 ,3-diol, cyclobutane-1 ,2- diol, cyclobutane-1 ,1-diol, 2-hydroxymethylcyclopropan-1-ol, 1-methylcyclopropane-1 ,2-diol, 3- methylcyclopropane-1 ,2-diol, 2-methylcyclopropane-1 ,1-diol, 1-hydroxymethylcyclopropan-1-ol, 1- cyclopropylmethane-1 ,1-diol), a C5 alkylene (pentane-1 ,5-diol, pentane-1 ,4-diol, pentane-1 ,3-diol, pentane-1 ,2-diol, pentane-1 ,1-diol, pentane-2,4-diol, pentane-2,3-diol, pentane-2,2-diol, pentane- 3,3-diol, 2-methylbutane-1 ,4-diol, 2-methylbutane-1 ,3-diol, 2-methylbutane-1 ,2-diol, 2- methylbutane-1 ,1-diol, 2-ethylpropan-1 ,3-diol, 3-methylbutane-1 ,3-diol, 3-methylbutane-1 ,2-diol, 3- methylbutane-1 ,1-diol, 2-methylbutane-2,3-diol, 3-methylbutane-2,2-diol, 2,2-dimethylpropane-1 ,3- diol, 2, 2-dimethylpropane-1 ,1-diol, cyclopentane-1 ,3-diol, cyclopentane-1 ,2-diol, cyclopentane-1 ,1- diol, 3-hydroxymethylcyclobutan-1-ol, 1-methylcyclobutane-1 ,3-diol, 3-methylcyclobutane-1 ,2-diol, 3-methylcyclobutane-1 ,1-diol, 2-hydroxymethylcyclobutan-1-ol, 1-methylcyclobutane-1 ,2-diol, 2- methylcyclobutane-1 ,1-diol, 1-hydroxymethylcyclobutan-1-ol, 1-cyclobutylmethane-1 ,1-diol, 2-(2- hydroxyethyl)cyclopropan-1 -ol, 2-(1-hydroxyethyl)cyclopropan-1-ol, 1-ethylcyclopropan-1 ,2-diol, 3- ethylcyclopropan-1 ,2-diol, 2-ethylcyclopropan-1 ,1-diol, 1-ethylcyclopropan-1 ,2-diol, 1-(2- hydroxyethyl)cyclopropan-1 -ol, 1 -(1 -hydroxyethyl)cyclopropan-1 -ol, 1 -cyclopropylethane-1 ,2-diol, 1 - cyclopropylethane-1 ,1-diol, or 2-cyclopropylethane-1 ,1-diol) and a Ce alkylene diol (hexane-1 ,6-diol, hexane-1 ,5-diol, hexane-1 ,4-diol, hexane-1 ,3-diol, hexane-1 ,2-diol, hexane-1 ,1-diol, hexane-2,5- diol, hexane-2,4-diol, hexane-2,3-diol, hexane-2,2-diol, hexane-3,4-diol, hexane-3,3-diol, 2- methylpentane-1 ,5-diol, 2-methylpentane-1 ,4-diol, 2-methylpentane-1 ,3-diol, 2-methylpentane-1 ,2- diol, 2-propylpropane-1 ,3-diol, 2-methylpentane-1 ,1-diol, 2-methylpentane-1 ,5-diol, 2- methylpentane-2,5-diol, 2-methylpentane-2,4-diol, 2-methylpentane-2,3-diol, 4-methylpentane-1 ,3- diol, 4-methylpentane-1 ,2-diol, 4-methylpentane-1 ,1-diol, 4-methylpentane-2,2-diol, 4- methylpentane-2,3-diol, 4-methylpentane-3,3-diol, 3-methylpentane-1 ,5-diol, 3-methylpentane-1 ,4- diol, 3-methylpentane-1 ,3-diol, 3-methylpentane-1 ,2-diol, 3-methylpentane-1 ,1-diol, 3- methylpentane-1 ,5-diol, 3-methylpentane-2,4-diol, 2-ethyl-1-methylpropane-1 ,3-diol, 3- methylpentane-2,3-diol, 3-methylpentane-2,2-diol, 1 ,1 -diethylethane-1 ,2-diol, 2-ethylbutane-1 ,1-diol,

[0056] 2.2-dimethylbutane-1 ,4-diol, 2,2-dimethylbutane-1 ,3-diol, 2, 2-dimethylbutane-1 ,1-diol, 2-ethyl-2- methylpropane-1 ,3-diol, 3.3-dimethylbutane-1 ,2-diol, 3.3-dimethylbutane-2,2-diol, 3.3- dimethylbutane-1 ,1-diol, 2.3-dimethylbutane-1 ,4-diol, 2.3-dimethylbutane-1 ,3-diol, 2.3- dimethylbutane-1 ,2-diol, 2-(1 -methylethyl)propane-1 ,3-diol, 2,3-dimethylbutane-2,4-diol, 2,3- dimethylbutane-2,3-diol, 2-ethylbutane-1 ,4-diol, 2-ethylbutane-1 ,3-diol, 2-ethylbutane-1 ,2-diol, 2- ethylbutane-1 ,1-diol, 2-propylpropan-1 ,3-diol, 3-ethylbutane-1 ,3-diol, 3-ethylbutane-1 ,2-diol, 3- ethylbutane-1 ,1-diol, 2-ethylbutane-2,3-diol, 3-ethylbutane-2,2-diol, cyclohexane-1 ,4-diol, cyclohexane-1 ,3-diol, cyclohexane-1 ,2-diol, cyclohexane-1 ,1-diol, 3-hydroxymethylcyclopentan-1-ol, 3-methylcyclopentan-1 ,3-diol, 3-methylcyclopentan-1 ,2-diol, 3-methylcyclopentan-1 ,1-diol, 4- methylcyclopentan-1 ,3-diol, 4-methylcyclopentan-1 ,2-diol, 2-hydroxymethylcyclopentan-1-ol, 2- methylcyclopentan-1 ,3-diol, 2-methylcyclopentan-1 ,2-diol, 2-methylcyclopentan-1 ,1-diol, 1- hydroxymethylcyclopentan-1-ol or 1-cyclopentylmethane-1 ,1-diol) and any L or R isomers thereof. Preferably each Ci-Ce alkylene diol is independently selected from the group consisting of: a Ci alkylene diol (methylene glycol), a C2 alkylene diol (ethylene glycol), a C3 alkylene diol (propane-1 ,3- diol or propane-1 ,2-diol), a C4 alkylene diol (butane-1 ,4-diol, butane-1 ,3-diol, butane-1 ,2-diol, butane-

[0057] 2.3-diol, 2-methyl-propan-1 ,3-diol or 2-methyl-propan-1 ,2-diol), a C5 alkylene (pentane-1 ,5-diol, pentane-1 ,4-diol, pentane-1 ,3-diol, pentane-1 ,2-diol, pentane-2,4-diol, pentane-2,3-diol, 2- methylbutane-1 ,4-diol, 2-methylbutane-1 ,3-diol, 2-methylbutane-1 ,2-diol, 2-ethylpropan-1 ,3-diol, 3- methylbutane-1 ,3-diol, 3-methylbutane-1 ,2-diol, 2-methylbutane-2,3-diol or 2,2-dimethylpropane- 1 .3-diol) and a Ce alkylene diol (hexane-1 ,6-diol, hexane-1 ,5-diol, hexane-1 ,4-diol, hexane-1 ,3-diol, hexane-1 ,2-diol, hexane-2,5-diol, hexane-2,4-diol, hexane-2,3-diol, hexane-3,4-diol, 2- methylpentane-1 ,5-diol, 2-methylpentane-1 ,4-diol, 2-methylpentane-1 ,3-diol, 2-methylpentane-1 ,2- diol, 2-propylpropane-1 ,3-diol, 2-methylpentane-1 ,1-diol, 2-methylpentane-1 ,5-diol, 2- methylpentane-2,5-diol, 2-methylpentane-2,4-diol, 2-methylpentane-2,3-diol, 4-methylpentane-1 ,3- diol, 4-methylpentane-1 ,2-diol, 4-methylpentane-2,3-diol, 3-methylpentane-1 ,5-diol, 3- methylpentane-1 ,4-diol, 3-methylpentane-1 ,3-diol, 3-methylpentane-1 ,2-diol, 3-methylpentane-1 ,5- diol, 3-methylpentane-2,4-diol, 2-ethyl-1-methylpropane-1 ,3-diol, 3-methylpentane-2,3-diol, 1 ,1- diethylethane-1 ,2-diol, 2, 2-dimethylbutane-1 ,4-diol, 2, 2-dimethylbutane-1 ,3-diol, 2-ethyl-2- methylpropane-1 ,3-diol, 3, 3-dimethylbutane-1 ,2-diol, 2, 3-dimethylbutane-1 ,4-diol, 2,3- dimethylbutane-1 ,3-diol, 2, 3-dimethylbutane-1 ,2-diol, 2-(1-methylethyl)propane-1 ,3-diol, 2,3- dimethylbutane-2,4-diol, 2,3-dimethylbutane-2,3-diol, 2-ethylbutane-1 ,4-diol, 2-ethylbutane-1 ,3-diol, 2-ethylbutane-1 ,2-diol, 2-propylpropan-1 ,3-diol, 3-ethylbutane-1 ,3-diol, 3-ethylbutane-1 ,2-diol or 2- ethylbutane-2,3-diol). More preferably, each Ci-Ce alkylene diol is independently selected from the group consisting of: a Ci alkylene diol (methylene glycol), a C2 alkylene diol (ethylene glycol), a C3 alkylene diol (propane-1 ,3-diol or propane-1 ,2-diol) and a C4 alkylene diol (butane-1 ,4-diol, butane-

[0058] 1 .3-diol, butane-1 ,2-diol or butane-2,3-diol). Even more preferably, each Ci-Ce alkylene diol is a C1- C3 alkylene diol, wherein each C1-C3 alkylene diol is independently selected from the group consisting of: a Ci alkylene diol (methylene glycol), a C2 alkylene diol (ethylene glycol) and a C3 alkylene diol (propane-1 ,3-diol or propane-1 ,2-diol). In preferred embodiments of the present invention, each Ci-Ce alkylene diol and C1-C3 alkylene diol is independently selected from methylene glycol and ethylene glycol.

[0059] Each method uses a hydrolase enzyme for formation of a diester of terephthalic acid from said PET. In a preferred embodiment of the methods of the present invention, the formation of said diester of terephthalic acid from PET is mediated (as defined below) by said hydrolase enzyme. Likewise, the formation of said monoester of terephthalic acid from said PET can also be mediated by said hydrolase enzyme. Preferably, said hydrolase enzyme is an enzyme capable of hydrolysing an ester [-C(O) - O-] bond with the addition of water to form the corresponding carboxylic acid and alcohol moieties, ideally an alpha / beta hydrolase. More preferably, said hydrolase enzyme is a carboxylic ester hydrolase, peptidase, lipase or protease. Even more preferably, said hydrolase enzyme is an EC 3.1 .1 hydrolase enzyme ( / .e. a carboxylic ester hydrolase) such as a carboxylesterase (including a polyesterase, EC. 3.1.1.1), lipase (e.g. EC 3.1.1.3, EC 3.1.1 .4, EC 3.1.1.5, EC 3.1.1.23, EC 3.1.1.26, EC 3.1.1.32, EC 3.1.1.34, EC 3.1.1.79 or EC 3.1.1.116), cutinase (e.g. EC 3.1.1.74), acetylxylan esterase (e.g. EC 3.1.1.72), carboxymethylenebutenolidase (e.g. EC 3.1.1.45). In a preferred embodiment of the methods of the present invention, said hydrolase enzyme is a carboxylesterase (EC 3.1 .1.1), a triacylglycerol lipase (EC 3.1.1.3), an acetylxylan esterase (EC 3.1.1.72), a cutinase (EC 3.1.1 .74) or a carboxymethylenebutenolidase (EC 3.1.1.45). Still more preferably, said hydrolase enzyme is a PETase (EC 3.1.1.101), yet more preferably a bacterial PETase, an insect PETase, or a fungal PETase. Said bacterial PETase is preferably a naturally occurring PETase found in ( / .e. isolated from) a bacterium of a genus selected from the group consisting of: Bacillus, Nocardia and Ideonella. More preferably, said bacterial PETase is selected from the group consisting of SEQ ID Nos: 1 to 30: FAST PETase [SEQ ID NO: 1 : Lu, H. et al. Nature (2022) 604, 662-667]; LCCJCCG PETase [SEQ ID NO: 2: Tournier, V. et al.; Nature (2020) 580, 216-219]; bacterium HR29 PETase (BhrPETase) (SEQ ID NO: 3); Nocardia species esterase (Sharon C. et al.; J. Microbiol. Biotech. Res. (2012) 2(2) 248-257); Ideonella sakaiensis PETase (SEQ ID NO: 4)]; uncultured bacterium dienelactone hydrolase “liplAF5-2” (SEQ ID NO: 5); Marinactinospora thermotolerans cutinase (SEQ ID NO: 6); Saccharopolyspora flava cutinase (SEQ ID NO: 7); Thermobifida fusca acetylxylan esterase (SEQ ID NO: 8); Thermobifida fusca cutinase (SEQ ID NO: 9); Thermobifida alba cutinase (SEQ ID NO: 10); Thermobifida fusca dienelactone hydrolase (SEQ ID NO: 11); Thermobifida cellulosilytica cutinase (SEQ ID NO: 12); Thermobifida alba cutinase (SEQ ID NO: 13); Thermomonospora curvata lipase (SEQ ID NO: 14); Thermomonospora catenispora alpha / beta hydrolase (SEQ ID NO: 15); Thermobifida fusca dienelactone hydrolase (SEQ ID NO: 16); Thermobifida alba alpha / beta hydrolase (SEQ ID NO: 17); Thermobifida alba alpha / beta hydrolase (SEQ ID NO: 18); Cys-FAST PETase (SEQ ID NO: 19); CalB (Pseudozyma antarctica (also known as Candida antarctica) lipase B) (SEQ ID NO: 20); Novozym 51032 (Humicola insolens cutinase, HiC, SEQ ID NO: 21); CalA (Pseudozyma antarctica (also known as Candida antarctica) lipase A, 2VEO_1 chains A,B, SEQ ID NO: 22); Thermobifida fusca KW3 cutinase (Tfcut2) (SEQ ID NO: 23); Thermobifida cellulosilytica cutinase 1 (Thc_cut1) (SEQ ID NO: 24); Thermomyces lanuginosus lipase (TIL; SEQ ID NO: 25); Rhizomucor miehei lipase (RmL, SEQ ID NO: 26); Thermobifida cellulosilytica cutinase 2, chain A (TFC_CUT2) (SEQ ID NO: 27); Thermobifida fusca cutinase 1 , partial (Thf42_Cut1) (SEQ ID NO: 28); Dura PETase [Cui, Y. et al.; ACS Catal. (2021) 11 , 1340-1350] (SEQ ID NO: 29); and Thermo PETase [Son, H. F. et al.; ACS Catal. (2019) 9(4), 3519-3526] (SEQ ID NO: 30), or an enzyme having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto. LCCJCCG is an extremely thermostable enzyme capable at working at the glass transition temperature of PET, where polymer chains become more labile and available to the enzyme. Preferred enzymes include SEQ ID NO:1 , SEQ ID NO:2 and SEQ ID NO:7 or an enzyme having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto

[0060] FAST PETase (SEQ ID NO: 1) MQTNPYARGPNPTAASLEASAGPFTVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQ SSIKWWGPRLASHGFWITIDTNSTLDQPESRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGV MGWSMGGGGSLISAANNPSLKAAAPQAPWHSSTNFSSVTVPTLIFACENDSIAPVNSSALPIYDS MSQNAKQFLEIKGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTAVSDFR TANCSLA

[0061] LCCJCCG PETase (SEQ ID NO: 2)

[0062] MSNPYQRGPNPTRSALTADGPFSVATYTVSRLSVSGFGGGVIYYPTGTSLTFGGIAMSPGYTADA SSLAWLGRRLASHGFVVLVINTNSRFDGPDSRASQLSAALNYLRTSSPSAVRARLDANRLAVAGH SMGGGGTLRIAEQNPSLKAAVPLTPWHTDKTFNTSVPVLIVGAEADTVAPVSQHAIPFYQNLPSTT PKVYVELCNASHIAPNSNNAAISVYTISWMKLWVDNDTRYRQFLCNVNDPALCDFRTNNRHCQ Bacterium HR29 PETase (B / ? / PETase) (SEQ ID NO: 3)

[0063] MQ WLG RVRSAGLLAALLALAAWALVWASPSAEAQSN PYQRG PN PTRSALTTDG PFSVATYS VS

[0064] RLSVSGFGGGVIYYPTGTTLTFGGIAMSPGYTADASSLAWLGRRLASHGFWIVINTNSRLDFPDS

[0065] RASQLSAALNYLRTSSPSAVRARLDANRLAVAGHSMGGGATLRISEQIPTLKAGVPLTPWTDKTFN

[0066] TPVPQLIVGAEADTVAPVSQHAIPFYQNLPSTTPKVYVELDNATHFAPNSPNAAISVYTISWMKLWV

[0067] DNDTRYRQFLCNVNDPALSDFRSNNRHCQ

[0068] Ideonella sakaiensis PETase ( / sPETase) (SEQ ID NO: 4)

[0069] MNFPRASRLMQAAVLGGLMAVSAAATAQTNPYARGPNPTAASLEASAGPFTVRSFTVSRPSGYG

[0070] AGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPSSRSSQQMAA

[0071] LRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNPSLKAAAPQAPWDSSTNFSSV

[0072] TVPTLIFACENDSIAPVNSSALPIYDSMSRNAKQFLEINGGSHSCANSGNSNQALIGKKGVAWMKR

[0073] FMDNDTRYSTFACENPNSTRVSDFRTANCS

[0074] Uncultured bacterium dienelactone hydrolase “liplAF5-2” (SEQ ID NO: 5)

[0075] MANPPGGDPDPGCQTDCNYQRGPDPTDAYLEAASGPYTVSTIRVSSLVPGFGGGTIHYPTNAGG

[0076] GKMAGIVVIPGYLSFESSIEWWGPRLASHGFWMTIDTNTIYDQPSQRRDQIEAALQYLVNQSNSS

[0077] SSPISGMVDSSRLAAVGWSMGGGGTLQLAADGGIKAAIALAPWNSSINDFNRIQVPTLIFACQLDAI

[0078] APVALHASPFYNRIPNTTPKAFFEMTGGDHWCANGGNIYSALLGKYGVSWMKLHLDQDTRYAPFL

[0079] CGPNHAAQTLISEYRGNCPY

[0080] Marinactinospora thermotolerans cutinase (SEQ ID NO: 6)

[0081] MSNPYERGPAPTESSVTAVRGYFDTDTDTVSSLVSGFGGGTIYYPTDTSEGTFGGWIAPGYTAS

[0082] QSSMAWMGHRIASQGFVVFTIDTITRYDQPDSRGRQIEAALDYLVEDSDVADRVDGNRLAVMGHS

[0083] MGGGGTLAAAENRPELRAAIPLTPWHLQKNWSDVEVPTMIIGAENDTVASVRTHSIPFYESLDEDL

[0084] ERAYLELDGASHFAPNISNTVIAKYSISWLKRFVDEDERYEQFLCPPPDTGLFSDFSDYRDSCPHT

[0085] T

[0086] Saccharopolyspora flava cutinase (SEQ ID NO: 7)

[0087] MAEPADVHGPDPTEESITAPRGPFEVDEESVSRLSVSGFGGGTIYYPTDTTDGLFSAVSISPGFTG

[0088] TQETMAWYGPRLASQGFVVFTIDTITTTDQPDSRARQLQASLDYLVNDSDVKDIIDPARLGVMGHS

[0089] MGGGGSLKAALDNPALKAAIPLTPWHTTKDFSGVQTPTLIIGAQNDTVAPVSQHAKPFYESLPDDP

[0090] GKAYLELAGASHLAPNTDNTTIAKFSIAWLKRFLDDDTRYDQFLCPPPENDDSISDYQSTCPY

[0091] Thermobifida fusca acetylxylan esterase (SEQ ID NO: 8)

[0092] MAANPYERGPNPTDALLEARSGPFSVSEENVSRLSASGFGGGTIYYPRESNTYGAVAISPGYTGT

[0093] EASIAWLGERIASHGFVVITIDTITTLDQPDSRAEQLNAALNHMINRASSTVRSRIDSSRLAVMGHS

[0094] MGGGGTLRLASQRPDLKAAIPLTPWHLNKNWSSVTVPTLIIGADLDTIAPVATHAKPFYNSLPSSIS

[0095] KAYLELDGATHFAPNIPNKIIGKYSVAWLKWFVDNDTRYTQFLCPGPRDGLFGEVEEYRSTCPF Thermobifida fusca cutinase (SEQ ID NO: 9)

[0096] MANPYERGPNPTDALLEARSGPFSVSEENVSRLSASGFGGGTIYYPRENNTYGAVAISPGYTGTE

[0097] ASIAWLGERIASHGFVVITIDTITTLDQPDSRAEQLNAALNHMINRASSTVRSRIDSSRLAVMGHSM

[0098] GGGGSLRLASQRPDLKAAIPLTPWHLNKNWSSVRVPTLIIGADLDTIAPVLTHARPFYNSLPTSISK

[0099] AYLELDGATHFAPNIPNKIIGKYSVAWLKRFVDNDTRYTQFLCPGPRDGLFGEVEEYRSTCPF

[0100] Thermobifida alba cutinase (SEQ ID NO: 10)

[0101] MANPYERGPNPTESMLEARSGPFSVSEERASRLGADGFGGGTIYYPRENNTYGAIAISPGYTGTQ

[0102] SSIAWLGERIASHGFVVIAIDTNTTLDQPDSRARQLNAALDYMLTDASSSVRNRIDASRLAVMGHS

[0103] MGGGGTLRLASQRPDLKAAIPLTPWHLNKSWRDITVPTLIIGADLDTIAPVSSHSEPFYNSIPSSTDK

[0104] AYLELNNATHFAPNITNKTIGMYSVAWLKRFVDEDTRYTQFLCPGPRTGLLSDVDEYRSTCPF

[0105] Thermobifida fusca dienelactone hydrolase (SEQ ID NO: 11)

[0106] MANPYERGPNPTDALLEARSGPFSVSEENVSRLSASGFGGGTIYYPRENNTYGAVAISPGYTGTE

[0107] ASIAWLGERIASHGFVVITIDTITTLDQPDSRAEQLNAALNHMINRASSTVRSRIDSSRLAVMGHSM

[0108] GGGGSLRLASQRPDLKAAIPLTPWHLNKNWSSVTVPTLIIGADLDTIAPVATHAKPFYNSLPSSISK

[0109] AYLELDGATHFAPNIPNKIIGKYSVAWLKRFVDNDTRYTQFLCPGPRDGLFGEVEEYRSTCPFY

[0110] Thermobifida cellulosilytica cutinase (SEQ ID NO: 12)

[0111] MANPYERGPNPTDALLEARSGPFSVSEENVSRFGADGFGGGTIYYPRENNTYGAVAISPGYTGTQ

[0112] ASVAWLGERIASHGFVVITIDTNTTLDQPDSRARQLNAALDYMINDASSAVRSRIDSSRLAVMGHS

[0113] MGGGGTLRLASQRPDLKAAIPLTPWHLNKNWSSVRVPTLIIGADLDTIAPVLTHARPFYNSLPTSIS

[0114] KAYLELDGATHFAPNIPNKIIGKYSVAWLKRFVDNDTRYTQFLCPGPRDGLFGEVEEYRSTCPFAL E

[0115] Thermobifida alba cutinase (SEQ ID NO: 13)

[0116] MANPYERGPNPTESMLEARSGPFSVSEERASRFGADGFGGGTIYYPRENNTYGAIAISPGYTGTQ

[0117] SSIAWLGERIASHGFVVIAIDTNTTLDQPDSRARQLNAALDYMLTDASSAVRNRIDASRLAVMGHS

[0118] MGGGGTLRLASQRPDLKAAIPLTPWHLNKSWRDITVPTLIIGAEYDTIASVTLHSKPFYNSIPSPTDK

[0119] AYLELDGASHFAPNITNKTIGMYSVAWLKRFVDEDTRYTQFLCPGPRTGLLSDVEEYRSTCPF

[0120] Thermomonospora curvata lipase (SEQ ID NO: 14)

[0121] MPTEASITAARGPFNTAEITVSRLSVSGFGGGKIYYPTTTSEGTFGAIAISPGFTAYWSSLEWLGHR

[0122] LASQGFVVIGIETNTTLDQPDQRGQQLLAALDYLTQRSAVRDRVDASRLAVAGHSMGGGGSLEAA

[0123] KARTSLKAAIPLAPWNLDKTWPEVRTPTLIIGGELDAVAPVATHSIPFYNSLSNAPEKAYLELDNAS

[0124] HFFPNITNTQMAKYMIAWMKRFIDDDTRYTQFLCPPPSTGLLSDFSDARFTCPM

[0125] Thermomonospora catenispora alpha / beta hydrolase (SEQ ID NO: 15)

[0126] MADNPYERGPDPTEQLIESARGPFATAQTRVSSLSVTGFGGGVIYYPTDTSQGTFGAVAISPGFTA

[0127] DWTSLDWLGPRLASHGFWIGIDTITRLDQPDSRGRQLLAALDYLTQRSSVRSRVDASRLAVAGH SMGGGGSLEAAKSRTSLKAAIPLAPWNLDKTWPEVVTPTMIFGGELDTVAPVSTHAIPFYNSLTNA

[0128] REKAYLEIDNGSHFFPNVSNTLVAKYMISWLKRYVDNDTRYEQFLCPVPDDRGLSDSRGTCPGS

[0129] Thermobifida fusca dienelactone hydrolase (SEQ ID NO: 16)

[0130] MANPYERGPNPTDALLEARSGPFSVSEENVSRLSASGFGGGTIYYPRENNTYGAVAISPGYTGTE

[0131] ASIAWLGERIASHGFVVITIDTITTLDQPDSRAEQLNAALNHMINRASSTVRSRIDSSRLAVMGHSM

[0132] GGGGTLRLASQRPDLKAAIPLTPWHLNKNWSSVTVPTLIIGADLDTIAPVATHAKPFYNSLPSSISKA

[0133] YLELDGATHFAPNIPNKIIGKYSVAWLKRFVDNDTRYTQFLCPGPRDGLFGEVEEYRSTCPF

[0134] Thermobifida alba alpha / beta hydrolase (SEQ ID NO: 17)

[0135] MANPYERGPYPTESMLEARSGPFSVSEERASRFGADGFGGGTIYYPRENNTYGAIAISPGYTGTQ

[0136] SSIAWLGERIASHGFVVIAIDTNTTLDQPDSRARQLNAALDYMLTDASSAVRNRIDASRLAVMGHS

[0137] MGGGGTLRLASQRPDLKAAIPLTPWHLNKSWRDITVPTLIIGAEYDTIASVTLHSKPFYNSIPSSTDK

[0138] AYLELDGASHFAPNITNKTIGMYSVAWLKRFVDEDTRYTQFLCPGPRTGLLSDVEEYRSTCPF

[0139] Thermobifida alba alpha / beta hydrolase (SEQ ID NO: 18)

[0140] MANPYERGPNPTQSMLEARSGPFSVSEERASRLGADGFGGGTIYYPRENNTYGAIAISPGYTGTQ

[0141] SSIAWLGERIASHGFVVIAIDTNTTLDQPDSRARQLNSALDYMLTDASSSVRNRIDASRLAVMGHS

[0142] MGGGGTLRLASQRPDLKAAIPLTPWHLNKSWRDITVPTLIIGADLDTIAPVSSHSEPFYNSIPSSTDK

[0143] AYLELNNATHFAPNITNKTIGMYSVAWLKRFVDEDTRYTQFLCPGPRTGLLSDVDEYRSTCPF

[0144] Cys-FAST PETase (SEQ ID NO: 19)

[0145] MQTNPYARGPNPTAASLEASAGPFTVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQ

[0146] SSIKWWGPRLASHGFWITIDTNSTLDQPESRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGV

[0147] MGWCMGGGGSLISAANNPSLKAAAPQAPWHSSTNFSSVTVPTLIFACENDSIAPVNSSALPIYDS

[0148] MSQNAKQFLEIKGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTAVSDFR

[0149] TANCSLA

[0150] CalB (Pseudozyma antarctica lipase B fragment, B6DAC2_PSEA2) (SEQ ID NO: 20)

[0151] MALPSGSDPAFSQPKSVLDAGLTCQGASPSSVSKPILLVPGTGTTGPQSFDSNWIPLSTQLGYTP

[0152] CWISPPPFMLNDTQVNTEYMVNAITALYAGSGNNKLPVLTWSQGGLVAQWGLTFFPSIRSKVDRL

[0153] MAFAPDYKGTVLAGPLDALAVSAPSVWQQTTGSALTTALRNAGGLTQIVPTTNLYSATDEIVQPQV

[0154] SNSPLDSSYLFNGKNVQAQAVCGPLFVIDHAGSLTSQFSYVVGRSALRSTTGQARSADYGITDCN

[0155] PLPANDLTPEQKVAAAALLAPAAAAIVAGPKQNCEPDLMPYARPFAVGKRTXSGIVTPSL

[0156] Novozym 51032 (Humicola insolens cutinase, A0A075B5G4_HUMIN) (SEQ ID NO: 21)

[0157] QLGAIENGLESGSANACPDAILIFARGSTEPGNMGITVGPALANGLESHIRNIWIQGVGGPYDAALA

[0158] TNFLPRGTSQANIDEGKRLFALANQKCPNTPVVAGGYSQGAALIAAAVSELSGAVKEQVKGVALF

[0159] GYTQNLQNRGGIPNYPRERTKVFCNVGDAVCTGTLIITPAHLSYTIEARGEAARFLRDRIRA CalA (Pseudozyma antarctica lipase A, 2VEO_1 chains A,B) (SEQ ID NO: 22)

[0160] APATETLDRRAALPNPYDDPFYTTPSNIGTFAKGQVIQSRKVPTDIGNANNAASFQLQYRTTNTQN EAVADVATVWIPAKPASPPKIFSYQVYEDATALDCAPSYSYLTGLDQPNKVTAVLDTPIIIGWALQQ GYYVVSSDHEGFKAAFIAGYEEGMAILDGIRALKNYQNLPSDSKVALEGYSGGAHATVWATSLAD

[0161] SYAPELNIVGASHGGTPVSAKDTFTFLNGGPFAGFALAGVSGLSLAHPDMESFIEARLNAKGQQTL

[0162] KQIRGRGFCLPQVVLTYPFLNVFSLVNDTNLLNEAPIAGILKQETVVQAEASYTVSVPKFPRFIWHAI

[0163] PDEIVPYQPAATYVKEQCAKGANINFSPYPIAEHLTAEIFGLVPSLWFIKQAFDGTTPKVICGTPIPAI

[0164] AGITTPSADQVLGSDLANQLRSLNGKQSAFGKPFGPITPP

[0165] Thermobifida fusca KW3 cutinase (Tfcut2) (SEQ ID NO: 23) MAVMTPRRERSSLLSRALQVTAAAATALVTAVSLAAPAHAANPYERGPNPTDALLEASSGPFSVS

[0166] EENVSRLSASGFGGGTIYYPRENNTYGAVAISPGYTGTEASIAWLGERIASHGFVVITIDTITTLDQP

[0167] DSRAEQLNAALNHMINRASSTVRSRIDSSRLAVMGHSMGGGGTLRLASQRPDLKAAIPLTPWHLN

[0168] KNWSSVTVPTLIIGADLDTIAPVATHAKPFYNSLPSSISKAYLELDGATHFAPNIPNKIIGKYSVAWLK

[0169] RFVDNDTRYTQFLCPGPRDGLFGEVEEYRSTCPF

[0170] Thermobifida cellulosilytica cutinase 1 (Thc_cut1) (SEQ ID NO: 24)

[0171] MANPYERGPNPTDALLEASSGPFSVSEENVSRLSASGFGGGTIYYPRENNTYGAVAISPGYTGTE

[0172] ASIAWLGERIASHGFVVITIDTITTLDQPDSRAEQLNAALNHMINRASSTVRSRIDSSRLAVMGHSM GGGGTLRLASQRPDLKAAIPLTPWHLNKNWSSVTVPTLIIGADLDTIAPVATHAKPFYNSLPSSISKA YLELDGATHFAPNIPNKIIGKYSVAWLKRFVDNDTRYTQFLCPGPRDGLFGEVEEYRSTCPFALE

[0173] Thermomyces lanuginosus lipase (LIP_THELA lipase) (SEQ ID NO: 25)

[0174] MRSSLVLFFVSAWTALASPIRREVSQDLFNQFNLFAQYSAAAYCGKNNDAPAGTNITCTGNACPE

[0175] VEKADATFLYSFEDSGVGDVTGFLALDNTNKLIVLSFRGSRSIENWIGNLNFDLKEINDICSGCRGH

[0176] DGFTSSWRSVADTLRQKVEDAVREHPDYRVVFTGHSLGGALATVAGADLRGNGYDIDVFSYGAP

[0177] RVGNRAFAEFLTVQTGGTLYRITHTNDIVPRLPPREFGYSHSSPEYWIKSGTLVPVTRNDIVKIEGID ATGGNNQPNIPDIPAHLWYFGLIGTCL

[0178] Rhizomucor miehei lipase (LIP_RHIMI lipase) (SEQ ID NO: 26)

[0179] MVLKQRANYLGFLIVFFTAFLVEAVPIKRQSNSTVDSLPPLIPSRTSAPSSSPSTTDPEAPAMSRNG

[0180] PLPSDVETKYGMALNATSYPDSWQAMSIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWD

[0181] CIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVH

[0182] KGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYT

[0183] QGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSD LETSDCSNSIVPFTSVLDHLSYFGINTGLCT

[0184] Thermobifida cellulosilytica cutinase 2, chain A (TFC_CUT2) (SEQ ID NO: 27)

[0185] MANPYERGPNPTDALLEARSGPFSVSEERASRFGADGFGGGTIYYPRENNTYGAVAISPGYTGTQ

[0186] ASVAWLGERIASHGFWITIDTNTTLDQPDSRARQLNAALDYMINDASSAVRSRIDSSRLAVMGHS

[0187] MGGGGTLRLASQRPDLKAAIPLTPWHLNKNWSSVRVPTLIIGADLDTIAPVLTHARPFYNSLPTSIS KAYLELDGATHFAPNIPNKIIGKYSVAWLKRFVDNDTRYTQFLCPGPRDGLFGEVEEYRSTCPFAL

[0188] E

[0189] Thermobifida fusca cutinase 1 , partial (Thf42_Cut1) (SEQ ID NO: 28) MANPYERGPNPTDALLEARSGPFSVSEENVSRLSASGFGGGTIYYPRENNTYGAVAISPGYTGTE ASIAWLGERIASHGFVVITIDTITTLDQPDSRAEQLNAALNHMINRASSTVRSRIDSSRLAVMGHSM GGGGSLRLASQRPDLKAAIPLTPWHLNKNWSSVRVPTLIIGADLDTIAPVLTHARPFYNSLPTSISK AYLELDGATHFAPNIPNKIIGKYSVAWLKRFVDNDTRYTQFLCPGPRDGLFGEVEEYRSTCPF

[0190] Dura PETase (SEQ ID NO: 29)

[0191] QTNPYARGPNPTAASLEASAGPFTVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQS SIKWWGPRLASHGFVVITIDTNSTFDYPSSRSSQQMAALRQVASLNGDSSSPIYGKVDTARMGVM GHSMGGGASLRSAANNPSLKAAIPQAPWDSQTNFSSVTVPTLIFACENDSIAPVNSHALPIYDSMS RNAKQFLEINGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTAVSDFRTA NCS

[0192] Thermo PETase (SEQ ID NO: 30)

[0193] QTNPYARGPNPTAASLEASAGPFTVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQS SIKWWGPRLASHGFVVITIDTNSTLDQPESRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVM GWSMGGGGSLISAANNPSLKAAAPQAPWHSSTNFSSVTVPTLIFACENDSIAPVNSSALPIYDSMS RNAKQFLEINGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTAVSDFRTA NCS

[0194] The formation of said diester and / or monoester may be performed under any conditions at which said hydrolase enzyme is active. Said hydrolysis is preferably carried out in a medium, more preferably an aqueous medium. Ideally, said aqueous medium does not contain more than 50 % volume / volume (% v. / v.) of said alcohol, preferably not more than 30 % v. / v. of said alcohol, more preferably between 1 and 20 % v. / v. of said alcohol, even more preferably between 5 and 15 % v. / v of said alcohol. Use of an aqueous medium containing not more than 50 % volume / volume (% v. / v.) of said alcohol, as defined above, allows transesterification to be conducted using reduced amounts of starting material, which not only represents a saving in materials and cost, but also renders it less energy intensive, faster and safer to separate the diester and / or monoester from any unreacted alcohol.

[0195] The formation of said diester and / or monoester may be carried out at a temperature of between 0 and 100 °C. In a preferred embodiment of the methods of the present invention, the formation of said diester is performed at between 0 and 100 °C. More preferably, formation of said diester and / or monoester is carried out at between 15 and 95 °C. In an even more preferred embodiment of the methods of the present invention, the formation of said diester is performed at between 30 and 80 °C. Still more preferably, formation of said diester and / or monoester is carried out at between 45 and 75 °C. The use of said hydrolase enzyme to effect formation of said diester and / or monoester permits transesterification to take place at a much lower temperature than required for conventional transesterification, thereby making the methods described herein cheaper and more energy efficient, as well as safer and suitable for conducting on an industrial scale.

[0196] Without wishing to be bound by any theory, it is believed that the transesterification with said hydrolase enzyme proceeds by enzyme catalysed formation of the acyl intermediate, rather than the diester (or indeed hydrolysis itself). Thus, the formation of the diester may be described as mediated by said enzyme insofar as not all steps in the transesterification with said hydrolase enzyme may be catalysed by said enzyme. Therefore, mediated by said enzyme means at least one step in the transesterification with said hydrolase enzyme is catalysed by said enzyme. Indeed, in a preferred embodiment of the methods of the present invention, the formation of said diester of terephthalic acid from said PET is mediated by said hydrolase enzyme. Taking the example of a serine hydrolase: the enzyme contains three amino acid moieties - an acidic aspartate or glutamate, a basic histidine, and a serine residue (a so-called catalytic triad: represented by the residues in the upper left of Scheme 1) - which are utilized in the formation of said diester. The function of the triad is to activate said serine residue as a nucleophile. For this to occur, said serine residue must be deprotonated by said basic histidine residue, which it is only capable of if activated by said adjacent acidic residue. Said serine residue then attacks the activated carbonyl carbon of an ester moiety of the substrate to form a tetrahedral intermediate (upper step of Scheme 1 , wherein the substrate may represent PET or an initial transesterification product formed from PET by base-catalysed transesterification). This then rearranges to expel an alcohol and form an acyl intermediate in which the remainder of said substrate and the nucleophilic serine residue are linked by an ester moiety (right step of Scheme 1). This acyl intermediate is then alcoholysed, in turn, by an alcohol (or rather, by the corresponding alkoxide ion formed by deprotonation of said alcohol), to yield the corresponding ester, regenerating the enzyme (lower two steps of Scheme 1 , where said alcohol is methanol), thereby overall resulting in alcoholysis of said substrate. It is possible that alcoholysis of the acyl intermediate is also activated by the catalytic triad of the hydrolase enzyme rather than by said alcohol (or alkoxide ion).

[0197]

[0198] Scheme 1 . Mechanism of formation of an ester of polyethylene terephthalate (PET) wherein the Ci-Ce alcohol is methanol. When water is present, the acyl intermediate may be hydrolysed by water (or, rather, by a hydroxide ion), to yield the corresponding carboxylic acid and regenerate the enzyme, thereby overall resulting in hydrolysis of said substrate ( / .e. said PET or initial transesterification product). It is possible that hydrolysis of the acyl intermediate is also activated by the catalytic triad of the hydrolase enzyme rather than by said water (or hydroxide ion). Thus, if water and an alcohol are both present, the formation of the corresponding ester may outcompete the formation of the corresponding carboxylic acid and vice versa.

[0199] The thermodynamic profile of this reaction, where the resulting ester (or carboxylic acid) and alcohol are more energetically stable than the preceding ester moiety of the substrate ( / .e. said PET or initial transesterification product), means that theoretically this reaction should occur spontaneously. In practice, the high activation energy of this reaction ( / .e. the instability of the tetrahedral intermediate) means that the rate of this reaction is extremely slow without the presence of said hydrolase enzyme as a catalyst. Thus, the role of the hydrolase enzyme in this reaction is to lower the activation energy of the reaction by stabilising the tetrahedral intermediate, which is largely down to an “oxyanion hole”, a collection of residues that stabilise the negative charge on the oxygen when the tetrahedral intermediate is formed by nucleophilic attack. This is the reason that when the substrate is PET, a plastic that will typically take hundreds of years to decompose in landfill, it may be fully depolymerised in a few hours by said hydrolase enzyme, as defined herein.

[0200] The diester formed when performing the methods described herein is dimethyl terephthalate (DMT) when said Ci -Ce alkyl alcohol is methanol, diethyl terephthalate (DET) when said Ci -Ce alkyl alcohol is ethanol, and bis(2-hydroxyethyl) terephthalate (BHET) when said Ci - Ce alkylene diol is ethylene glycol. Any monoester formed when performing the methods described herein is monomethyl terephthalic acid (MMT) when said Ci - Ce alkyl alcohol is methanol, monoethyl terephthalic acid (MET) when said Ci - Ce alkyl alcohol is ethanol, and monohydroxyethyl terephthalic acid (MHET) when said Ci - Ce alkylene diol is ethylene glycol.

[0201] As DMT, DET and BHET are each esters, they are less stable and more easily repolymerised than terephthalic acid (TPA). Producing the DMT, DET or BHET product (as opposed to the corresponding monoester product, MMT, MET or MHET, respectively) helps to prevent said hydrolase enzyme from being inhibited by said monoester products.

[0202] Polymerisation of the diester product (as described in the method of repolymerisation of PET of the present invention) takes place once the diester resulting from step (i) has been isolated in a step (ii). Isolation of the diester resulting from step (i) may involve, by way of example and without limitation: removal of waste (e.g. adhesive or natural fibre, alcohol, water, enzyme or medium) from said diester; and / or removal of by-products (e.g. substances present in said material with said PET such as dye or metal, ethylene glycol, oligomeric products, monoesters or terephthalic acid) from said diester. Isolation may be achieved without limitation, by, for example, extraction, filtration, centrifugation, evaporation, adsorption, chromatography, refining, distillation, decontamination, trituration, or any combination thereof. The use of a Ci-Ce alkyl alcohol as the alcohol starting material in the method for formation of a diester of terephthalic acid from PET renders it less energy intensive, faster and safer to separate the diester and / or monoester from any unreacted alcohol than when the alcohol is a Ci-Ce alkylene diol. As a result, when an excess of said alcohol is employed, it is less energy intensive to use a Ci-Ce alkyl alcohol as opposed to a Ci-Ce alkylene diol.

[0203] Being non-charged at basic pH means that said diester product also possesses a very different solubility profile to hydrolysis products ( / .e. said monoester or said TPA), and can be isolated from said hydrolysis products. Thus, step (ii) of the method for repolymerisation of PET preferably comprises isolating said diester by extraction, more preferably by extraction of diester from the aqueous medium in which it is formed into an organic solvent that is not miscible with water. Isolation may be effected using, for example an aqueous biphasic solvent system [e.g. an aqueous solution and methyl tert-butyl ether (MTBE) or diethyl ether (EE)], more preferably in a continuous process while step (i) is being carried out.

[0204] Consequently, the formation of said diester may take place in a biphasic solvent system. However, it may also take place in a monophasic solvent system. Indeed, in a preferred embodiment of the methods of the present invention, the formation of said diester is performed in a monophasic solvent system or biphasic solvent system. Said monophasic solvent system or biphasic solvent system may comprise water. In order to ensure that any carboxylic acid remains in the aqueous phase during extraction of the diester, said aqueous phase preferably has a pH which is greater than the highest pKa of terephthalic acid ( / .e. a pH > 4.3). More preferably, said aqueous phase has a pH > 5.0, even more preferably, a pH > 6.0. In a preferred embodiment of the methods of the present invention, the formation of said diester is performed in a monophasic solvent system or biphasic solvent system which comprises an aqueous solution having a pH in the range of 7.0 to 12.0.

[0205] In another preferred embodiment of the methods of the present invention, dye(s) and / or monoester(s) and / or terephthalic acid are also removed during step (ii) and subsequently isolated using any of the aforementioned techniques.

[0206] When the methods described herein form a monoester of terephthalic acid rather than or in addition to said diester, said monoester can be subsequently hydrolysed to terephthalic acid. Said hydrolysis may be enzymatic, acid-catalysed or base-promoted hydrolysis. Alternatively, when the methods described herein form a monoester of terephthalic acid rather than or in addition to said diester, said monoester can be subsequently esterified to a diester with an alcohol selected from the group consisting of: a Ci-Ce alkyl alcohol and a Ci-Ce alkylene diol, as defined herein [using e.g. the method of tin (II) glucarate-catalysed esterification of terephthalic acid disclosed in US20200190046 A1 , wherein the monoester replaces said terephthalic acid and said alcohol replaces methanol, said method being hereby incorporated in its entirety by reference]. Said esterification of the monoester to diester preferably results in a diester wherein each ester moiety is the same (e.g. when said monoester is monomethylterephthalate, the alcohol is methanol).

[0207] In addition to being more easily recovered than TPA or said monoester when formed on an industrial scale, said diester product inhibits said hydrolase enzyme to a lesser extent and has lower energy requirements for polymerisation, as described according to step (iii), than do TPA or said monoester.

[0208] Step (iii) (as described in the method for repolymerisation of PET of the present invention) involves polymerising said diester product with ethylene glycol or bis(2-hydroxyethyl)terephthalate to form new PET, once said diester resulting from step (i) has been isolated in step (ii). Thus, the method for repolymerisation of PET of the present invention is a method for preparation of PET (PETn; i.e. new PET), wherein the method comprises the following steps: (i) forming a diester of terephthalic acid from PET (PETm; i.e. PET comprised in a material, as defined herein) using a hydrolase enzyme in the presence of at least one alcohol selected from the group consisting of: a Ci-Ce alkyl alcohol and a Ci-Ce alkylene diol;

[0209] (ii) isolating the diester resulting from step (i); and

[0210] (iii) polymerising the diester with ethylene glycol or bis(2-hydroxyethyl)terephthalate to form said PETn. Said polymerisation may be polycondensation, condensative chain polymerisation or demethanolisation polymerisation.

[0211] The use of a Ci-Ce alkyl alcohol as the alcohol starting material in the method for formation of a diester of terephthalic acid from PET also has an effect upon the method for re-polymerisation of PET in that it renders it less energy intensive, faster and safer to separate the repolymerised PET (PETn) from the corresponding alcohol that is formed as a by-product of polymerising the diester with ethylene glycol or bis(2-hydroxyethyl)terephthalate, than when the alcohol is a Ci-Ce alkylene diol.

[0212] In some embodiments, step (iii) involves polymerising said diester with said diol (i.e. ethylene glycol or bis(2-hydroxyethyl)terephthalate) to form said PETn under conditions (e.g. at a temperature and / or pressure) that selectively drive off (i.e. remove e.g. by evaporation) methanol but do not drive off ethylene glycol, the molar ratio of diester : diol is in the range of 1 :9 to 9:1 , preferably 3:7 to 7:3, more preferably 2:3 to 3:2, even more preferably 9:1 1 to 11 :9 or 99:101 to 101 :99. Most preferably, the molar ratio of diester : diol is approximately 1 :1. Conditions that selectively drive off methanol without driving off ethylene glycol include heating said diester with said diol to between 150 °C and 170 °C, e.g. between 155 °C and 165 °C, at between 100 and 102 kPa (e.g. between 101 and 101.6 Pa).

[0213] However, when step (iii) involves polymerising said diesterwith said diol (i.e. ethylene glycol or bis(2- hydroxyethyl)terephthalate) to form said PETn under conditions (e.g. at a temperature and / or pressure) that drive off methanol and ethylene glycol, a molar excess of said diol relative to said diester is used. Conditions that drive off methanol without driving off ethylene glycol include heating said diester with said diol at between 195 °C and 300 °C, e.g. between 197.3 °C and 275 °C, at between 100 and 102 kPa (e.g. between 101 and 101.6 Pa).

[0214] When a method of the present invention comprises formation of a diester of terephthalic acid from PET via an initial transesterification product or products (i.e. via a step comprising an initial basecatalysed transesterification of said PET with a C1-C10 alcohol), said C1-C10 alcohol is selected from the group consisting of a Ci-Ce alkyl alcohol, as defined herein, a Ci-Ce alkylene diol, as defined herein, and a C7-C10 alcohol. Said C7-C10 alcohol may be a C7 alcohol, a Cs alcohol, a C9 alcohol or a Cw alcohol. Preferably, said C1-C10 alcohol is an alkyl alcohol (e.g. hexan-1-ol), an aryl-substituted alkyl alcohol or alkylene diol having a boiling point greater than that of water (at equivalent pressure). More preferably, said C1-C10 alcohol is selected from the group consisting of: a C7-C10 alkyl alcohol, a C7-C10 aryl-substituted alkyl alcohol and a C2-C4 alkylene diol. Said C7-C10 alkyl alcohol may be selected from the group consisting of: heptan-1-ol, 2-methyl-hexan-1-ol, 2-ethyl-pentan-1-ol, 2,2- dimethyl-pentan-1-ol, 2-ethyl-2-methyl-butan-1-ol, octan-1-ol, 2-methyl-heptan-1-ol, 2-ethyl-hexan- 1-ol, 2-ethyl-2-methyl-pentan-1-ol, 2,2-diethyl-butan-1-ol, 2-propyl-pentan-1-ol, nonan-1-ol, 2- methyl-octan-1-ol, 2-ethyl-heptan-1-ol, 2,2-dimethyl-heptan-1-ol, 2-propyl-hexan-1-ol, 2-ethyl-2- methyl-hexan-1-ol, 2,2-diethyl-pentan-1-ol, 2-methyl-2-propyl-pentan-1-ol, decan-1 -ol, 2-methyl- nonan-1-ol, 2-ethyl-octan-1-ol, 2,2-dimethyl-octan-1-ol, 2-propyl-heptan-1-ol, 2-ethyl-2-methyl- heptan-1-ol, 2-butyl-hexan-1-ol, 2-propyl-2-methyl-hexan-1-ol and 2,2-diethyl-hexan-1-ol. Said C2-C4 alkylene diol may be selected from the group consisting of: ethylene glycol, propan-1 ,2-diol, propan-1 ,3-diol, butan-1 ,2-diol, butan-1 ,3-diol, butan-2,3-diol and butan-1 ,4-diol. Said C7-C10 arylsubstituted alkyl alcohol may be selected from the group consisting of: benzyl alcohol, 2-methyl benzyl alcohol, 3-methyl benzyl alcohol, 4-methyl benzyl alcohol, alpha-methyl benzyl alcohol, 2- ethyl benzyl alcohol, 3-ethyl benzyl alcohol, 4-ethyl benzyl alcohol, alpha-ethyl benzyl alcohol, 2,3- dimethyl benzyl alcohol, 2,4-dimethyl benzyl alcohol, 2,5-dimethyl benzyl alcohol, 2,6-dimethyl benzyl alcohol, 2-methyl-alpha-methyl benzyl alcohol, 4-methyl-alpha-methyl benzyl alcohol, 3- methyl-alpha-methyl benzyl alcohol, and alpha, alpha-dimethyl benzyl alcohol. In an even more preferred embodiment, the C1-C10 alcohol is selected from the group consisting of: benzyl alcohol, heptan-1-ol, octan-1-ol, 2-ethyl-hexan-1-ol and ethylene glycol.

[0215] When a method of the present invention comprises formation of a diester of terephthalic acid from PET via an initial transesterification product or products ( / .e. via a step comprising an initial basecatalysed transesterification of said PET with a C1-C10 alcohol), said C1-C10 alcohol is not the same as the Ci-Ce alkyl alcohol subsequently used for formation of a diester of terephthalic acid from polyethylene terephthalate (PET) using a hydrolase enzyme. Thus, in the embodiment where the formation of a diester of terephthalic acid from polyethylene terephthalate (PET) using a hydrolase enzyme is carried out in the presence of a Ci-Ce alkyl alcohol which is a C1-C3 alkyl alcohol, the C1- Cw alcohol used in the initial transesterification may be selected from the group consisting of: a C4 alkyl alcohol, a C5 alkyl alcohol, a Ce alkyl alcohol, a C7-C10 alkyl alcohol, a C7-C10 aryl-substituted alkyl alcohol and a C2-C4 alkylene diol, as defined herein. When said C1-C10 alcohol used in the formation of an initial transesterification product or products is an alkyl alcohol, it preferably has a longer chain length than said Ci-Ce alkyl alcohol subsequently used for formation of a diester of terephthalic acid from polyethylene terephthalate (PET) using a hydrolase enzyme (e.g. when said C1-C10 alcohol used in the formation of an initial transesterification product or products is a Ce alkyl alcohol, said Ci-Ce alkyl alcohol subsequently used for formation of a diester of terephthalic acid from polyethylene terephthalate (PET) using a hydrolase enzyme may be a Ci alkyl alcohol, C2 alkyl alcohol, C3 alkyl alcohol, C4 alkyl alcohol or C5 alkyl alcohol). Similarly, when said C1-C10 alcohol used in the formation of an initial transesterification product or products is an alkylene diol, it preferably has a longer chain length than said Ci-Ce alkylene diol subsequently used for formation of a diester of terephthalic acid from polyethylene terephthalate (PET) using a hydrolase enzyme.

[0216] The C1-C10 alcohol used will typically be present in a molar excess of that required to transesterify the entire mass of PET being used. For example, the C1-C10 alcohol may be used in at least a 2:1 , or 3:1 or 4:1 molar excess relative to PET. In one embodiment, the base catalyst used to form the initial transesterification product or products is an alkali metal base, preferably at least one alkali metal hydroxide. Examples of suitable alkali metal hydroxides include, but are not limited to, lithium hydroxide, sodium hydroxide and potassium hydroxide. In a preferred embodiment, the base catalyst used to form the initial transesterification product or products is sodium hydroxide or potassium hydroxide. There is no particular limitation on the amount of base catalyst that can be used. For example, the amount of base catalyst may be present in an amount ranging from 1 :20 to 5:1 (mass of base catalyst : mass of PET).

[0217] In another embodiment, the base-catalysed initial transesterification reaction is performed at a temperature of less than 200 °C, for example at a temperature ranging from about 60 °C to about 180 °C, or from about 100 °C to 175 °C, preferably from about 120 °C to 160 °C. More preferably, the temperature at which the base-catalysed initial transesterification reaction is performed is less than the boiling point of the C1-C10 alcohol used to form the initial transesterification product.

[0218] In another embodiment, the base-catalysed initial transesterification reaction is performed in the presence of one or more other liquid reaction media, for example an inert solvent. An inert solvent is defined as a liquid that does not take part in the initial transesterification reaction or prevent said reaction from taking place. Examples of such liquid reaction media include, but are not limited to, dimethyl sulfoxide (DMSO), dimethyl formamide (DMF) and Cyrene™.

[0219] The initial transesterification product(s) present in the composition formed in the base-catalysed initial transesterification of PET with a C1-C10 alcohol may be isolated from said composition or purified prior to being treated with said hydrolase enzyme in the presence of said at least one alcohol selected from the group consisting of: a Ci-Ce alkyl alcohol and a Ci-Ce alkylene diol, as defined herein. For example, in one embodiment, the composition is subjected to one or more of solvent extraction, solvent washing (including washing with water or an aqueous solution), filtration, distillation, solvent evaporation, column chromatography and crystallisation in order to isolate or purify said initial transesterification product(s). Once formed, said initial transesterification products can be subsequently converted into the diester of terephthalic acid using said hydrolase enzyme in the presence of said at least one Ci-Ce alkyl alcohol.

[0220] EXAMPLES

[0221] Example 1: Subjecting PET to a hydrolase enzyme in the presence of methanol

[0222] Reactions 1 & 2 were conducted according to the following generic methodology using the materials, amounts and conditions specified in Table 1 : amorphous cryomilled PET film (Goodfellow, ES30- FM-000145), methanol and, where relevant, PETase were dispersed at the specified amounts in the specified buffer and maintained at the specified temperature for 3 days.

[0223] FAST = FAST PETase (SEQ ID NO: 1)

[0224] Table 1. Specific reaction conditions.

[0225] A first portion of each reaction mixture was extracted into MTBE (methyl fe / Y-butyl ether) and analysed by GC-MS. The methanol-containing reactions were overlaid with an extracted ion of mass 194 (dimethyl terephthalate) and the ethanol reactions with an extraction ion mass of 222 (diethyl terephthalate).

[0226] In the full total ion chromatogram (TIC) for Reaction 1 , peaks for both dimethyl terephthalate (DMT) and monomethyl terephthalate (MMT) were observed (see Table 2). Integrating the peaks gives a 5:4 ratio in favour of the dimethyl ester over the monomethyl ester.

[0227] Table 2. Peak data for full TIC of Reaction 1 .

[0228] To a second portion of each reaction was added equal parts methanol and the resulting solutions were analysed by LC-MS. For Reaction 1 , peaks for terephthalic acid (TPA), monohydroxyethyl terephthalate (MHET) and monomethyl terephthalate (MMT) were observed. On integration of these peaks, an almost equal amount of MHET and MMT is observed (see Table 3). This is a very significant result, considering the proportion of MMT and DMT observed via GC-MS. The peak areas are also large, suggesting the reaction proceeded at a good rate.

[0229] Table 3. Peak data for LC-MS of Reaction 1 .

[0230] Analysis of reaction 2 revealed that transesterification did not take place without the presence of an enzyme, confirming that the reaction is enzyme catalysed. Example 2: Repolymerising PET from DMT and Ethylene Glycol

[0231] The DMT diester formed from Reaction 1 of Example 1 was isolated by extraction into MTBE and said ether and any unreacted methanol were removed by evaporation. Said diester was subsequently combined with ethylene glycol (with the latter in molar excess) in the presence of antimony trioxide and calcium hydride as catalytic precursors according to the method disclosed in US2647885 A which is hereby incorporated by reference. The initial reaction was performed at 1 18 °C at atmospheric pressure for 3.5 h, with methanol removed via distillation. At this point, the reaction mixture consisted mainly of DMT and bis(hydroxyethyl)terephthalate (BHET) in an approximately 1 :1 molar ratio of DMT : BHET. The temperature was increased to 275 °C and the reaction was placed under vacuum (0.5 mmHg) for 5.1 hours. Re-polymerisation was confirmed by identification of liquid PET as the product.

[0232] Example 3: Repolymerising PET directly from DMT and BHET

[0233] The DMT diester formed from Reaction 1 of Example 1 was isolated by extraction into MTBE and said ether removed by evaporation. Said diester was subsequently polymerised with BHET (in a 1 :1 molar ratio of DMT : BHET) in the presence of antimony trioxide and calcium hydride as catalytic precursors. The temperature was increased to 275 °C and the reaction was placed under vacuum for 5.1 hours. Re-polymerisation was confirmed by identification of liquid PET as the product.

[0234] Example 4: Screening of homologous enzymes

[0235] To a 96 deep well plate was added 10 mg of cryomilled amorphous PET film (Goodfellow, ES30-FM- 000145) per well. Sodium phosphate buffer (700 pL of pH 8 100 mM) was added to each well, followed by purified enzyme solution (200 pL, as specified in Table 5, below) and methanol (100 pL). The plate was sealed and incubated with shaking at 50 °C for 16 hours. Following this, MTBE (500 pL) was added to each well and the plate was vortexed to form an emulsion. The plate was then centrifuged to separate the layers and the organic layer was transferred to a HPLC vial for GC-MS analysis.

[0236] Once all samples had run, the ion extracted chromatograms for a mass of 194 (the mass of dimethyl terephthalate) were overlaid. From this data, it is seen that there are a number of transesterification hits of varied activity (see DMT integration in Table 5, below).

[0237] Table 5: Tabulation of DMT integration for the ion extracted chromatograms. Example 5: Transesterification with FAST PETase cysteine mutant

[0238] Replacing the nucleophilic serine residue in a PET hydrolase with cysteine leads to a thioester acyl intermediate. As this thioester is more unstable, an energetic driving force is introduced to the transesterification step. The Ser / Cys mutant of FAST PETase was tested as an enzyme lysate.

[0239] BL21 (DE3) E. coll expressing Cys-FAST PETase (SEQ ID NO: 19) was suspended to a concentration of 200 mg / mL in B-PER™ Bacterial Protein Extraction Reagent (ThermoFisher). The Suspension was incubated at 30 °C with shaking for thirty minutes. The lysed cell pellet was then separated with centrifugation at 21 ,300 g for two minutes. 100 pL of supernatant was combined with 800 pL of 100 mM pH 8 sodium phosphate buffer and 100 pL of methanol. 10 mg of cryo-milled amorphous PET film (Goodfellow) was added to the reaction. The reaction was incubated for 16 hours with shaking at 50 °C. 500 pL of MTBE was added to the reaction and the mixture was vortexed to form an emulsion. The emulsion was separated via centrifugation at 21 ,300 g for two minutes. The organic layer was transferred to a HPLC vial for GC-MS analysis. Dimethyl terephthalate was detected in the sample.

[0240] Example 6 (comparative): Transesterification of PET with ethylene glycol

[0241] Demonstrating transesterification of PET to BHET is advantageous as BHET and DMT may be polymerised back to PET, or BHET may be transesterified into DMT (see Example 7). Incubation of PET with a PETase and ethylene glycol may theoretically form BHET through either transesterification or hydrolysis. Therefore, a control reaction with no ethylene glycol present was performed to demonstrate this product resulted from transesterification.

[0242] To a microcentrifuge tube was added a 1000 pL combination of sodium phosphate buffer (pH 8, 100 mM), ethylene glycol, and an aqueous solution of LCCJCCG PET hydrolase (SEQ ID NO: 2) in a combination that resulted in the % v / v of ethylene glycol and enzyme concentration outlined for each reaction in Table 6. For reaction 2, which contained no enzyme, 900 pL of sodium phosphate buffer (pH 8, 100 mM) was simply combined with 100 pL of ethylene glycol. The tubes were sealed and incubated with shaking at 50 °C for 16 hours. 100 pL of reaction mixture was transferred to a fresh microcentrifuge tube and combined with 400 pL of ethanol. The mixture was centrifuged at 21 ,300 g for 2 minutes to pellet any precipitated protein and the supernatant was transferred to a HPLC vial for LC-MS analysis. BHET presence was quantified by integration of the positive mode peak observed in the extracted ion chromatogram for a mass of 255.

[0243] From this experiment, the integration results shown in Table 6 were obtained.

[0244] Table 6: Integration Values This work was then repeated with the isotopically labelled substrate ethylene-d4 glycol (Santa Cruz Biotechnology, sc-228089). The presence of product mass signatures corresponding to the incorporation of this substrate confirmed the ability of the enzyme to utilise ethylene glycol for transesterification.

[0245] Example 7: Transesterification of BHET with methanol

[0246] A. Novozym 51032

[0247] B / s(2-hydroxyethyl)terephthalate (2.5 mg) was combined with methanol (500 pL) and 500 pL of Novozym 51032 solubilised in 100 mM aqueous pH 7.4 sodium phosphate buffer. The reaction was maintained at a temperature of 37 °C for 5 hours. After this period, a 50 pL sample of the reaction mixture was combined with methanol (950 pL) to quench the reaction. The quenched sample was then subjected to analysis via GC-MS.

[0248] An extracted ion chromatogram for a mass of 194 confirmed the presence of dimethyl terephthalate (DMT) therein.

[0249] When 100 mM aqueous pH 7.4 sodium phosphate buffer (500 pL) was used in place of the enzyme solution, no transesterification was observed.

[0250] B. Lipase B

[0251] B / s(2-hydroxyethyl)terephthalate (2.5 mg) was combined with methanol (500 pL), 100 mM aqueous pH 7.4 sodium phosphate buffer (500 pL) and Lipase B from Candida antarctica immobilized on Immobead 150 (10 mg). The reaction was maintained at a temperature of 37 °C for 5 hours. After this period, a 50 pL sample of the reaction mixture was combined with methanol (950 pL) to quench the reaction. The quenched sample was then subjected to analysis via GC-MS.

[0252] An extracted ion chromatogram for a mass of 194 confirmed the presence of dimethyl terephthalate (DMT) therein.

[0253] When no enzyme was added, no transesterification was observed.

[0254] Example 8: Transesterification of PET with methanol via an initial transesterification reaction

[0255] A. Dibenzyl terephthalate

[0256] Benzyl alcohol (10 mL) was heated to 175 °C and treated with sodium hydroxide (150 mg) and PET (1.5 g). The reaction mixture was stirred for 10 minutes, before being cooled to 20 to 25 °C and filtered to remove remaining solids. The resulting filtrate was washed with water, and unreacted benzyl alcohol was removed in vacuo to afford dibenzyl terephthalate (DBT) as an initial transesterification product.

[0257] Said initial transesterification product (3.5 mg) was added to a reaction vessel and combined with methanol (500 pL) and Novozym 51032 solubilised in 100 mM aqueous pH 7.4 sodium phosphate buffer (500 pL). The resulting mixture was maintained at a temperature of 37 °C for 5 hours. After this period, a 50 pL sample was obtained from the reaction mixture and combined with methanol (950 pL) to quench the reaction. The quenched sample was then subjected to analysis via GC-MS. An extracted ion chromatogram for a mass of 194 confirmed the presence of dimethyl terephthalate (DMT) therein.

[0258] When 100 mM aqueous pH 7.4 sodium phosphate buffer (500 pL) was used in place of the enzyme solution, no transesterification was observed.

[0259] B. b / s(2-ethylhexyl)terephthalate

[0260] 2-Ethyl hexanol (10 mL) was heated to 175 °C and treated with sodium hydroxide (150 mg) and PET (1.5 g). The reaction mixture was stirred for 10 minutes, before being cooled to 20 to 25 °C and filtered to remove remaining solids. The resulting filtrate was washed with water, and unreacted 2- ethyl hexanol was removed in vacuo to afford b / s(2-ethylhexyl)terephthalate (BEHT) as an initial transesterification product.

[0261] Said initial transesterification product (3.9 mg) was added to a reaction vessel and combined with methanol (500 pL) and Novozym 51032 solubilised in 100 mM aqueous pH 7.4 sodium phosphate buffer (500 pL). The resulting mixture was maintained at a temperature of 37 °C for 5 hours. After this period, a 50 pL sample was obtained from the reaction mixture and combined with methanol (950 pL) to quench the reaction. The quenched sample was then subjected to analysis via GC-MS. An extracted ion chromatogram for a mass of 194 confirmed the presence of dimethyl terephthalate (DMT) therein.

[0262] When 100 mM aqueous pH 7.4 sodium phosphate buffer (500 pL) was used in place of the enzyme solution, no transesterification was observed.

[0263] C. b / s(2-hydroxyethyl)terephthalate

[0264] Ethylene glycol (10 mL) was heated to 175 °C and treated with sodium hydroxide (150 mg) and PET (1.5 g). The reaction mixture was stirred for 10 minutes, before being cooled to 20 to 25 °C and filtered to remove remaining solids. The resulting filtrate was washed with water, and unreacted 2- ethylene glycol was removed in vacuo to afford b / s(2-hydroxyethyl)terephthalate (BHET) as an initial transesterification product.

[0265] Said initial transesterification product (2.5 mg) was converted into dimethyl terephthalate (DMT) as per Example 7A or 7B.

Claims

CLAIMS1 . A method for formation of a diester of terephthalic acid from polyethylene terephthalate (PET) using a hydrolase enzyme in the presence of at least one Ci-Ce alkyl alcohol, wherein said hydrolase enzyme is an enzyme selected from the group consisting of: a carboxylesterase, a lipase, a cutinase, an acetylxylan esterase and a carboxymethylenebutenolidase.

2. The method according to claim 1 , wherein the formation of said diester of terephthalic acid from said PET is mediated by said hydrolase enzyme.

3. The method according to any one of claim 1 and claim 2, wherein said hydrolase enzyme is an esterase.

4. The method according to any one of claims 1 to 3, wherein said hydrolase enzyme is a carboxylesterase (EC 3.1.1.1), a triacylglycerol lipase (EC 3.1.1.3), an acetylxylan esterase (EC 3.1 .1 .72), a cutinase (EC 3.1 .1 .74), or a carboxymethylenebutenolidase (EC 3.1 .1 .45).

5. The method according to any one of claims 1 to 4, wherein said PET is a treated PET obtained by subjecting a first PET to at least one of a mechanical treatment, a thermomechanical treatment and a chemical treatment, wherein said treated PET has lower crystallinity and greater surface area : volume ratio than said first PET.

6. The method according to any one of claims 1 to 5, wherein said method of forming a diester of terephthalic acid additionally forms a monoester of terephthalic acid.

7. The method according to claim 6, wherein said method forms less monoester than diester.

8. The method according to any one of claims 1 to 7, wherein said method is performed in a monophasic solvent system or biphasic solvent system.

9. The method according to claim 8, wherein said monophasic solvent system or biphasic solvent system comprises an aqueous solution having a pH in the range of 7 to 12.

10. The method according to any one of claims 1 to 9, wherein said method is performed at between 0 and 100 °C.

11. The method according to claim 10, wherein said method is performed at between 30 and 80 °C.

12. The method according to any one of claims 1 to 11 , wherein said PET is subjected to basecatalysed transesterification with a Ce-Cw alcohol to produce a composition comprising a corresponding initial transesterification product, before treating said initial transesterification product with said hydrolase enzyme in the presence of said at least one Ci-Ce alkyl alcohol.

13. A method for re-polymerisation of polyethylene terephthalate (PET), wherein the method comprises the following steps:(i) forming a diester of terephthalic acid from said PET using a hydrolase enzyme in the presence of at least one Ci-Ce alkyl alcohol, wherein said hydrolase enzyme is an enzyme selected from the group consisting of: a carboxylesterase, a lipase, a cutinase, an acetylxylan esterase and a carboxymethylenebutenolidase;(ii) isolating the diester resulting from step (i); and(iii) polymerising the diester with ethylene glycol or bis(2-hydroxyethyl)terephthalate to form new PET.

14. The method according to claim 13, wherein the step (i) of forming said diester of terephthalic acid from said PET is mediated by said hydrolase enzyme.

15. The method according to any one of claims 13 and 14, wherein said hydrolase enzyme is an esterase.

16. The method according to any one of claims 13 to 15, wherein said hydrolase enzyme is a carboxylesterase (EC 3.1.1.1), a triacylglycerol lipase (EC 3.1.1.3), an acetylxylan esterase (EC 3.1 .1 .72), a cutinase (EC 3.1 .1 .74), or a carboxymethylenebutenolidase (EC 3.1 .1 .45).

17. The method according to any one of claims 13 to 16, wherein said PET from which a diester of terephthalic acid is formed in step (i) is a treated PET obtained by subjecting a first PET to at least one of a thermomechanical treatment and a chemical treatment, wherein said treated PET has lower crystallinity and greater surface area : volume ratio than said first PET.

18. The method according to any one of claims 13 to 17, wherein step (i) additionally forms a monoester of terephthalic acid.

19. The method according to claim 18, wherein step (i) forms less monoester than diester.

20. The method according to any one of claims 13 to 19, wherein said step of forming a diester of terephthalic acid is performed in a monophasic solvent system or biphasic solvent system.21 . The method according to claim 20, wherein said monophasic solvent system or biphasic solvent system comprises an aqueous solution having a pH in the range of 7 to 12.

22. The method according to any one of claims 13 to 21 , wherein said transesterification is performed at between 0 and 100 °C.

23. The method according to claim 22, wherein said transesterification is performed at between 30 and 80 °C.

24. The method according to any one of claims 13 to 23, wherein in said step of forming a diester of terephthalic acid, said PET is subjected to base-catalysed transesterification with a Ce-Cw alcohol to produce a composition comprising a corresponding initial transesterification product, before treating said initial transesterification product with said hydrolase enzyme in the presence of said at least one Ci-Ce alkyl alcohol.