Pet hydrolase mutants and implementations thereof

Abstract

The present disclosure provides mutant hydrolase enzymes with improved efficiency and stability, which is capable of functioning at reduced pH and high temperatures. The mutant hydrolases, as described herein, are capable of being stable and active for a period of 10 days or more, and are capable of hydrolyzing or depolymerizing polyester and polyester based material efficiently. Also provided is a method of degrading polyester and polyester material.

Description

PET HYDROLASE MUTANTS AND IMPLEMENTATIONS THEREOFFIELD OF INVENTION

[0001] The present disclosure broadly relates to the field hydrolases. The present disclosure particularly relates to hydrolase mutants, a method of producing the mutant, and a method of degrading polyester material and waste using the mutant.BACKGROUND OF THE INVENTION

[0002] Polyester, including polyethylene terephthalate, usually abbreviated as PET or PETE, is created by polymerizing ester monomers. In that, PET is a thermoset plastic and one of the most widely used and versatile plastic polymers. However, the short useful life, large production volume and non-biodegradability of PET have received attention from various researchers, as PET waste has become a major portion of plastic pollution worldwide. Conventional recycling techniques pose several limitations. For instance, mechanical recycling (majorly used) produces deteriorated quality of recycled products leading to microplastic pollution. On the other hand, chemical recycling or pyrolysis, a process with high carbon footprint requires chemical catalysts working at high temperatures demanding high energy usage resulting in increased greenhouse gas emissions. These prevent efficient recycling of PET and requires the continuous production of virgin PET from fossil fuel, which is again a process with high carbon footprint.

[0003] PET hydrolases (polyethylene terephthalate hydrolases), an esterase class of enzymes belonging to the serine hydrolase family are an excellent alternative. PET is polymerized by the formation of an ester bond between terephthalic acid (TP A) and ethylene glycol (EG). Depolymerising post consumer PET or other kind of PET waste back to obtain terephthalatic acid and ethylene glycol, the building blocks for further PET production can be beneficial in reducing the overall carbon footprint. Although the naturally occurring enzymes has the ability to depolymerise PET, it is extremely slow and inefficient, and additionally the energy consumption and the reaction temperature and pH are considerably high. Attempts have been made toimprove the enzymatic activity of PET hydrolase through protein engineering, directed evolution, site-directed mutagenesis, despite which the decomposition activities thereof are still low at moderate temperatures and regulation of the corresponding reaction parameters have been challenging.

[0004] Therefore, there is an unmet need to develop a hydrolase with enhanced efficiency in polyester degradation.SUMMARY OF INVENTION

[0005] In an aspect of the present disclosure, there is provided a hydrolase mutant comprising a polypeptide having an amino acid sequence of at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 1 or SEQ ID NO. 25; and wherein the polypeptide comprises a mutation selected from the group consisting of: (a) an amino acid substitution in at least one amino acid position selected from a group consisting of 136, 35, 163, 170, 42, 147, and 161 of SEQ ID NO. 1; (b) an amino acid substitution in at least one amino acid position selected from a group consisting of 148, 114, 13, 125, 141, 20, and 139 of SEQ ID NO. 25; (c) an amino acid insertion at a position adjacent to at least one amino acid position selected from a group consisting of 55 and 56 of the SEQ ID NO. 1; (d) an amino acid insertion at a position adjacent to at least one amino acid position selected from a group consisting of 33 and 34 of the SEQ ID NO. 25; (e) an amino acid substitution at a position corresponding to 55 of SEQ ID NO. 1, and an amino acid insertion adjacent to at least one amino acid position selected from 55 and / or 56 of SEQ ID NO. 1; and (f) an amino acid substitution at a position corresponding to 33 of SEQ ID NO. 25, and an amino acid insertion adjacent to at least one amino acid position selected from 33 and / or 34 of SEQ ID NO. 25.

[0006] In an aspect of the present disclosure, there is provided a polynucleotide encoding the PET (polyethylene terephthalate) hydrolase mutant as disclosed herein.

[0007] In another aspect of the present disclosure, there is provided a recombinant vector comprising the polynucleotide as disclosed herein.

[0008] In yet another aspect of the present disclosure, there is provided a host cell comprising the recombinant vector as disclosed herein.

[0009] In an aspect of the present disclosure, there is provided a method of producing the hydrolase mutant as disclosed herein, the method comprising, culturing the host cell as disclosed herein under condition suitable to express the hydrolase mutant encoded by the polynucleotide as disclosed herein.

[0010] In another aspect of the present disclosure, there is provided a composition comprising a hydrolase mutant as disclosed herein, and optionally an additive.

[0011] In yet another aspect of the present disclosure, there is provided a method of degrading polyester or polyester material, the method comprising: contacting polyester or polyester material with the hydrolase mutant as disclosed herein or the composition as disclosed herein in a reaction mixture; and incubating to allow degradation of the polyester or polyester material.

[0012] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the disclosed subject matter, nor is it intended to be used to limit the scope of the disclosed subject matter.BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

[0013] The following drawings form a part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

[0014] Figure 1 depicts an exemplary recombinant vector for expressing the hydrolase mutants, in accordance with the embodiments herein.

[0015] Figure 2 depicts the gel images of agarose gel electrophoresis, verifying the mutation at the potential residues using site directed mutagenesis, in accordance with the embodiments herein.

[0016] Figures 3(a) to 3(c) depicts the results of the differential scanning calorimetry analysis of the post-consumer PET to determine its crystallinity, in accordance with the embodiments herein.

[0017] Figures 4(a) to 4(p) depicts the results of the PET degradation assay and HPLC analysis of the degraded products along with standards carried out using the hydrolase mutants and the amorphous PET sample, in accordance with the embodiments herein.

[0018] Figure 5 depicts the degradation trend of all mutants, i.e. PM-29, PM-31, PM-36, PM-37, PM-44, PM-47, PM-53, PM-59, PM-60, PM-916, and PM-917 wrt concentration, in accordance with the embodiments herein

[0019] Figure 6 depicts effect of buffer pH on the activity of the mutants PM-29, PM- 31, PM-36, PM-37, PM-44, PM-47, PM-53, PM-59, PM-60, PM-916, and PM- 917 on the PET sample, in accordance with the embodiments herein.

[0020] Figure 7 depicts the comparison of the degradation efficiency of PM-916 wrt to Wt and the poorly performing mutants PM- 10, PM- 13 and PM-55, in accordance with the embodiments herein.

[0021] Figure 8 shows temperature dependence of the hydrolase mutants, in accordance with the embodiments herein.DETAILED DESCRIPTION OF THE INVENTION

[0022] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps, features, compositions, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features. Definitions

[0023] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are delineated here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have themeanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.

[0024] The articles “a”, “an” and “the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0025] The terms “comprise” and “comprising” are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as “consists of only”.

[0026] Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated element or step or group of elements or steps but not the exclusion of any other element or step or group of elements or steps.

[0027] The term “including” is used to mean “including but not limited to”. “Including” and “including but not limited to” are used interchangeably.

[0028] Ratios, concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.

[0029] Unless defined otherwise, 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 disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference.

[0030] As discussed in the background, there is a need in the art to provide a PET hydrolase enzyme with improved efficiency and better stability, which is capable of functioning at reduced pH and high temperatures. The present inventors have observed that the mutants of the present disclosure are stable and active for a periodof about 10 days and more, and is capable of hydrolyzing or depolymerizing certain post-consumer PET in about 12 to 17 hours, preferably 17 hours, upto 80% to 100%, preferably 85% with or without pre-processing of substrate to 95%, preferably 90%.

[0031] Accordingly, embodiments herein provide a hydrolase mutant, comprising a mutation selected from amino acid substitution, an amino acid insertion, or both.Mutant

[0032] Embodiments herein provide hydrolase mutants. The term “hydrolase”, “PET hydrolase” or “PETase”, used interchangeably herein, refers to an enzyme under “carboxylic ester hydrolases” or “esterase” which belong to the class of hydrolases classified under enzyme classification number E.C. 3.1.1 that catalyzes the hydrolysis of esters bonds to form alcohol and carboxylic acids. “PETase” belonging to Enzyme classification number 3.1.1.101, from Piscinibacter sakaiensis (Ideonella sakaiensis) corresponding to the uniprot ID: A0A0K8P6T7 catalyzes the hydrolysis of polyethylene terephthalate (PET) plastic to monomeric mono-2-hydroxy ethyl terephthalate (MEET), terephthalic acid (TPA and trace amounts of bi s(2 -hydroxy ethyl) terephthalate (BHET). For example, the hydrolase comprises a polypeptide having an amino acid sequence as set forth in SEQ ID NO. 1 or SEQ ID NO. 25. The term “mutant” as used herein, refers to a variant of the corresponding wildtype. Accordingly, “hydrolase mutant” or “PET hydrolase mutant” as used interchangeably herein refers to a hydrolase having one or more variations such as amino acid substitutions and / or one or more amino acid insertions, in respect of the corresponding wildtype. The hydrolase mutant, according to embodiments herein, is capable of efficiently degrading polyester material, including PET or PET material. The term “polyethylene terephthalate” or “PET”, as used herein, refers to a polyester. The term “polyester” as used herein refers to a category of polymers that contain one or two ester linkages in every repeat unit of their main chain. Examples of polyesters include, but not limited to, polyethylene terephthalate (PET), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PELT), polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylene succinate(PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), polycaprolactone (PCL), polyethylene adipate) (PEA), and / or polyethylene naphthalate (PEN), and blends / mixtures of these polymers. The term “polyester material”, as used herein refers, to a material comprising at least one polyester. The term “PET material”, as used herein refers, to a material comprising a polyethylene terephthalate. In an embodiment, the polyester material encompasses, but is not limited to, a material comprising polyethylene terephthalate (PET), polybutylene adipate terephthalate (PBAT), polybutylene terephthalate (PBT), polytrimethylene terephthalate (PTT), polyethylene isosorbide terephthalate (PELT), polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polyethylene furanoate (PEF), polycaprolactone (PCL), polyethylene adipate) (PEA), and / or polyethylene naphthalate (PEN) polymers.

[0033] Substitution mutations, according to embodiments herein, are also described herein by use of the following notation: amino acid residue substituted: amino acid position in the sequence: amino acid residue substitute. Accordingly, an amino acid substitution with, for instance, if the amino acid substitution is of phenylalanine (F) with proline (P), at position 111 in a sequence, it may be indicated as Fl I IP. Further, alternative amino acid residues substitutes are represented by the notation: amino acid substitute / amino acid substitute / amino acid substitute. For instance, if amino acid substitution of phenylalanine (F) is with proline (P) or serine (S), at position 111 in a sequence, it may be indicated as Fl 11P / S. Similarly, when more than one alternatives are possible, it may be indicated as F111P / S / A, and so on. Such notations are generally known to a person skilled in the art and generally used in representing amino acid substitutions in a given sequence. Also, amino acids are generally represented by single letter and three letter abbreviations, for example “Alanine” is represented by single letter code “A” and three letter code “ala” or “Ala”. Similarly, the single letter code include R (Arginine), N (Asparagine), D (Aspartic acid), C (Cysteine), E (Glutamic acid), Q (Glutamine), G (Glycine), H (Histidine), I (Isoleucine), L (Leucine), K (Lysine), M (Methionine), F (Phenylalanine), P (Proline), S (Serine), T (Threonine), W (Tryptophan), Y(Tyrosine), and V (Valine). Such representations are generally used and well understood by a person skilled in the art. The present disclosure in describing the present invention employs such representations or phrases which is intended to mean the generally acceptable meaning in the art.

[0034] In an embodiment of the present disclosure, the hydrolase mutant comprises a polypeptide having an amino acid sequence of at least 80%, at least 85%, at least 90%, at least 95%, or at least 96% sequence identity to an amino acid sequence selected from SEQ ID NO. 1 or SEQ ID NO. 25; and comprising a mutation selected from at least one amino acid substitution, at least one amino acid insertion, or combinations thereof. The polypeptide of SEQ ID NO. 1, according to the present disclosure, comprises a polypeptide having the sequences as depicted in SEQ ID NO. 25; and a tag, preferably His-tag, at the N-terminal end.

[0035] In an embodiment, the hydrolase mutant comprises a polypeptide having an amino acid sequence of at least 80%, at least 85%, at least 90%, at least 95%, or at least 96% sequence identity to an amino acid sequence as set forth in SEQ ID NO. 1 ; and wherein the polypeptide comprises a mutation selected from the group consisting of: (a) an amino acid substitution in at least one amino acid position selected from a group consisting of 136, 35, 163, 170, 42, 147, 161, and 55 of the polypeptide (SEQ ID NO. 1); (b) an amino acid insertion at a position adjacent to at least one amino acid position selected from a group consisting of 55 and 56 of the polypeptide (SEQ ID NO. 1); and (c) an amino acid substitution in at least one amino acid position selected from a group consisting of 136, 35, 163, 170, 42, 147, 161 and 55 of the polypeptide, and an amino acid insertion adjacent to at least one amino acid position selected from 55 and / or 56 of polypeptide (SEQ ID NO.l).

[0036] In an embodiment, the hydrolase mutant comprises a polypeptide having an amino acid sequence of at least 80%, at least 85%, at least 90%, at least 95%, or at least 96% sequence identity to an amino acid sequence as set forth in SEQ ID NO. 25; and wherein the polypeptide comprises a mutation selected from the group consisting of: (a) an amino acid substitution in at least one amino acid position selected from a group consisting of 148, 114, 13, 125, 141, 20, 139, and 33 of the polypeptide (SEQ ID NO. 25); (b) an amino acid insertion at a position adjacent toat least one amino acid position selected from a group consisting of 33 and 34 of the polypeptide (SEQ ID NO. 25); and (c) an amino acid substitution in at least one amino acid position selected from a group consisting of 148, 114, 13, 125, 141, 20, 139, and 33 of the polypeptide, and an amino acid insertion adjacent to at least one amino acid position selected from 33 and / or 34 of polypeptide (SEQ ID NO. 25).

[0037] In an embodiment, the hydrolase mutant comprises a polypeptide having an amino acid sequence of at least 80%, at least 85%, at least 90%, at least 95%, or at least 96% sequence identity to an amino acid sequence as set forth in SEQ ID NO. 25; and wherein the polypeptide comprises a mutation selected from the group consisting of: (a) an amino acid substitution in at least one amino acid position selected from a group consisting of 148, 114, 13, 141, 20, 125, and 139 of the polypeptide (SEQ ID NO. 25); (b) an amino acid substitution in an amino acid position 33 of the polypeptide (SEQ ID NO. 25), and an amino acid insertion at a position adjacent to at least one amino acid position selected from 33 and / or 34 of the polypeptide (SEQ ID NO. 25); and (c) an amino acid substitution in an amino acid position 33 of the polypeptide (SEQ ID NO. 25), and an amino acid substitution in at least one amino acid position selected from a group consisting of 148, 114, 13, 141, 20, 125, 139, and 33 of the polypeptide, and an amino acid insertion adjacent to at least one amino acid position selected from 33 and / or 34 of polypeptide (SEQ ID NO. 25).

[0038] In an embodiment, the hydrolase mutant comprises a polypeptide having an amino acid sequence of at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 25 or SEQ ID NO. 1; and wherein the polypeptide comprises a mutation selected from the group consisting of: a. an amino acid substitution in at least one amino acid position selected from a group consisting of 136, 35, 163, 170, 42, 147, and 161 of SEQ ID NO. 1; b. an amino acid substitution in at least one amino acid position selected from a group consisting of 148, 114, 13, 125, 141, 20, and 139 of SEQ ID NO. 25; c. an amino acid insertion at a position adjacent to at least one amino acid position selected from a group consisting of 55 and 56 of the SEQ ID NO. 1;d. an amino acid insertion at a position adjacent to at least one amino acid position selected from a group consisting of 33 and 34 of the SEQ ID NO. 25; e. an amino acid substitution at a position corresponding to 55 of SEQ ID NO. 1, and an amino acid insertion adjacent to at least one amino acid position selected from 55 and / or 56 of SEQ ID NO. 1; and f. an amino acid substitution at a position corresponding to 33 of SEQ ID NO. 25, and an amino acid insertion adjacent to at least one amino acid position selected from 33 and / or 34 of SEQ ID NO. 25.

[0039] In an embodiment of the present disclosure, the hydrolase mutant comprises a polypeptide selected from a group consisting of: (a) a polypeptide having an amino acid sequence of at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO.l, comprising an amino acid substitution in at least one amino acid position selected from a group consisting of 136, 35, 163, 170, 42, 147, 161, and 55; and (b) a polypeptide having an amino acid sequence of at least 90% sequence identity to the amino acid sequence as set forth in SEQ ID NO.l, comprising an amino acid substitution in at least one amino acid position selected from a group consisting of 136, 35, 163, 170, 42, 147, 161, and 55, and an amino acid insertion adjacent to at least one amino acid position selected from 55 and / or 56, in respect of the sequence of SEQ ID NO. 1.

[0040] In an embodiment of the present disclosure, the hydrolase mutant comprises a polypeptide having an amino acid sequence of at least 90% sequence identity to the amino acid sequence as set forth in SEQ ID NO.l; and wherein the polypeptide comprises a mutation selected from the group consisting of: (a) an amino acid substitution in at least one amino acid position selected from a group consisting of 136, 35, 163, 170, 42, 147, and 161 of SEQ ID NO. l; and (b) an amino acid substitution in at least one amino acid position 55 of SEQ ID NO.l, and an amino acid insertion at a position adjacent to at least one amino acid position selected from 55 and / or 56 of SEQ ID NO.1.

[0041] In an embodiment of the present disclosure, there is provided a PET hydrolase mutant, wherein the mutation is an amino acid substitution in at least oneamino acid position selected from the group consisting of 136, 35, 163, 170, 42, 147, 161, and 55 of the polypeptide as set forth in SEQ ID NO. l.

[0042] In an embodiment of the present disclosure, there is provided a hydrolase mutant, wherein the amino acid substitution is selected from the group consisting of: (i) substitution of amino acid residue at position 136 with D, T, N, Q or E; (ii) substitution of amino acid residue at position 35 with S, V, L, I, T, or N; (iii) substitution of amino acid residue at position 163 with R, V, A, L, K or H; (iv) substitution of amino acid residue at position 170 with N, D, T, Q, or E; (v) substitution of amino acid residue at position 42 with R, V, L, I, T, or K; (vi) substitution of amino acid residue at position 147 with N, S, V, L, I, T, or N; (vii) substitution of amino acid residue at position 161 with A, V, I, L, C, M, or T; and (viii) substitution of amino acid residue at position 55 with L, T, S, A, V, or I, at positions corresponding to SEQ ID NO. 1.

[0043] In an embodiment of the present disclosure, there is provided a hydrolase mutant, wherein the amino acid substitution is selected from the group consisting of: (i) substitution of amino acid residue at position 114 with D, T, N, Q or E; (ii) substitution of amino acid residue at position 13 with S, V, L, I, T, or N; (iii) substitution of amino acid residue at position 141 with R, V, A, L, K or H; (iv) substitution of amino acid residue at position 148 with N, D, T, Q, or E; (v) substitution of amino acid residue at position 20 with R, V, L, I, T, or K; (vi) substitution of amino acid residue at position 125 with N, S, V, L, I, T, or N; (vii) substitution of amino acid residue at position 139 with A, V, I, L, C, M, or T; and (viii) substitution of amino acid residue at position 33 with L, T, S, A, V, or I, at positions corresponding to SEQ ID NO. 25.

[0044] In an embodiment of the present disclosure, there is provided a hydrolase mutant comprising a polypeptide having an amino acid sequence of at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 1 or 25; and an amino acid substitution selected from (i) an amino acid substitution selected from the group consisting of S136D / T / N / Q / E, A35S / V / L / I / T / N / , I163R / V / A / L / K / H, S 170N / D / T / Q / E, A42R / V / L / I / T / K, A l 47N / S / V / L / I / T / N, S161A / V / I / L / T / C / M, and P55L / T / S / A / V / I or SEQ ID NO. 1; or (ii) an amino acidsubstitution selected from the group consisting of SI 14D / T / N / Q / E, A13S / V / L / I / T / N / , I141R / V / A / L / K / H, S148N / D / T / Q / E, A20R / V / L / I / T / K,A125N / S / V / L / I / T / N, S139A / V / I / L / T / C / M, and P33L / T / S / A / V / I of SEQ ID NO. 25. In an embodiment of the present disclosure, there is provided a hydrolase mutant comprising a polypeptide having an amino acid sequence of at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO.l; and a mutation selected from the group consisting of: (a) an amino acid insertion adjacent to position 55 in respect of the sequence of SEQ ID NO. 1, wherein the amino acid inserted is selected from Q, N, D, E, T, or S; and / or (b) an amino acid insertion adjacent to amino acid position 56 in respect of the sequence of SEQ ID NO. 1, wherein the amino acid inserted is selected from Q, N, D, E, T, or S.

[0045] In an embodiment of the present disclosure, there is provided a hydrolase mutant comprising a polypeptide having an amino acid sequence of at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 25; and a mutation selected from the group consisting of: (a) an amino acid insertion adjacent to position 33 in respect of the sequence of SEQ ID NO. 25, wherein the amino acid inserted is selected from Q, N, D, E, T, or S; and / or (b) an amino acid insertion adjacent to amino acid position 34 in respect of the sequence of SEQ ID NO. 25, wherein the amino acid inserted is selected from Q, N, D, E, T, or S.

[0046] In an embodiment of the present disclosure, there is provided a hydrolase mutant comprising a polypeptide having an amino acid sequence of at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO.l; and a mutation selected from the group consisting of: (a) amino acid substitution selected from the group consisting of (i) substitution of amino acid residue at position 136 with D, T, N, Q or E; (ii) substitution of amino acid residue at position 35 with S, V, L, I, T, or N; (iii) substitution of amino acid residue at position 163 with R, V, A, L, K or H; (iv) substitution of amino acid residue at position 170 with N, D, T, Q, or E; (v) substitution of amino acid residue at position 42 with R, V, L, I, T, or K; (vi) substitution of amino acid residue at position 147 with N, S, V, L, I, T, or N; (vii) substitution of amino acid residue at position 161 with A, V, I, L, C, M or T; and (viii) substitution of amino acid residue at position 55 with L, T, S, A,V, or I; and (b) an amino acid insertion adjacent to position 55, wherein the amino acid inserted is selected from Q, N, D, E, T, or S; and (c) an amino acid insertion adjacent to amino acid position 56, wherein the amino acid inserted is selected from Q, N, D, E, T, or S.

[0047] In an embodiment of the present disclosure, there is provided a PET hydrolase mutant comprising a polypeptide having an amino acid sequence of at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 25; and a mutation selected from the group consisting of: (a) amino acid substitution selected from the group consisting of (i) substitution of amino acid residue at position 114 with D, T, N, Q or E; (ii) substitution of amino acid residue at position 13 with S, V, L, I, T, or N; (iii) substitution of amino acid residue at position 141 with R, V, A, L, K or H; (iv) substitution of amino acid residue at position 148 with N, D, T, Q, or E; (v) substitution of amino acid residue at position 20 with R, V, L, I, T, or K; (vi) substitution of amino acid residue at position 125 with N, S, V, L, I, T, or N; (vii) substitution of amino acid residue at position 139 with A, V, I, L, C, M or T; and (viii) substitution of amino acid residue at position 33 with L, T, S, A, V, or I, in respect of SEQ ID NO. 25; and (b) an amino acid insertion adjacent to position 33, wherein the amino acid inserted is selected from Q, N, D, E, T, or S; and (c) an amino acid insertion adjacent to amino acid position 34, wherein the amino acid inserted is selected from Q, N, D, E, T, or S. The expression “adjacent to position 33”, in context to amino acid insertion, as used herein preferably refers to amino acid insertion between the position 33 and 34 of the amino acid sequence, also referred to herein as “after position 33”. Similarly, the expression “adjacent to position 34”, in context to amino acid insertion, as used herein preferably refers to amino acid insertion between the position 34 and 35 of the amino acid sequence, also referred to herein as “after position 34”. Accordingly, the expression “adjacent to position 33” may be used interchangeably with the expression “between the position 33 and 34” or, “after position 33”, similarly the expression “adjacent to position 34” may be used interchangeably with the expression “between the position 34 and 35” or “after position 34”. Similar expressions may be used inrespect of the term “adjacent to position”, “adjacent to position 55” and “adjacent to position 56”.

[0048] In an embodiment of the present disclosure, the hydrolase mutant comprises a polypeptide having an amino acid sequence of at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO.l; and wherein the polypeptide comprises a mutation selected from the group consisting of: (a) an amino acid substitution selected from a group consisting of S136D, A35S, I163R, S170N, S170D, A42R, A147S, A147N, and S161A; and (b) an amino acid substitution selected from the group consisting of: P55L and P55T, and an amino acid insertion at a position adjacent to at least one position selected from 55 and / or 56, in respect of SEQ ID NO.l, wherein the amino acid insertion is of amino acid Q and D adjacent to positions 55 and 56, respectively.

[0049] In an embodiment of the present disclosure, the hydrolase mutant comprises a polypeptide having an amino acid sequence of at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 25; and wherein the polypeptide comprises a mutation selected from the group consisting of: (a) an amino acid substitution selected from a group consisting of S114D, A13S, I141R, S148N, S148D, A20R, A125S, A125N, and S139A, and P33L; and (b) an amino acid substitution selected from the group consisting of: P33L and P33T, and an amino acid insertion at a position adjacent to at least one position selected from 33 and / or 34, in respect of SEQ ID NO. 25, wherein the amino acid insertion is of amino acid Q and D adjacent to positions 33 and 34, respectively.

[0050] In an embodiment of the present disclosure, there is provided a hydrolase mutant comprising a polypeptide having an amino acid sequence of at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 1; and an amino acid substitution of S136D / T / N / QZE, preferably S136D.

[0051] In an embodiment, the hydrolase mutant comprises a polypeptide having an amino acid sequence of at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO.l; and wherein the polypeptide comprises an amino acid substitution at position S170N / D / T / QZE, preferably S170N or S170D. In an embodiment, the hydrolase mutant comprises a polypeptide having an amino acidsequence of at least 85%, at least 90%, at least 95%, at least 96%, or at least 98% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 2; and wherein the polypeptide comprises an amino acid “N” at position 170.

[0052] In an embodiment, the hydrolase mutant comprises a polypeptide having an amino acid sequence of at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 1; and wherein the polypeptide comprises an amino acid substitution at position S136D / T / N / Q / E, preferably S136D. In an embodiment, the hydrolase mutant comprises a polypeptide having an amino acid sequence of at least 85%, at least 90%, at least 95%, at least 96%, or at least 98% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 3; and wherein the polypeptide comprises an amino acid “D” at position 136.

[0053] In an embodiment, the hydrolase mutant comprises a polypeptide having an amino acid sequence of at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 1; and wherein the polypeptide comprises an amino acid substitution at position A35S / V / L / I / T / N, preferably A35S. In an embodiment, the hydrolase mutant comprises a polypeptide having an amino acid sequence of at least 85%, at least 90%, at least 95%, at least 96%, or at least 98% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 4; and wherein the polypeptide comprises an amino acid “S” at position 35.

[0054] In an embodiment, the hydrolase mutant comprises a polypeptide having an amino acid sequence of at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 1; and wherein the polypeptide comprises an amino acid substitution at position A I47N / S / V / L / I / T / N, preferably A147S or A147N. In an embodiment, the hydrolase mutant comprises a polypeptide having an amino acid sequence of at least 85%, at least 90%, at least 95%, at least 96%, or at least 98% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 5; and wherein the polypeptide comprises an amino acid “S” at position 147. In an embodiment, the hydrolase mutant comprises a polypeptide having an amino acid sequence of at least 85%, at least 90%, at least 95%, at least 96%, or at least 98% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 9; and wherein the polypeptide comprises an amino acid “N” at position 147.

[0055] In an embodiment, the hydrolase mutant comprises a polypeptide having an amino acid sequence of at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO.l; and wherein the polypeptide comprises an amino acid substitution at position I163R / V / A / L / K / II, preferably I163R. In an embodiment, the hydrolase mutant comprises a polypeptide having an amino acid sequence of at least 85%, at least 90%, at least 95%, at least 96%, or at least 98% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 6; and wherein the polypeptide comprises an amino acid “R” at position 163.

[0056] In an embodiment, the hydrolase mutant comprises a polypeptide having an amino acid sequence of at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO.l; and wherein the polypeptide comprises an amino acid substitution at position S170N / D / T / Q / E, preferably S170N or S170D. In an embodiment, the PET hydrolase mutant comprises a polypeptide having an amino acid sequence of at least 85%, at least 90%, at least 95%, at least 96%, or at least 98% sequence identity to the amino acid sequence as set forth in SEQ ID NO.7; and wherein the polypeptide comprises amino acids “N” or “D” at position 170.

[0057] In an embodiment, the PET hydrolase mutant comprises a polypeptide having an amino acid sequence of at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 1; and wherein the polypeptide comprises an amino acid substitution at position A42R. / V / L / I / T / K, preferably A42R. In an embodiment, the hydrolase mutant comprises a polypeptide having an amino acid sequence of at least 85%, at least 90%, at least 95%, at least 96%, or at least 98% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 8; and wherein the polypeptide comprises an amino acid “R” at position 42.

[0058] In an embodiment, the PET hydrolase mutant comprises a polypeptide having an amino acid sequence of at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 1; and wherein the polypeptide comprises an amino acid substitution at position S161A / V / I / L / T / C / M, preferably S161A. In an embodiment, the hydrolase mutant comprises a polypeptide having an amino acid sequence of at least 85%, at least 90%, at least 95%, at least 96%, or at least 98%sequence identity to the amino acid sequence as set forth in SEQ ID NO. 10; and wherein the polypeptide comprises an amino acid “A” at position 161.

[0059] In an embodiment of the present disclosure, there is provided a hydrolase mutant comprising a polypeptide having an amino acid sequence of at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 1; wherein the polypeptide comprises an amino acid substitution at position 55, preferably P55L / T / S / A / V / I, more preferably P55L; an amino acid insertion adjacent to position 55, wherein the amino acid inserted is selected from Q, N, D, E, T, or S, preferably Q; and an amino acid insertion adjacent to amino acid position 56, wherein the amino acid inserted is selected from Q, N, D, E, T, or S, preferably D.

[0060] In an embodiment, the hydrolase mutant comprises a polypeptide having an amino acid sequence of at least 85%, at least 90%, at least 95%, at least 96%, or at least 98% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 11; wherein the polypeptide comprises an amino acid “L” at position 55; an amino acid insertion adjacent to position 55, wherein the amino acid inserted is Q; and an amino acid insertion adjacent to amino acid position 57 of SEQ ID NO.11, wherein the amino acid inserted is D.

[0061] In an embodiment, the hydrolase mutant comprises a polypeptide having an amino acid sequence of at least 85%, at least 90%, at least 95%, at least 98%, or at least 98% sequence identity to the amino acid sequence as set forth in SEQ ID NO.12; wherein the polypeptide comprises an amino acid “T” at position 55; an amino acid insertion adjacent to position 55, wherein the amino acid inserted is Q; and an amino acid insertion adjacent to amino acid position 57 of SEQ ID NO.12, wherein the amino acid inserted is D.

[0062] In an embodiment of the present disclosure, the hydrolase mutant exhibits increased degrading activity compared to polypeptide having amino acid sequence as set forth in SEQ ID NO. 1 or SEQ ID NO. 25.

[0063] In an embodiment of the present disclosure, there is provided a hydrolase mutant comprising a polypeptide having an amino acid sequence of at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 25or SEQ ID NO. 1; and wherein the polypeptide comprises a mutation selected from the group consisting of: a) an amino acid substitution in at least one amino acid position selected from a group consisting of 136, 35, 163, 170, 42, 147, and 161 of SEQ ID NO. 1; b) an amino acid substitution in at least one amino acid position selected from a group consisting of 148, 114, 13, 125, 141, 20, and 139 of SEQ ID NO. 25; c) an amino acid substitution at a position corresponding to 55 of SEQ ID NO. 1, and an amino acid insertion adjacent to at least one amino acid position selected from 55 and / or 56 of SEQ ID NO. 1; and d) an amino acid substitution at a position corresponding to 33 of SEQ ID NO. 25, and an amino acid insertion adjacent to at least one amino acid position selected from 33 and / or 34 of SEQ ID NO. 25.

[0064] In an embodiment of the present disclosure, there is provided a hydrolase mutant comprising a polypeptide having an amino acid sequence selected from a group consisting of:(a) at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 1; and an amino acid substitution of S136D / T / N / Q / E, preferably S136D ;(b) at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 1; and an amino acid substitution at position S170N / D / T / Q / E, preferably S170N or S170D, of SEQ ID NO. 1;(c) at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 1; and an amino acid substitution at position A35S / V / L / VT7N, preferably A35S, of SEQ ID NO. 1;(d) at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 1; and an amino acid substitution position A I47N / S / V / L / I / T / N, preferably A147S or A147N, of SEQ ID NO. 1;(e) at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 1; and an amino acid substitution at position I163R / V / A / L / K / H, preferably I163R, of SEQ ID NO. 1;(f) at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 1; and an amino acid substitution at position A42R / V / L / VT / K, preferably A42R, of SEQ ID NO. 1;(g) at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 1; and an amino acid substitution at position S161A / V / I / L / T / C / M, preferably S161A, of SEQ ID NO. 1;(h) at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 1; and an amino acid mutation, wherein the amino acid mutation comprises an amino acid substitution at position P55L / T / S / A / V / I, more preferably P55L, an amino acid insertion of Q, N, D, E, T, or S, preferably Q, after the amino acid position 55 , and an amino acid insertion of Q, N, D, E, T, or S, preferably D after the amino acid position 56, of SEQ ID NO. 1;(i) at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 25; and an amino acid substitution at position SI 14D / T / N / Q / E, preferably SI 14D, of SEQ ID NO. 25;(j) at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 25; and an amino acid substitution at position S148N / D / T / Q / E, preferably S148N or S148D, of SEQ ID NO. 25;(k) at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 25; and an amino acid substitution at position A13S / V / L / VT7N, preferably A13S, of SEQ ID NO. 25;(l) at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 25; and an amino acid substitution at position A125N / S / V / L / VT7N, preferably A125S or A125N, of SEQ ID NO. 25;(m)at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 25; and an amino acid substitution at position I141R / V / A / L / K / H, preferably I141R, of SEQ ID NO. 25;(n) at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 25; and an amino acid substitution at position A20R / V / L / I / T / K, preferably A20R, of SEQ ID NO. 25;(o) at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 25; and an amino acid substitution at position S139A / V / I / L / T, preferably S139A, of SEQ ID NO. 25; and(p) at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 25; and an amino acid mutation, wherein the amino acid mutation comprises an amino acid substitution at position P33L / T / S / A / V / I, more preferably P33L, an amino acid insertion of Q, N, D, E, T, or S, preferably Q, after the amino acid position 33, and an amino acid insertion of Q, N, D, E, T, or S, preferably D after the amino acid position 34, of SEQ ID NO. 25.

[0065] In an embodiment of the present disclosure, there is provided a hydrolase mutant comprising a polypeptide, wherein the polypeptide comprises an tag, preferably His-tag (Histidine Tag). The tag, His-tag or any other tag, may be attached at the N-terminal or C-terminal end of the polypeptide. In an embodiment, the His-tag is attached at the N-terminal end of the polypeptide. The tag may facilitate preparation, purification, and / or secretion of the polypeptide or hydrolase mutant.

[0066] In an embodiment of the present disclosure, there is provided a hydrolase mutant comprising a polypeptide having an amino acid sequence selected from a group consisting of:(a) at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 25; and an amino acid substitution at position SI 14D / T / N / Q / E, preferably SI 14D, of SEQ ID NO. 25;(b) at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 25; and an amino acid substitution at position S148N / D / T / Q / E, preferably S148N or S148D, of SEQ ID NO. 25;(c) at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 25; and an amino acid substitution at position A13S / V / L / VT / N, preferably A13S, of SEQ ID NO. 25;(d) at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 25; and an amino acid substitution at position A125N / S / V / L / VT / N, preferably A125S or A125N, of SEQ ID NO. 25;(e) at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 25; and an amino acid substitution at position I141R / V / A / L / K / H, preferably I141R, of SEQ ID NO. 25;(f) at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 25; and an amino acid substitution at position A20R / V / L / I / T / K, preferably A20R, of SEQ ID NO. 25;(g) at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 25; and an amino acid substitution at position S139A / V / I / L / T / C / M, preferably S139A, of SEQ ID NO. 25; and(h) at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 25; and an amino acid mutation, wherein the amino acid mutation comprises an amino acid substitution at position P33L / T / S / A / V / I, more preferably P33L, an amino acid insertion of Q, N, D, E, T, or S, preferably Q, after the amino acid position 33, and an amino acid insertion of Q, N, D, E, T, or S, preferably D after the amino acid position 34, of SEQ ID NO. 25; and wherein the polypeptide comprises a His-tag.

[0067] In an embodiment of the present disclosure, the hydrolase mutant comprises a polypeptide having a sequence of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, or at least 98% sequence identity to the sequence selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36.

[0068] The hydrolase mutant, according to embodiments herein may further comprise a His-tag, optionally one or more protease cleavage site, combinationthereof. In an embodiment, the hydrolase mutant comprises a polypeptide comprising a His-tag and a protease cleavage site, attached at the N-terminal end of the polypeptide. The His-tag and a protease cleavage site, may facilitate secretion of the hydrolase mutant outside the cell. In an embodiment of the present disclosure, the hydrolase mutant comprises a polypeptide having a sequence of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, or at least 98% sequence identity to the sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, wherein each of the polypeptides comprises a His-tag.

[0069] Accordingly, in an embodiment of the present disclosure, the hydrolase mutant comprises a polypeptide having a sequence of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, or at least 98% sequence identity to the sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36.

[0070] In an embodiment of the present disclosure, the mutant comprises a polypeptide having an amino acid sequence of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, or at least 98% sequence identity to the sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, wherein SEQ ID NO: 2 comprises an amino acid substitution of 170N; SEQ ID NO: 3 comprises an amino acid substitution of 136D; SEQ ID NO: 4 comprises an amino acid substitution of 35S; SEQ ID NO: 5 comprises an amino acid substitution of 147S; SEQ ID NO: 6 comprises an amino acid substitution of 163R; SEQ ID NO:7 comprises an amino acid substitution of 170D; SEQ ID NO: 8 comprises an amino acid substitution of 42R; SEQ ID NO: 9 comprises an amino acid substitution of 147N; SEQ ID NO: 10 comprises an amino acid substitution of 161 A; SEQ ID NO: 11 comprises an amino acid substitution of 55L, an insertion of Q adjacent to 55thposition, and an insertion of D adjacent to 57thposition on SEQ ID NO: 11; SEQ ID NO: 12 comprises an amino acid substitution of 55T, an insertion of Q adjacent to 55thposition; and an insertion of D adjacent to 57thposition on SEQ ID NO: 12, SEQ ID NO: 26 comprises an amino acid substitution of 148N; SEQ ID NO: 27 comprises an amino acid substitution of 114D; SEQ ID NO: 28 comprises an amino acid substitution of 13S; SEQ ID NO: 29 comprises an amino acid substitution of 125S; SEQ ID NO: 30 comprises an amino acid substitution of 141R; SEQ ID NO: 31 comprises an amino acid substitution of 148D; SEQ ID NO: 32 comprises an amino acid substitution of 20R; SEQ ID NO: 33 comprises an amino acid substitution of 125N; SEQ ID NO: 34 comprises an amino acid substitution of 139A; SEQ ID NO: 35 comprises an amino acid substitution of 33L, an insertion of Q after 33rdposition, and an insertion of D after 35thposition on SEQ ID NO: 35; and SEQ ID NO: 36 comprises an amino acid substitution of 33T, an insertion of Q after 33rdposition; and an insertion of D after 35thposition on SEQ ID NO: 36.

[0071] Table 1 depicts the details of the sequences of the hydrolase mutants, in accordance with the embodiments herein.

[0072] Table 1: Polypeptide sequences, in accordance with the embodiments herein

[0073] Table 2 depicts the mutations in the amino acid sequences, in accordance with embodiments herein.Table 2:

[0074] In an embodiment of the present disclosure, the mutant is stable at a pH range of 5.0 to 10, preferably pH 6.0 to pH 9, more preferably pH 7.0 to pH 9. and temperature in a range of 30°C to 50°C.

[0075] In an embodiment of the present disclosure, the mutant is stable at a temperature in the range of 20°C to 60°C, preferably 30°C to 50°C.

[0076] In an embodiment of the present disclosure, the mutant is stable at a temperature in the range of 20°C to 60°C, preferably 30°C to 50°C, and at a pH range of 5.0 to 10.

[0077] Embodiments herein provide nucleic acid / polynucleotides encoding the PET hydrolase mutant. The term “nucleic acid” or “polynucleotide”, used interchangeably herein, refers to a combination of nucleotide monomers which are connected to each other through covalent bonds. The term includes to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and mRNA (messenger RNA). In an embodiment of the present disclosure, there is provided a nucleic acid encoding the PET hydrolase mutant as disclosed herein.

[0078] In an embodiment of the present disclosure, there is provided a nucleic acid, wherein the nucleic acid encodes a polypeptide having an amino acid sequence of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, or at least 98% sequence identity to the sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ IDNO: 12, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36.

[0079] In an embodiment of the present disclosure, there is provided a polynucleotide having a sequence of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48.

[0080] In an embodiment of the present disclosure, the polynucleotide has a nucleotide sequence selected from SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50.

[0081] In an embodiment of the present disclosure, there is provided nucleic acid having a sequence of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to a sequence as set forth in SEQ ID NO: 46, wherein the nucleic acid has a sequence as depicted in SEQ ID NO: 49 or SEQ ID NO: 50. The nucleic acid having sequences as depicted in SEQ ID NO: 49 and SEQ ID NO: 50 have a percentage sequence identity of about 55.5 %, and may be used to encode the hydrolase mutant having a polypeptide as set forth in SEQ ID NO: 36. Similarly, it is understood that nucleic acid sequences having percentage identity of about 50% to 60% with the recited nucleic acid sequences such as SEQ ID NO: 38 to 49 may be used to encode the hydrolase mutants as described herein.

[0082] Embodiments herein provide a recombinant vector for expressing the hydrolase mutant. In an embodiment of the present disclosure, there is provided a recombinant vector comprising the nucleic acid encoding the hydrolase mutant as disclosed herein.

[0083] The term “recombinant vector” as used herein, refers to an engineered or artificially synthesized expression vector capable of carrying the nucleic acid encoding the hydrolase mutant, and expressing the protein encoded by the nucleic acid in a host cell. Vectors may be derived from plasmids, bacteriophages, viruses, cosmids, and the like. Typically, the choice of the vector depends on the compatibility of the vector with the host cell into which the vector is to be introduced. Examples of vectors include, but is not limited to, pET vector selected from pET-3 series, pET-11 series, pET-15 series, pET28 series, pET-32 series, pET41 series, pET43 series, and pET-duet vectors.

[0084] In an embodiment of the present disclosure, there is provided a recombinant vector comprising a nucleic acid, wherein the nucleic acid encodes a polypeptide having an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, or at least 98% sequence identity to the sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36.

[0085] In an embodiment of the present disclosure, there is provided a recombinant vector comprising a nucleic acid having a nucleotide sequence of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80% at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50.

[0086] The vector may further comprise a selectable marker gene, to facilitate selectable marker genes, for example antibiotic resistance genes which confer the ability to grow in the presence of the appropriate antibiotic. Examples of such selectable marker genes include genes resistance to antibiotic such as hygromycin, ampicillin, tetracycline, kanamycin, puromycin, bleomycin, streptomycin, and so on. The recombinant vector comprising the nucleic acid may further comprise a promoter operably linked to the nucleic acid for expression of the hydrolase mutant according to embodiments herein. In an example, the promoter may be capable of expression in prokaryotic cells or eukaryotic cells. Examples of prokaryotic promoters include, but are not limited to, SP6, T7, T5, tac, bla, trp, gal, lac, or maltose promoters. Examples of eukaryotic promoters include, but are not limited to, viral promoters such as CMV, SV40, or hsp70 promoter.

[0087] The term “host cell” as used herein, refers to a cell into which the recombinant vector is introduced for the expression of the protein encoded by the nucleic acid. In an example, the host cell is a prokaryotic cell or eukaryotic cell. In an embodiment, the host cell is selected from E. coli, Streptomyces spp., Bacillus spp., Staphylococcus spp., Saccharomyces cerevisea, Pichia pastoris, A. niger, A. oryzae, Trichoderma species such as T. reesei or mammalian cell.

[0088] In an embodiment of the present disclosure, there is provided a host cell comprising the recombinant vector or the nucleic acid as described herein, wherein the nucleic acid encodes a polypeptide having an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, or at least 98% sequence identity to the sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36.

[0089] In an embodiment of the present disclosure, there is provided a host cell comprising the recombinant vector or the nucleic acid as described herein, wherein the nucleic acid has a nucleotide sequence of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80% at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48.

[0090] In an embodiment of the present disclosure, the host cell is selected from bacteria, fungi, insect cell, plant cell, or animal cell.

[0091] In an embodiment of the present disclosure, there is provided a host cell comprising the recombinant vector as disclosed herein.Method

[0092] Embodiments herein provide a method of producing the hydrolase mutant as disclosed herein.

[0093] In an embodiment of the present disclosure, the method of producing the PET hydrolase mutant comprises culturing the host cell as described herein under condition suitable to express the hydrolase mutant encoded by the nucleic acid as disclosed herein. In another embodiment of the present disclosure, the host cell is bacteria. In yet another embodiment of the present disclosure, the host cell is selected from Escherichia coli, like E.coli BL21(DE3), BL21(DE3) codon+REL, BL21(DE3) codon+RP, BL21(DE3) codon+RIPL, T7 Shuffle, T7 Shuffle express, Origami(DE3), or Rosettagami (DE3).

[0094] In an embodiment of the present disclosure, the method comprises culturing the host cell as described herein in the presence of the recombinant vector comprising the nucleic acid encoding the hydrolase mutant. In another embodiment of the present disclosure, the recombinant vector is a pET vector.

[0095] In an embodiment of the present disclosure, the recombinant vector comprising the nucleic acid encoding the hydrolase mutant is obtained using site directed mutagenesis.

[0096] The term “site directed mutagenesis” refers to a polymerase chain reaction (PCR)-based method to create specific, targeted changes in specified nucleotides of a sequence within a plasmid vector, using custom designed oligonucleotide primers.

[0097] In an example, the custom designed oligonucleotide primer comprises the mutation to the nucleic acid encoding the hydrolase, in respect of the amino acid substitutions as disclosed herein, and / or the amino acid insertions as disclosed herein, in the polypeptide of the hydrolase having amino acid sequence as set forth in SEQ ID NO. 1 or SEQ ID NO. 25.

[0098] In an embodiment of the present disclosure, the recombinant vector comprising the nucleic acid encoding the hydrolase is subjected to site directed mutagenesis using the custom designed oligonucleotide primers to obtain the recombinant vector comprising the nucleic acid encoding the hydrolase mutants.

[0099] In an embodiment of the present disclosure, there is provided a method of producing the hydrolase mutant as disclosed herein, comprising culturing the host cell, wherein the host cell comprises the nucleic acid encoding the hydrolase mutant, wherein the nucleic acid has a sequence of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80% at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48, to express the hydrolase mutant. In an embodiment, the hydrolase mutant is secreted outside the cell.

[0100] The hydrolase mutant, according to embodiments herein, may be dried, spray-dried, lyophilized, granulated, and / or stabilized for storage. In an embodiment, the hydrolase mutant may be solubilized in water or buffer. Thehydrolase mutant may also be crystallized or isolated or purified in accordance with conventional methods, such as filtration, extraction, precipitation, chromatography, affinity chromatography, or electrophoresis.

[0101] In an embodiment of the present disclosure, there is provided a method of producing the hydrolase mutant as disclosed herein, comprising culturing the host cell, as described herein, comprising the recombinant vector having the nucleic acid encoding the hydrolase mutant, and recovering the hydrolase enzyme from the host cell culture. The recovery of the hydrolase enzyme from the host cell culture may be performed by downstream processing.

[0102] The term “downstream processing” refers to a series of purification steps, including, for example, separation of the solid-liquid phase, release of intracellular products or separation from extracellular products, purification, and concentration to derive a pure and homogeneous protein product (mutant) from the host cell. Such methods are generally known to a person skilled in the art and may be used in purification of the mutant enzyme.

[0103] In an embodiment of the present disclosure, the obtained cell culture was subjected to downstream processing, wherein separation of the solid-liquid phase is through centrifugation or filtration followed by separation of the solid phase (cells), release of intracellular products is through cell lysis aided by sonication or homogenisation or freeze thaw or other chemical or enzymatic methods purification is through differential solubility (ammonium sulfate precipitation) or chromatographic techniques (affinity, ion exchange, hydrophobic, and size exclusion chromatography), preferably affinity chromatography, and concentration is through precipitation or ultrafiltration using semipermeable membrane or centrifugal force or freeze-crying or spray drying to obtain pure hydrolase mutants.

[0104] The hydrolase mutant enzyme may be used directly or in purified form, either alone or in combination with other enzymes for catalysing a reaction involving degradation of polyester or polyester material. The hydrolase mutant may be used in soluble form or may be immobilised on a support like resin, glass, polymer, nanostructures, etc or bound to lipid vesicles. Embodiments herein provide a composition comprising the hydrolase mutant as described herein, andoptionally an additive, wherein the additive is selected from stabilizers, buffers, solvent, or combinations thereof.

[0105] In another embodiment, there is provided a composition, wherein the composition comprises at least one hydrolase mutant as described herein, and at least one additive.

[0106] In an embodiment, there is provided a composition, wherein the composition comprises at least one hydrolase mutant; and at least one additive, wherein the hydrolase mutant comprises a polypeptide having a sequence of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, or at least 98% sequence identity to the sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36.

[0107] In an embodiment, there is provided a composition, wherein the composition comprises a combination of at least 2 hydrolase mutant; and at least one additive, wherein the hydrolase mutant comprises a polypeptide having a sequence of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, or at least 98% sequence identity to the sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36.

[0108] The additive may be selected from buffers, stabilisers, a preservatives, salts, surfactants, amphipathic agents, or combination thereof.

[0109] In an embodiment, the hydrolase mutant is solubilized in water or buffer.

[0110] Accordingly, in an embodiment, there is provided a composition, wherein the composition comprises the hydrolase mutant, and at least one solvent, preferably an aqueous solvent, more preferably water.

[0111] In an embodiment, there is provided a composition, wherein the stabiliser is selected from glycerol, sucrose, trehalose, polyethylene glycol (PEG), arginine, glycine, imidazole, EDTA or combination thereof.

[0112] In an embodiment, there is provided a composition, wherein the salt is selected from sodium chloride, calcium chloride, magnesium chloride, calcium chloride, nickle sulphate, or combination thereof.

[0113] In an embodiment, there is provided a composition, wherein the amphipathic agent is selected from sodium dodecyl sulfate (SDS), Tween-20, or combination thereof.

[0114] In an embodiment, there is provided a composition, wherein the buffer is selected from 2-(N-morpholino)ethanesulfonic acid (MES), BIS-Tris, 2,2'-[(2- amino-2-oxoethyl)azanediyl]diacetic acid (ADA), N-(2-acetamido)-2- aminoethanesulfonic acid (ACES), piperazine-N,N'-bis(2-ethanesulfonic acid (PIPES), 3 -morpholinopropanesulfonic acid (MOPSO), Bis-Tris Propane, N,N- Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 3-(N- morpholino)propanesulfonic acid (MOPS), Tris (hydroxymethyl) aminomethane (TES), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 3-(N- morpholino)propanesulfonic acid (MOBS), 3 -(Bi s(2 -Hydroxy ethyl) Amino)-2- Hydroxypropane-1 -Sulfonic Acid (DIPSO), TRIS, 2-Hydroxy-3- [tris(hydroxymethyl)methylamino]-l-propanesulfonic acid, N-[Tris(hydroxymethyl)methyl]-3-amino-2-hydroxypropanesulfonic acid (TAPSO), 2 -Hydroxy-3 -(4-(2-hydroxyethyl)piperazin- 1 -yl)propane- 1 -sulfonic acid(HEPPSO), Piperazine-l,4-bis(2-hydroxy-3-propanesulfonic acid), dihydrate (POPSO), Tris-acetate-EDTA (TEA), Phosphate, phosphate buffered saline (PBS), glycine, bicine, N-(2-Hydroxyethyl)piperazine-N'-(4-butanesulfonic acid (HEPBS), tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS), 2- amino-2-methyl-l -propanol (AMPD), N-tris(Hydroxymethyl)methyl-4- aminobutanesulfonic acid (TABS), N-(l,l-Dimethyl-2-hydroxyethyl)-3-amino-2- hydroxypropanesulfonic acid (AMPSO), 2-(Cyclohexylamino)ethanesulfonic acid (CHES), 3 -(Cyclohexylamino)-2-hydroxy-l -propanesulfonic acid (CAPSO), Adenosine 5 ’-monophosphate disodium salt (AMP), or combinations thereof

[0115] In another embodiments, there is provided a composition, wherein the composition comprises the hydrolase mutant; and an additive selected from glycerol, sorbitol, dextrin, starch, glycol (for eg: propanediol), salt, or combination thereof. Table 3 depicts an exemplary composition, in accordance with embodiments herein.Table 3: depicts an exemplary composition:

[0116] The composition, according to embodiments herein, may be dried, spray- dried or lyophilized, granulated, and / or stabilized for storage. In an embodiment, the may be present in the form of a liquid composition or a solid composition, selected from solution, dispersion, paste, powder, granule, coated granules, tablet, cake, crystal, slurry, gel, or pellet.

[0117] In an embodiment, the composition is a dry composition. For example, the composition may be a lyophilised composition, freeze-dried composition, spray- dried composition, etc. Various methods for drying are well known to the one skilled in the art and may be used in embodiments herein. Examples of such methods include, but is not limited to, lyophilisation, freeze-drying, spray-drying, supercritical drying, down-draught evaporation, thin-layer evaporation, centrifugal evaporation, conveyer drying, fluidized bed drying, drum drying, or any combination thereof.

[0118] Embodiments herein provide use of the hydrolase mutants as disclosed herein, or the composition comprising a hydrolase mutant as disclosed herein, in degradation of polyester or polyester material. In an embodiment, the polyester is PET and the polyester material is PET material. In an embodiment of the present disclosure, the PET material is a post-consumer polyester or polyester material. Inanother embodiment of the present disclosure, the PET material is post-consumer PET waste, having a crystallinity in the range of 1% to 50%, preferably 15.4%.

[0119] Embodiments herein provide a method of degrading polyester or polyester material.

[0120] In an embodiment of the present disclosure, the method of degrading polyester or polyester material comprises contacting the polyester or polyester material with at least one of the hydrolase mutants as disclosed herein, or the composition comprising at least one of the hydrolase mutants as disclosed herein, in a reaction mixture, and allowing degradation of the polyester or polyester material.

[0121] In an embodiment of the present disclosure, the method of degrading polyester or polyester material comprises contacting the polyester or polyester material with at least one of the hydrolase mutants as disclosed herein, or the composition comprising at least one of the hydrolase mutants as disclosed herein, in a reaction mixture, and incubating to allow degradation of the polyester or polyester material.

[0122] In an embodiment of the present disclosure, there is provided a method for degradation of the polyester or polyester material, wherein the reaction mixture comprises a buffer selected from 2-(N-morpholino)ethanesulfonic acid (MES) buffer, BIS-Tris buffer, 2,2'-[(2-amino-2-oxoethyl)azanediyl]diacetic acid (ADA) buffer, N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) buffer, piperazine- N,N'-bis(2-ethanesulfonic acid (PIPES) buffer, 3 -morpholinopropanesulfonic acid (MOPSO) buffer, Bis-Tris Propane buffer, N,N-Bis(2-hydroxyethyl)-2- aminoethanesulfonic acid (BES) buffer, 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, Tris (hydroxymethyl) aminomethane (TES) buffer, N-2- hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer, 3-(N- morpholino)propanesulfonic acid (MOBS) buffer, 3 -(Bis(2 -Hydroxy ethyl)Amino)- 2-Hydroxypropane-l -Sulfonic Acid (DIPSO) buffer, TRIS buffer, 2-Hydroxy-3- [tris(hydroxymethyl)methylamino]-l-propanesulfonic acid buffer, N- [Tris(hydroxymethyl)methyl]-3-amino-2-hydroxypropanesulfonic acid (TAPSO) buffer, 2-Hydroxy-3-(4-(2-hydroxyethyl)piperazin-l-yl)propane-l-sulfonic acid(HEPPSO) buffer, Piperazine- l,4-bis(2-hydroxy-3 -propanesulfonic acid) dihydrate (POPSO) buffer, Tris-acetate-EDTA (TEA) buffer, Phosphate buffer, phosphate buffered saline (PBS) buffer, glycine buffer, bicine buffer, N-(2- Hydroxyethyl)piperazine-N'-(4-butanesulfonic acid (HEPBS) buffer, tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS) buffer, 2-amino-2- m ethyl- 1 -propanol (AMPD) buffer, N-tris(Hydroxymethyl)methyl-4- aminobutanesulfonic acid (TABS) buffer, N-(l,l-Dimethyl-2-hydroxyethyl)-3- amino-2-hydroxypropanesulfonic acid (AMPSO) buffer, 2-(Cyclohexylamino)ethanesulfonic acid (CHES) buffer, 3-(Cyclohexylamino)-2- hydroxy-1 -propanesulfonic acid (CAPSO) buffer, Adenosine 5 ’-monophosphate disodium salt (AMP) buffer, or combinations thereof.

[0123] In an embodiment of the present disclosure, the method of degrading polyester or polyester material comprises contacting polyester or polyester material with the hydrolase mutant as disclosed herein, in a reaction mixture; and incubating to allow degradation of the polyester or polyester material.

[0124] In an embodiment of the present disclosure, there is provided a method of degrading polyester or polyester material, wherein the method is performed at a temperature in the range of 20°C to 60°C, preferably 30°C to 50°C, and pH in the range of pH 5.0 to pH 10, preferably pH 7.0 to pH 9.0.

[0125] In an embodiment of the present disclosure, there is provided a method of degrading polyester or polyester material, wherein the hydrolase mutant and polyester or polyester material is in a weight ratio range of lng: lmg or 75ng: lmg to 6pg: Img, or wherein the hydrolase mutant and polyester or polyester material is in a weight ratio range of of 1 x IO"6: 1 to 6 x 10"3: 1, or 75 x IO"6: 1 to 6 x 10"3: 1.

[0126] In an embodiment of the present disclosure, the method further comprises recovering monomers and / or oligomers resulting from degradation of polyester or polyester material, for reuse. In an embodiment of the present disclosure, the monomers are selected from ethylene glycol, terephthalate mono-2-hydroxy ethyl terephthalate (MEET), bis (2-hydroxyethyl) terephthalate peaks, or combinations thereof.

[0127] Although the subject matter has been described with reference to specific embodiments, this description is not meant to be construed in a limiting sense. Various modifications of the disclosed embodiments, as well as alternate embodiments of the subject matter, will become apparent to persons skilled in the art upon reference to the description of the subject matter. It is therefore contemplated that such modifications can be made without departing from the spirit or scope of the present subject matter as defined.EXAMPLES

[0128] The disclosure will now be illustrated with following examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.Materials

[0129] The wild type PET hydrolase gene (SEQ ID NO. 25) cloned in pET- 28a(+)-TEV with N-terminal His tag, sequence was synthesised from Genscript and obtained via Biotech Desk Pvt. Ltd. The plasmid extraction kit was procured from Macherey -Nagel. The Ni-NTA agarose resins were procured from Genetix Biotech Asia Pvt. Ltd. The centrifugal filter units (centricons) were procured from Merck. The chemicals used were procured from Sisco Research Laboratory India Pvt. Ltd., Sigma Aldrich, HiMedia, GBioscience, Thermo Fisher, Genetix Biotech Asia Pvt. Ltd., VNIR Biotechnologies Pvt. Ltd., Goodfellow, BLD Pharmatech Pvt. Ltd., Qualigens, Changshu Hongsheng Fine chemical. Pippettes from Eppendorf, Central Drug House Ltd., Gilson and pFact, plastic wares from Tarsons and glass wares from Borosil.EXAMPLE 1: Method of producing the hydrolase mutant

[0130] This example describes the production of the hydrolase mutants, comprising a mutation as described herein. The mutants were generated using site directed mutagenesis (SDM) of the wild type sequence (SEQ ID NO. 25). A machine learning pipeline developed in-house, as also described in patent application no: IN 202441010189 which is incorporated herein in its entirety, was used to discern a list of potential residues that can be mutated using SDM.Site Directed Mutagenesis:

[0131] Cloning. The synthetic gene cloned in commercially available plasmid pET-28a(+)-TEV (5.4kb), (Figure 1) was used as a template for the PCR reactions. The wild type PET hydrolase gene (SEQ ID NO. 25) was cloned into the plasmid as per the manufacturer’s protocol and was further subjected to PCR using standard protocol to obtain the PET hydrolase mutants.

[0132] PCR: Forward and reverse primers for the corresponding mutants were designed using the free version of Snapgene Viewer and a lOpM stock was used for all the PCR reactions. The PCR cycling conditions were denaturation at 98°C for 30s, annealing, for 60s (35 cycles), extension at 72°C for 2.5 min and a final extension at 72°C for 15 min to obtain the corresponding mutants. The obtained mutants were confirmed using agarose-based electrophoresis (Figure 2).

[0133] Transformation (DH5a): Commercially available E. coli DH5a cells were transformed with the wild type gene and mutants using standard transformation protocol. The obtained mutants were subjected to Dpnl (0.5pl) digestion at 37°C, overnight and 5 pl of the digested product was transformed into the DH5a cells using heat shock method and plated on LB plate with 50pg / ml Kanamycin, incubated at 37°C, overnight. The obtained single colonies were further inoculated in LB with 50pg / ml Kanamycin and incubated at 37°C, overnight. Plasmids were isolated using the NucleoSpin Plasmid, Mini kit for plasmid DNA from Macherey-Nagel and were sequenced to re-confirm.

[0134] Protein expression: Protein expression was carried out in a E. coli expression strain (Shuffle T7). The sequence confirmed plasmids were transformed into E. coli expression strain using standard protocol and were plated onto LB with 50pg / ml Kanamycin and 25pg / ml streptomycin, incubated overnight at 30°C. Single colonies were further inoculated in LB with 50pg / ml Kanamycin and incubated at 30°C overnight, at 180rpm until the ODeoo reached 0.6-0.8. At 0.6-0.8 OD, the culture was induced using 0.5mM IPTG and incubated at 30°C overnight, at 180rpm to obtain cells containing the expressed mutant protein.

[0135] Downstream processing: The cells containing the expressed mutant protein were harvested (centrifugation at 5000 rpm for 20 minutes at 4°C) and resuspended in 10ml of lysis buffer containing 50mM Tris (pH 7.5), 300mM NaCl and vortexed thoroughly. The cells were sonicated (5s ON / lOs OFF) until complete cell lysis and centrifuged at 9000 rpm for 60 minutes at 4°C, to obtain a supernatant. The supernatant was further purified using affinity chromatography, with NiNTA (nickel -nitrilotri cetic acid) agarose resins as matrix at a flow rate of ~0.5ml per minute and the bound proteins were eluted using an elution buffer containing 50mM Tris (pH 7.5), 300mM NaCl and 300 mM Imidazole to obtain eluted proteins. The eluted proteins were further concentrated using lOkDa cut off centrifugal filter units and the imidazole was subsequently removed using an exchange buffer containing 50mM Tris (pH 7.5) and 300mM NaCl. The eluted proteins (hydrolase mutants) were verified using SDS-PAGE. The PET hydrolase mutants were further evaluated for their PET degradation activity.

[0136] The nucleotide of sequence as depicted in: SEQ ID NO: 13 was used to express PM-29 mutant, SEQ ID NO: 14 was used to express PM-31, SEQ ID NO: 15 was used to express the PM-36, SEQ ID NO: 16 was used to express PM-37, SEQ ID NO: 17 was used to express PM-44, SEQ ID NO: 18 was used to express PM-47, SEQ ID NO: 19 was used to express PM-53, SEQ ID NO: 20 was used to express PM-59, SEQ ID NO:21 was used to express SEQ ID NO: 22 was used to express PM-60, SEQ ID NO: 23 was used to express PM-916, and SEQ ID NO: 24 was used to express PM-917.EXAMPLE 2: Method of degrading PET using the hydrolase mutants.

[0137] The hydrolase mutants produced using the method described in Example 1 was used in a PET degradation assay to evaluate their efficiency in degrading a post-consumer PET waste. The post-consumer PET waste (PET material) was obtained from a local vendor. The crystallinity of the post-consumer PET material and an amorphous PET (used as a standard) was determined using differential scanning calorimetry analysis, before carrying out the degradation assay. The crystallinity of the amorphous PET, the post-consumer cake container PET and post consumer biscuit container PET material was found to be 7.5% (Figure 3(a)), 13.9% (Figure 3(b), and 15.4% (Figure 3(c) respectively (Table 4). 100Table 4: depicts crystallinity of the post-consumer PET material and amorphousPET.PET degradation assay:

[0138] The assay is an absorbance-based method measured at 240nm, using UV spectroscopy. The amorphous PET from GoodFellows was used for screening experiments. The absorbance indicates the amount of the degradation corresponding to the degraded product release (terephthalic acid equivalents).

[0139] For the assay, ~5mg (5mm disc) of PET material was contacted with 400nM of the PM-29 mutant in 50mM Tris (pH 7.5) and 300mM NaCl buffer in the presence of IM phosphate buffer pH 8 containing in a reaction volume of 500pL at 40°C. The reaction was incubated at a constant temperature with continuous mixing at 500rpm in an Eppendorf thermal mixer. The absorbance at 240nm corresponding to the release of the products was measured after contact with thePM-29 mutant after 8days. The reaction mixture on 8thday was filtered to separate any remaining PET sample, heat treated, spinned and sent for HPLC analysis using Cl 8 column.

[0140] Similarly, the assay was performed for using the mutants: PM-31, PM-36, PM-37, PM-44, PM-47, PM-53, PM-59, PM-60, PM-916, and PM-917, by contacting with the PET material, and absorbance was measured for each mutant after 8thday.Results

[0141] It was observed that mutants of the present disclosure, represented as PM- 29, PM-31, PM-36, PM-37, PM-44, PM-47, PM-53, PM-59, PM-60, PM-916, and PM-917 showed degradation better than the wild type (Figure 4(a)), without preprocessing of the substrate, with PM-916 and PM-917 showing complete degradation on 8thday and having the highest degradation efficiency (Figure 4(a)). The Figures 4(b), 4(c), 4(d), 4(e), 4(f), 4(g), 4(h), 4(i), 4(j), 4(k), 4(1), 4(m), 4(n), 4(o), and 4(p) are HPLC chromatograms depicting results of degradation assay, wherein Figure 4(b)-4(d) depicts standard terephthalic acid peak, mono (2- hydroxyethyl) terephthalate peak, and bis (2 -hydroxy ethyl) terephthalate peaks, respectively, and wherein Figure 4(e)-4(o) depicts HPLC data for samples PM-29, PM-31, PM-36, PM-37, PM-44, PM-47, PM-53, PM-59, PM-60, PM-916, and PM- 917, respectively, and Figure 4(p) depicts HPLC data for the wildtype enzyme (Wt).EXAMPLE 3: Effect of mutant enzyme concentration dependencies on Enzyme activity

[0142] The assay is an absorbance-based method measured at 240nm, using UV spectroscopy. The amorphous PET from GoodFellows was used for screening experiments. The absorbance indicates the amount of the degradation corresponding to the degraded product release (terephthalic acid equivalents).

[0143] Procedure: For the assay, ~5mg (5mm disc) of PET material was contacted with 25nM to lOOOnM of the PM-29 mutant in 50mM Tris (pH 7.5) and 300mM NaCl buffer in the presence of IM phosphate buffer pH 8 containing in a reaction volume of 500pL at 40°C. The reaction was incubated at a constant temperaturewith continuous mixing at 500rpm in an Eppendorf thermal mixer. The absorbance at 240nm corresponding to the release of the products was measured after contact with the PM-29 mutant every 24h. Similarly, the assay was performed for using the mutants: PM-31, PM-36, PM-37, PM-44, PM-47, PM-53, PM-59, PM-60, PM-916, and PM-917, by contacting with the PET material, and absorbance was measured for each mutant after 24h.

[0144] Result: Figure 5 depicts the degradation trend of all mutants, i.e. PM-29, PM-31, PM-36, PM-37, PM-44, PM-47, PM-53, PM-59, PM-60, PM-916, and PM- 917 wrt concentration. All the mutants show degradation activity starting from 25nM upto lOOOnM (Figure 5), with the highest activity ranging between 200nM and lOOOnM for different mutants.EXAMPLE 4: Effect of DH on Enzyme activity

[0145] The assay is an absorbance-based method measured at 240nm, using UV spectroscopy. The amorphous PET from GoodFellows was used for screening experiments. The absorbance indicates the amount of the degradation corresponding to the degraded product release (terephthalic acid equivalents).

[0146] Procedure: For the assay, ~5mg (5mm disc) of PET material was contacted with 400nM of the PM-29 mutant in 50mM Tris (pH 7.5) and 300mM NaCl buffer in a IM phosphate buffer with pH 5, pH 6, pH 7, pH 8, pH 9 and pH 10 in a reaction volume of 500pL at 40°C. The reaction was incubated at a constant temperature with continuous mixing at 500rpm in an Eppendorf thermal mixer. The absorbance at 240nm corresponding to the release of the products was measured after contact with the PM-29 mutant after 24h. Similarly, the assay was performed for using the mutants: PM-31, PM-36, PM-37, PM-44, PM-47, PM-53, PM-59, PM-60, PM-916, and PM-917, by contacting with the PET material, and absorbance was measured for each mutant after 24h.

[0147] Result: Figure 6 depicts effect of buffer pH on the activity of the mutants PM-29, PM-31, PM-36, PM-37, PM-44, PM-47, PM-53, PM-59, PM-60, PM-916, and PM-917 on the PET sample. All the mutants show trace activity at pH 5 and pH 6 and significant activity between pH 7 and 9.

[0148] Comparative studies were performed with other Mutant enzymes which performed poorly compared to Wt, having a deletion mutation in positions 134- 138 (depicted as PM-10) position in respect of SEQ ID NO. 1, a substitution mutant E269P position in respect of SEQ ID NO. 1 (depicted as PM- 13), or a substitution mutant Ti l ID position in respect of SEQ ID NO. 1 (depicted as PM-55), independently, the wild type enzyme (depicted as Wt) and PM-916.

[0149] Procedure: The assay is an absorbance-based method measured at 240nm, using UV spectroscopy. The amorphous PET from GoodFellows was used for screening experiments. The absorbance indicates the amount of the degradation corresponding to the degraded product release (terephthalic acid equivalents).

[0150] For the assay, ~5mg (5mm disc) of PET material was contacted with 400nM of the PM-10 mutant in 50mM Tris (pH 7.5) and 300mM NaCl buffer in a IM phosphate buffer with pH 8 in a reaction volume of 500pL at 40°C. The reaction was incubated at a constant temperature with continuous mixing at 500rpm in an Eppendorf thermal mixer. The absorbance at 240nm corresponding to the release of the products was measured after contact with the PM-10 mutant after 8 days. Similarly, the assay was performed for using the mutants: PM-13, PM-55, Wt, PM- 916, by contacting with the PET material, and absorbance was measured for each mutant after 8 days.

[0151] Results: Figure 7 depicts the comparison of the degradation efficiency of PM-916 wrt to Wt and the poorly performing mutants PM-10, PM-13 and PM-55. PM-916 degraded the sample completely and performed best.EXAMPLE 6: Effect of temperature on PET degradation

[0152] Amorphous PET sheet procured from GoodFellows with crystallinity less than 15% was used as sample. The sheet was washed with filtered (MilliQ) water, dried, cut into small discs and used for degradation reaction. The degradation reaction was carried out in a IM phosphate buffer of pH 8.

[0153] For the assay, ~5mg (5mm disc) of PET material was contacted with 400nM of the PM-10 mutant in 50mM Tris (pH 7.5) and 300mM NaCl buffer in a IM phosphate buffer with pH 8 in a reaction volume of 500pL at 20°C, 30°C, 40°C, 50°C and 60°C. The reaction was incubated at a constant temperature with continuous mixing at 500rpm in an Eppendorf thermal mixer. The absorbance at 240nm corresponding to the release of the products was measured after contact with the PM-10 mutant after 24h. Similarly, the assay was performed for using the mutants: PM-13, PM-55, Wt, PM-916, by contacting with the PET material, and absorbance was measured for each mutant after 24h.

[0154] Results: Figure 8 shows that the majority of PET hydrolase mutants are active between 20°C to 50°C, while some are active at 60°C.

[0155] Observation: All mutants have higher activity than the wild type enzyme at all temperatures studied.EXAMPLE 7: Weight reduction test of PM-916 on a post-consumer PET

[0156] The PM-916 mutant was used to test the degradation and weight reduction of a post-consumer two PET samples from an FMCG which included material such as cookie, cake container tray. Degradation conditions are as mentioned earlier. Preprocessing of samples was performed by cleaning the post-consumer PET samples and washing using filtered (MilliQ) water to remove dirt and manually cutting into small pieces. The weight of the post-consumer PET samples was measured before the start of the reaction. 750Mg of post consumer PET sample was added to a reaction volume of 100ml containing IM phosphate buffer at pH 8, to which the PET hydrolase mutant PM-916 was added to a final concentration of 400nM. The reaction was kept at constant temperature of 40°C on a magnetic stirrer, with constant stirring at 500rpm. After 17 hrs of reaction, the reaction mixtures was filtered to separate the remaining sample. The remaining sample was dried on a blotting paper and kept at 37°C for 30min to Ihr for complete drying before checking the weight. The weight of remaining sample was 49mg. The percentage reduction in the weight was at least 93.4% (as depicted in Table 5) .Table 5: depicts percentage reduction in the weightEXAMPLE 8: Assay for the comparative quantification of the activity of the hydrolase mutant with respect to the wild type enzyme.

[0157] Procedure: The assay is an absorbance-based method measured at 240nm, using UV spectroscopy. A post-consumer PET sample from an FMCG was used for specific activity experiments. The absorbance indicates the amount of the degradation corresponding to the degraded product release (terephthalic acid equivalents).

[0158] For the assay, ~500mg of PET material was contacted with 400nM of the PM-916 mutant in 50mM Tris (pH 7.5) and 300mMNaCl buffer in a IM phosphate buffer with pH 8 in a reaction volume of 50ml at 40°C. The reaction was incubated at a constant temperature with continuous mixing at 500rpm on a magnetic stirrer. The absorbance at 240nm corresponding to the release of the products was measured after contact with the PM-916 mutant every hour. Similarly, the assay was performed for using the wild type enzyme by contacting with the PET material, and absorbance was measured every hour. Both experiments were conducted in triplicates.

[0159] Standard TPA plot: Different concentration of terephthalic acid dissolved in the same buffer used for the assay was prepared for standard plot. The absorbance of each was measured on a nanodrop at 240nm. Absorbance vs concentration plot was plotted.

[0160] Using the standard plot, the concentration and amount of TPA-equivalent in the sample (i.e. PM-916 and Wt) reaction mixture were calculated.

[0161] Results: The mean and standard deviation of the specific activity of PM- 916 was 242.97 ± 6.3 pmolTPA-eqh'1rngenzyme'1and that of Wt enzyme was 20.89 ± 4.1 pmol pA-eq h'1rngenzyme'1. PM-916 is ~11 times or more efficient than the wildtype enzyme or the specific activity of PM-916 compared to wild type is 1163.09% ± 30.1.ADVANTAGES OF THE PRESENT DISCLOSURE

[0162] The present disclosure provides a PET (polyethylene terephthalate) hydrolase mutant, with the following advantages: a. Enzyme was capable of degrading post-consumer PET waste at 40°C, which consumes less energy than enzymes which functions at higher temperature or other PET recycling technologies. b. It can depolymerize certain post-consumer PET, with a crystallinity of up to 15.4%, in 17 hours up to 90% to 100% without amorphization of substrate. c. The enzyme exhibited tolerance to lower pH. d. Enzyme was stable for at least 10 days.

Claims

I / We claim:

1. A hydrolase mutant, comprising a polypeptide having an amino acid sequence of at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 25 or SEQ ID NO. 1; and wherein the polypeptide comprises a mutation selected from the group consisting of: a) an amino acid substitution in at least one amino acid position selected from a group consisting of 136, 35, 163, 170, 42, 147, and 161 of SEQ ID NO. 1; b) an amino acid substitution in at least one amino acid position selected from a group consisting of 148, 114, 13, 125, 141, 20, and 139 of SEQ ID NO. 25; c) an amino acid substitution at a position corresponding to 55 of SEQ ID NO. 1, and an amino acid insertion adjacent to at least one amino acid position selected from 55 and / or 56 of SEQ ID NO. 1; and d) an amino acid substitution at a position corresponding to 33 of SEQ ID NO. 25, and an amino acid insertion adjacent to at least one amino acid position selected from 33 and / or 34 of SEQ ID NO. 25.

2. The hydrolase mutant as claimed in claim 1, wherein the mutant has increased polyester degrading activity compared to polypeptide having an amino acid sequence as set forth in SEQ ID NO. 1 or SEQ ID NO. 25.

3. The hydrolase mutant as claimed in claim 1, wherein the amino acid substitution is selected from the group consisting of: i) substitution of amino acid residue at position 136 with D, T, N, Q or E, at position corresponding to SEQ ID NO. 1; ii) substitution of amino acid residue at position 35 with S, V, L, I, T, or N, at position corresponding to SEQ ID NO. 1; iii) substitution of amino acid residue at position 163 with R, V, A, L, K or H, at position corresponding to SEQ ID NO. 1; iv) substitution of amino acid residue at position 170 with N, D, T, Q, or E, at position corresponding to SEQ ID NO. 1;v) substitution of amino acid residue at position 42 with R, V, L, I, T, or K, at position corresponding to SEQ ID NO. 1; vi) substitution of amino acid residue at position 147 with N, S, V, L, I, T, or N, at position corresponding to SEQ ID NO. 1; vii) substitution of amino acid residue at position 161 with A, V, I, L, C, M or T, at position corresponding to SEQ ID NO. 1; viii) substitution of amino acid residue at position 114 with D, T, N, Q or E, at position corresponding to SEQ ID NO. 25; ix) substitution of amino acid residue at position 13 with S, V, L, I, T, or N, at position corresponding to SEQ ID NO. 25; x) substitution of amino acid residue at position 141 with R, V, A, L, K or H, at position corresponding to SEQ ID NO. 25; xi) substitution of amino acid residue at position 148 with N, D, T, Q, or E, at position corresponding to SEQ ID NO. 25; xii) substitution of amino acid residue at position 20 with R, V, L, I, T, or K, at position corresponding to SEQ ID NO. 25; xiii) substitution of amino acid residue at position 125 with N, S, V, L, I, T, or N, at position corresponding to SEQ ID NO. 25; xiv) substitution of amino acid residue at position 139 with A, V, I, L, C, M or T, at position corresponding to SEQ ID NO. 25; and xv) substitution of amino acid residue at position 33 with L, T, S, A, V, or I, at position corresponding to SEQ ID NO. 25.

4. The hydrolase mutant as claimed in claim 1, wherein the polypeptide comprises a mutation selected from: i) an amino acid substitution at position 55 with L, T, S, A, V, or I, at position corresponding to SEQ ID NO. 1, an amino acid insertion adjacent to position 55 at position corresponding to SEQ ID NO. 1, wherein the amino acid inserted is selected from Q, N, D, E, T, or S, and an amino acid insertion adjacent to amino acid position 56 at position corresponding to SEQ ID NO. 1, wherein the amino acid inserted is selected from Q, N, D, E, T, or S; orii) an amino acid substitution at position 33 with L, T, S, A, V, or I, at position corresponding to SEQ ID NO. 25, an amino acid insertion adjacent to position 33 at position corresponding to SEQ ID NO. 25, wherein the amino acid inserted is selected from Q, N, D, E, T, or S, and an amino acid insertion adjacent to amino acid position 34 at position corresponding to SEQ ID NO. 25, wherein the amino acid inserted is selected from Q, N, D, E, T, or S.

5. The hydrolase mutant as claimed in claim 1, wherein the PET hydrolase mutant comprises a polypeptide having an amino acid sequence of at least 85%, at least 90%, at least 95%, at least 96%, or at least 98% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36.

6. A polynucleotide encoding the hydrolase mutant as claimed in any one of claims 1 to 5.

7. The polynucleotide as claimed in claim 6, wherein the polynucleotide has a nucleotide sequence of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50.

8. A recombinant vector comprising the polynucleotide as claimed in claim 6.

9. A host cell comprising the recombinant vector as claimed in claim 8.

10. A method of producing the hydrolase mutant as claimed in claim 1, the method comprising, culturing the host cell as claimed in claim 9 under condition suitable to express the hydrolase mutant and recovering the hydrolase enzyme from the host cell culture.

11. A composition comprising a hydrolase mutant as claimed in any one of claims 1 to 5, and optionally an additive.

12. A method of degrading polyester or polyester material, the method comprising: contacting the polyester or polyester material with at least one hydrolase mutant as claimed in any one of claims 1 to 5, or the composition as claimed in claim 11, in a reaction mixture; and incubating to allow degradation of the polyester or polyester material.

13. The method as claimed in claim 12, wherein said method is performed at a temperature in the range of 20°C to 60°C, preferably 30°C to 50°C, and pH in the range of pH 5.0 to pH 10.

14. The method as claimed in claim 12, wherein the hydrolase mutant and polyester or polyester material is in a weight ratio range of lng: lmg to 6pg: lmg, or wherein the hydrolase mutant and polyester or polyester material is in a weight ratio range of of 1 x 10'6: 1 to 6 x 10'3: 1.

15. The method as claimed in claim 12, wherein the method further comprises recovering monomers and / or oligomers resulting from degradation of at least one polyester or polyester material.

16. The method as claimed in claim 12, wherein the reaction mixture comprises a buffer, preferably selected from 2-(N-morpholino)ethanesulfonic acid (MES) buffer, BIS -Tris buffer, 2,2'-[(2-amino-2- oxoethyl)azanediyl]diacetic acid (ADA) buffer, N-(2-acetamido)-2- aminoethanesulfonic acid (ACES) buffer, piperazine-N,N'-bis(2- ethanesulfonic acid (PIPES) buffer, 3 -morpholinopropanesulfonic acid (MOPSO) buffer, Bis-Tris Propane buffer, N,N-Bis(2-hydroxyethyl)-2- aminoethanesulfonic acid (BES) buffer, 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, Tris (hydroxymethyl) aminomethane (TES) buffer, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer, 3-(N- morpholino)propanesulfonic acid (MOBS) buffer, 3-(Bis(2- Hydroxyethyl)Amino)-2-Hydroxypropane-l -Sulfonic Acid (DIPSO) buffer, TRIS buffer, 2-Hydroxy-3-[tris(hydroxymethyl)methylamino]-l- propanesulfonic acid buffer, N-[Tris(hydroxymethyl)methyl]-3-amino-2- hydroxypropanesulfonic acid (TAPSO) buffer, 2-Hydroxy-3-(4-(2- hydroxyethyl)piperazin-l-yl)propane-l -sulfonic acid (HEPPSO) buffer, Piperazine-l,4-bis(2-hydroxy-3-propanesulfonic acid) dihydrate (POPSO) buffer, Tris-acetate-EDTA (TEA) buffer, Phosphate buffer, phosphate buffered saline (PBS) buffer, glycine buffer, bicine buffer, N-(2- Hydroxyethyl)piperazine-N'-(4-butanesulfonic acid (HEPBS) buffer, tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS) buffer, 2- amino-2-methyl-l -propanol (AMPD) buffer, N- tris(Hydroxymethyl)methyl-4-aminobutanesulfonic acid (TABS) buffer, N- (l,l-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid (AMPSO) buffer, 2-(Cyclohexylamino)ethanesulfonic acid (CHES) buffer,3 -(Cyclohexylamino)-2-hydroxy-l -propanesulfonic acid (CAPSO) buffer, Adenosine 5 ’-monophosphate disodium salt (AMP) buffer, or combinations thereof.

17. Use of the hydrolase mutant as claimed in claim 1, or the composition as claimed in claim 11, for degrading polyester or polyester material.

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