Hydrolases and methods of use thereof

Abstract

Provided are hydrolases and methods of using hydrolases for degrading polyester-containing polymers, such as polyethylene terephthalate and polyester-polyurethane.

Description

HYDROLASES AND METHODS OF USE THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 63 / 621,751, filed January 17, 2024, entitled “HYDROLASES AND METHODS OF USE THEREOF,” which is hereby incorporated by reference in its entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (B170470000WO00-SEQ-JIB.xml; Size: 10,144 bytes; and Date of Creation: January 15, 2025) are herein incorporated by reference in its entirety.BACKGROUND

[0003] 100 million tons of polyethylene terephthalate (PET) are used annually to manufacture plastic products including beverage containers and textiles. PET is currently recycled using mechanical recycling, which requires elaborate sortation, melting, and extrusion to produce recycled resin. It is costly, energy intensive, and typically produces lower quality plastic resin that is downcycled into lower value products. Innovation is needed to improve the economics of plastic recycling, the quality of recycled resin, and bring plastics into the circular economy.

[0004] Bio-enzymatic plastic recycling uses enzymes as “molecular scissors” to hydrolyze plastic into monomers or oligomers that can be recovered and used as a drop-in replacement for plastic manufacturing. Bio-enzymatic recycling of PET could enable manufacture of 100% recycled plastic with the same performance properties as virgin plastic produced from petrochemicals, with significant reductions in energy consumption and greenhouse gas emissions.SUMMARY

[0005] The present disclosure, in some aspects, provides hydrolases and their uses, including identification of a novel hydrolase for use in plastic recycling. In some embodiments, hydrolases of the disclosure are useful in methods of degrading a plastic material, such as a polyester-containing polymer. In some embodiments, such methods produce one or more degradation products, which can be reused in the production of new plastic materials. Thus, hydrolases and methods described herein advantageously providemeans for recycling plastic products by degrading one polymer to produce monomers and / or oligomers that can be repolymerized to form another polymer.

[0006] In some aspects, the disclosure provides a hydrolase comprising a sequence that is at least 60% (e.g., at least 70%, at least 80%, at least 90%, 60-80%, 70-90%, 80-99%) identical to SEQ ID NO: 1 or 2. In some embodiments, the hydrolase comprises a sequence that is at least 60% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, 60-80%, 70-90%, or 80-99%) identical to SEQ ID NO: 1. In some embodiments, the hydrolase comprises a sequence that is at least 60% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, 60-80%, 70- 90%, or 80-99%) identical to SEQ ID NO: 2. In some embodiments, the hydrolase comprises the sequence of SEQ ID NO: 1 or 2 or a conservatively substituted version thereof.

[0007] In some aspects, the disclosure provides a hydrolase having an amino acid sequence that is at least 60% (e.g., at least 70%, at least 80%, at least 90%, 60-80%, 70-90%, 80-99%) identical to SEQ ID NO: 1 or SEQ ID NO: 2, where the amino acid sequence of the hydrolase comprises an amino acid substitution at one or more positions corresponding to positions N4, E7, A67, T70, QUO, E172, and N214 of SEQ ID NO: 2. In some embodiments, the amino acid substitution is selected from N4C, E7C, A67G, T70C, Q110R, E172L, and N214C relative to the sequence of SEQ ID NO: 2.

[0008] In some embodiments, the amino acid sequence of the hydrolase comprises an amino acid substitution at one or more positions corresponding to positions QUO, E172, and N214 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of the hydrolase comprises an amino acid substitution at positions corresponding to positions T70 and N214 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of the hydrolase comprises an amino acid substitution at positions corresponding to positions QUO and N214 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of the hydrolase comprises an amino acid substitution at positions corresponding to positions E172 and N214 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of the hydrolase comprises an amino acid substitution at positions corresponding to positions N4 and E7 of SEQ ID NO: 2.

[0009] In some embodiments, the amino acid sequence of the hydrolase comprises amino acid substitutions at positions corresponding to the following positions within SEQ ID NO: 2: N4 and E7; T70 and N214; QUO and N214; El 72 and N214; A67, QUO, and N214; N4, E7, A67, and El 72; N4, E7, QUO, and N214; A67, QUO, E 172, and N214; A67, T70, El 72, and N214; A67, T70, QUO, and N214; N4, E7, A67, QUO, and N214; N4, E7, QUO, E172, andN214; A67, T70, QUO, E172, and N214; or N4, E7, A67, QUO, E172, and N214. In some embodiments, the amino acid substitutions comprise relative to the sequence of SEQ ID NO: 2: N4C and E7C; T70C and N214C; Q110R and N214C; E172L and N214C; A67G, Q110R, and N214C; N4C, E7C, A67G, and E172L; N4C, E7C, Q110R, and N214C; A67G, Q110R, E172L, and N214C; A67G, T70C, E172L, and N214C; A67G, T70C, Q110R, and N214C; N4C, E7C, A67G, Q110R, and N214C; N4C, E7C, Q110R, E172L, and N214C; A67G, T70C, Q110R, E172L, and N214C; or N4C, E7C, A67G, Q110R, E172L, and N214C.

[0010] In some embodiments, a hydrolase of the disclosure exhibits increased depolymerization activity relative to a control hydrolase. In some embodiments, the hydrolase exhibits at least 1.5-fold, at least 2-fold, at least 5-fold, or at least 10-fold increased depolymerization activity relative to the control hydrolase. In some embodiments, the hydrolase exhibits at least 1.5-fold, at least 2-fold, at least 5-fold, or at least 10-fold increased depolymerization of PET relative to the control hydrolase. In some embodiments, the control hydrolase is a hydrolase that comprises the amino acid sequence of SEQ ID NO: 2.

[0011] In some embodiments, a hydrolase of the disclosure exhibits improved thermotolerance relative to a control hydrolase. In some embodiments, the hydrolase exhibits at least 1.5-fold, at least 2-fold, at least 5-fold, or at least 10-fold improved thermotolerance relative to the control hydrolase. In some embodiments, the control hydrolase is a hydrolase that comprises the amino acid sequence of SEQ ID NO: 2.

[0012] In some aspects, the disclosure provides a host cell that comprises a heterologous polynucleotide encoding a hydrolase described herein. In some embodiments, the host cell comprises a heterologous polynucleotide encoding a hydrolase comprising a sequence that is at least 60% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, 60-80%, 70-90%, or 80-99%) identical to SEQ ID NO: 1 or 2. In some embodiments, the hydrolase comprises the sequence of SEQ ID NO: 1 or 2 or a conservatively substituted version thereof.

[0013] In some aspects, the disclosure provides a nucleic acid (e.g., naturally occurring or non-naturally occurring) encoding a hydrolase described herein. In some embodiments, the nucleic acid encodes a hydrolase having a sequence that is at least 60% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, 60-80%, 70- 90%, or 80-99%) identical to SEQ ID NO: 1 or 2. In some embodiments, the sequence of the hydrolase comprises an amino acid substitution at one or more positions corresponding to positions N4, E7, A67, T70, QUO, E172, and N214 of SEQ ID NO: 2. In some embodiments, the hydrolase comprises the sequence of SEQ ID NO: 1 or 2.

[0014] In some aspects, the disclosure provides a vector comprising a nucleic acid described herein. In some aspects, the disclosure provides an expression cassette comprising a nucleic acid described herein.

[0015] In some aspects, the disclosure provides a composition comprising a hydrolase described herein. In some embodiments, the composition comprises: a first hydrolase comprising a polypeptide having a sequence that is at least 60% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, 60-80%, 70-90%, or 80-99%) identical to SEQ ID NO: 1 or 2; and a second hydrolase, where the first and second hydrolases are different. In some embodiments, the second hydrolase is a mono(2- hydroxyethyl) terephthalate hydrolase (MHETase). In some embodiments, the MHETase is from Ideonella sakaiensis. In some embodiments, the sequence of the first hydrolase comprises an amino acid substitution at one or more positions corresponding to positions N4, E7, A67, T70, QUO, E172, and N214 of SEQ ID NO: 2. In some embodiments, the first hydrolase comprises the sequence of SEQ ID NO: 1 or 2 or a conservatively substituted version thereof.

[0016] In some aspects, the disclosure provides a kit comprising a hydrolase described herein. In some embodiments, the kit comprises instructions for using the hydrolase in a method of degrading a polymer (e.g., a polyester-containing polymer as described herein). In some embodiments, the kit comprises: a first hydrolase comprising a polypeptide having a sequence that is at least 60% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, 60-80%, 70-90%, or 80-99%) identical to SEQ ID NO: 1 or 2; and a second hydrolase, where the first and second hydrolases are different. In some embodiments, the second hydrolase is a mono(2-hydroxyethyl) terephthalate hydrolase (MHETase). In some embodiments, the MHETase is from Ideonella sakaiensis. In some embodiments, the sequence of the first hydrolase comprises an amino acid substitution at one or more positions corresponding to positions N4, E7, A67, T70, QUO, E172, and N214 of SEQ ID NO: 2. In some embodiments, the first hydrolase comprises the sequence of SEQ ID NO: 1 or 2 or a conservatively substituted version thereof.

[0017] In some aspects, the disclosure provides methods of degrading a polyester-containing polymer (e.g., polyethylene terephthalate, polyester-polyurethane, or a combination thereof). In some embodiments, a method of degrading a polyester-containing polymer comprises contacting the polyester-containing polymer with a hydrolase of the disclosure under conditions to degrade the polyester-containing polymer.

[0018] In some embodiments, the hydrolase comprises a sequence that is at least 60% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, 60-80%, 70-90%, or 80-99%) identical to SEQ ID NO: 1 or 2. In some embodiments, the sequence of the hydrolase comprises an amino acid substitution at one or more positions corresponding to positions N4, E7, A67, T70, QUO, E172, and N214 of SEQ ID NO: 2. In some embodiments, the hydrolase comprises a sequence that is at least 60% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, 60- 80%, 70-90%, or 80-99%) identical to SEQ ID NO: 1. In some embodiments, the hydrolase comprises a sequence that is at least 60% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, 60-80%, 70-90%, or 80-99%) identical to SEQ ID NO: 2. In some embodiments, the hydrolase comprises the sequence of SEQ ID NO: 1 or 2 or a conservatively substituted version thereof.

[0019] In some embodiments, the polyester-containing polymer comprises polyethylene terephthalate, polyester-polyurethane, or a combination thereof. In some embodiments, the polyester-containing polymer comprises a polyether-polyurethane material comprising a plurality of ester bonds. In some embodiments, degradation of the polyester-containing polymer by the hydrolase produces one or more degradation products comprising monomers and / or oligomers. In some embodiments, a method of the disclosure includes recovering the one or more degradation products. In some embodiments, the one or more degradation products include polyols, polycarboxylic acids, and / or polyamines.

[0020] In some embodiments, the polyester-containing polymer comprises polyesterpolyurethane. Accordingly, in some aspects, the disclosure provides methods of degrading a polyester-polyurethane. In some embodiments, a method of degrading a polyesterpolyurethane comprises contacting the polyester-polyurethane with a hydrolase of the disclosure under conditions to degrade the polyester-polyurethane, where the hydrolase comprises a sequence that is at least 60% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, 60-80%, 70-90%, or 80-99%) identical to SEQ ID NO: 1 or 2. In some embodiments, the sequence of the hydrolase comprises an amino acid substitution at one or more positions corresponding to positions N4, E7, A67, T70, QUO, E172, and N214 of SEQ ID NO: 2. In some embodiments, the hydrolase comprises the sequence of SEQ ID NO: 1 or 2 or a conservatively substituted version thereof.

[0021] In some embodiments, a method of the disclosure comprises contacting a polyester- containing polymer with a hydrolase of the disclosure under conditions to degrade the polyester-containing polymer. In some embodiments, the hydrolase is a purified hydrolase(e.g., a purified recombinant hydrolase). For example, in some embodiments, the polyester- containing polymer is contacted with a purified hydrolase of the disclosure. In some embodiments, the polyester-containing polymer is contacted with a host cell expressing and secreting the hydrolase.

[0022] The details of certain embodiments of the disclosure are set forth in the Detailed Description. Other features, objects, and advantages of the disclosure will be apparent from the Examples, Drawings, and Claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying Drawings, which constitute a part of this specification, illustrate several embodiments of the disclosure and together with the accompanying description, serve to explain the principles of the disclosure.

[0024] FIG. 1 shows an example workflow for bioprospecting for novel putative PETase enzymes.

[0025] FIG. 2 shows assay results showing sum of monomers identified by HPLC in metagenome samples (sum of TPA+BHET+MHET area under the curve).

[0026] FIG. 3 shows taxonomic distribution and abundance of microbes by taxonomic class in metagenome samples selected for next-generation sequencing.

[0027] FIGs. 4A-4E show analyses of a novel hydrolase identified herein (JIIDDLBP_27442, also referred to as “CARLase” herein). FIG. 4A shows a percent identity matrix of CARLase (SEQ ID NO: 2) relative to leaf-branch compost cutinase (LCC; SEQ ID NO: 5) and Ideonella sakaiensis PETase ( / .sPETasc; SEQ ID NO: 6). FIG. 4B shows a percent similarity matrix of CARLase relative to LCC and / .sPETasc. FIG. 4C shows a predicted structure of CARLase (AlphaFold), with an arrow indicating the active site (shown as sticks). FIG. 4D shows an overlay of CARLase (dark shading) and LCC (light shading). FIG. 4E shows an overlay of CARLase (dark shading) and / .sPETasc (light shading).

[0028] FIGs. 5A-5D show results from experimental characterization of CARLase. FIG. 5A depicts a protein gel showing purified CARLase obtained following expression in E. coli. FIG. 5B depicts zone of clearing on PET agar plate with CARLase whole cell lysates at 37 °C and 50 °C. FIG. 5C depicts zone of clearing on polyester-polyurethane agar plate with purified CARLase at 37 °C. FIG. 5D shows results from a PET depolymerization reaction with CARLase.

[0029] FIG. 6 shows results from protein engineering of CARLase which identified variants having greater than 1.5-fold improvement over parent (“FIOP”) in PETase activity relative to the parent CARLase enzyme at 37 °C.

[0030] FIG. 7 is a flowchart showing an example process for bio-enzymatic degradation of PET.DETAILED DESCRIPTION

[0031] Among other aspects, the disclosure provides hydrolases, compositions comprising hydrolases, and methods of using hydrolases. In some embodiments, hydrolases and compositions of the disclosure can be useful for bio-enzymatic degradation of plastic materials. Accordingly, some embodiments of the disclosure relate to methods of degrading a plastic material, such as a polyester-containing polymer. In some embodiments, the methods comprise degrading a polyester-containing polymer to produce monomers and / or oligomers, which can be recycled into polymers for use in new plastic materials.Hydrolases

[0032] Aspects of the disclosure relate to the identification of a new hydrolase enzyme and hydrolase enzyme variants thereof for use in bio-enzymatic degradation of plastic materials. As described in Examples 1 and 2, an environmental discovery effort was undertaken to identify enzymes that hydrolyze different types of plastic. This effort resulted in the identification of a novel PET hydrolase, referred to herein as “CARLase” (JIIDDLBP_27442).

[0033] As used herein, a hydrolase (corresponding to EC 3 in the EC number classification of enzymes) refers to an enzyme that can use water to break a chemical bond. As used herein, a “poly(ethylene terephthalate) hydrolase” or “PET hydrolase” refers to a hydrolase enzyme that can degrade poly(ethylene terephthalate), abbreviated “PET.”

[0034] The amino acid sequence of CARLase, with and without its endogenous signal peptide, is provided in Table 1. As discussed in Example 2, and as shown in FIG. 4A, surprisingly, the sequence of CARLase does not show a high percent identity to previously- characterized enzymes for bio-enzymatic degradation of plastic materials. Accordingly, in some aspects, the disclosure provides a hydrolase comprising a sequence of SEQ ID NO: 1 or 2 or a conservatively substituted version thereof. In some embodiments, a hydrolase of the disclosure comprises a sequence that is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to SEQ ID NO: 1 or 2. In some embodiments, the hydrolase comprises a sequence that is 60-70%, 70-80%, 80-90%, 90-95%, 92-99%, 94-99%, 95-99%, 60-100%, 70-100%, 80-100%, 90-100%, 92-100%, 94-100%, 95-100%, 96-100%, or 100% identical to SEQ ID NO: 1 or 2.

[0035] In some embodiments, a hydrolase of the disclosure comprises a sequence that is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to SEQ ID NO: 1. In some embodiments, the hydrolase comprises a sequence that is 60-70%, 70-80%, 80-90%, 90-95%, 92-99%, 94-99%, 95-99%, 60-100%, 70-100%, 80-100%, 90-100%, 92-100%, 94- 100%, 95-100%, 96-100%, or 100% identical to SEQ ID NO: 1.

[0036] In some embodiments, a hydrolase of the disclosure comprises a sequence that is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to SEQ ID NO: 2. In some embodiments, the hydrolase comprises a sequence that is 60-70%, 70-80%, 80-90%, 90-95%, 92-99%, 94-99%, 95-99%, 60-100%, 70-100%, 80-100%, 90-100%, 92-100%, 94- 100%, 95-100%, 96-100%, or 100% identical to SEQ ID NO: 2.

[0037] As used herein, a “variant” hydrolase refers to a hydrolase in which the amino acid sequence contains at least one difference relative to the amino acid sequence of a control or reference hydrolase sequence. For example, in some embodiments, a variant hydrolase refers to a hydrolase having an amino acid sequence that contains at least one amino acid substitution, deletion, or insertion relative to the amino acid sequence of a control or reference hydrolase sequence.

[0038] As described in Example 3, and as shown in FIG. 6, library screening was performed to identify variants of the hydrolase CARLase that exhibited thermotolerance improvements as indicated by increased depolymerization activity under high-temperature conditions.These efforts resulted in the development of hydrolase variants with up to 10-fold improvements in depolymerization activity under high-temperature conditions relative to the parent enzyme comprising the amino acid sequence of SEQ ID NO: 2. Improvements in depolymerization activity under high-temperature conditions were observed with hydrolase variants having only one amino acid substitution or more than one amino acid substitution incombination at position(s) corresponding to positions N4, E7, A67, T70, QUO, E172, and / or N214 of SEQ ID NO: 2.

[0039] Accordingly, in some aspects, the disclosure provides a hydrolase having an amino acid sequence that is at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical to SEQ ID NO: 2, where the amino acid sequence of the hydrolase comprises an amino acid substitution at one or more positions corresponding to positions N4, E7, A67, T70, QI 10, E172, and N214 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of the hydrolase comprises an amino acid substitution at two or more (e.g., three or more, four or more, five or more, six or more, two, three, four, five, six, seven, 2-7, 2-6, 2-5, 2-4, 3-7, 3-6, 3-5, 4-7, or 5-7) positions corresponding to positions N4, E7, A67, T70, QI 10, E172, and N214 of SEQ ID NO: 2. In some embodiments, the amino acid substitution is selected from N4C, E7C, A67G, T70C, Q110R, E172L, and N214C relative to the sequence of SEQ ID NO: 2.

[0040] In some embodiments, the hydrolase comprises an amino acid sequence that is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to SEQ ID NO: 2, wherein the amino acid sequence of the hydrolase comprises an amino acid substitution at one or more positions described herein. In some embodiments, the hydrolase comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, wherein the amino acid sequence of the hydrolase comprises an amino acid substitution at one or more positions described herein. In some embodiments, the hydrolase comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 2, wherein the amino acid sequence of the hydrolase comprises an amino acid substitution at one or more positions described herein. In some embodiments, the hydrolase comprises an amino acid sequence that is 60-70%, 70- 80%, 80-90%, 90-95%, 92-99%, 94-99%, 95-99%, 60-99%, 70-99%, 80-99%, 90-99%, 92- 99%, 94-99%, 95-99%, 96-99%, or 97-99% identical to SEQ ID NO: 2, wherein the amino acid sequence of the hydrolase comprises an amino acid substitution at one or more positions described herein.

[0041] In some embodiments, the hydrolase comprises an amino acid substitution at a position corresponding to position N214 of SEQ ID NO: 2. In some embodiments, the amino acid substitution is N214C relative to the sequence of SEQ ID NO: 2. In some embodiments, the hydrolase comprises an amino acid substitution at a position corresponding to positionN214 of SEQ ID NO: 2 and at one or more positions corresponding to positions N4, E7, A67, T70, QI 10, and E172 of SEQ ID NO: 2. In some embodiments, the hydrolase comprises an amino acid substitution at positions corresponding to positions T70 and N214 of SEQ ID NO: 2 (e.g., T70C and N214C amino acid substitutions relative to the sequence of SEQ ID NO: 2).

[0042] As described in Example 3, a first round of screening of hydrolase variants identified a variant having cysteine substitutions at positions N214 and T70 of SEQ ID NO: 2 that exhibited an approximately 3-fold improvement in depolymerization activity under high- temperature conditions relative to the parent enzyme (FIOP). Without wishing to be bound by any theory, based on structural analyses, the improved performance of this variant (containing N214C, T70C amino acid substitutions relative to the sequence of SEQ ID NO: 2) may be attributable to increased thermostability provided by disulfide bonding and / or increased substrate affinity due to the proximity to the substrate binding region.

[0043] As also described in Example 3, a second round of screening identified variants having the N214C substitution relative to the sequence of SEQ ID NO: 2 without the T70C substitution relative to the sequence of SEQ ID NO: 2, which demonstrated that the improvements conferred by the N214C substitution relative to the sequence of SEQ ID NO: 2 were not dependent upon its combination with the T70C substitution relative to the sequence of SEQ ID NO: 2.

[0044] In some embodiments, the hydrolase comprises an amino acid substitution at a position corresponding to position QI 10 of SEQ ID NO: 2. In some embodiments, the amino acid substitution is QI 10R relative to the sequence of SEQ ID NO: 2. Without wishing to be bound by any theory, based on structural analyses, the improved thermotolerance conferred by QI 10R variants may be attributable to increased stability provided by favorable paired interaction with an aspartate residue, DI 14. In some embodiments, the hydrolase comprises an amino acid substitution at a position corresponding to position QI 10 of SEQ ID NO: 2 and at one or more positions corresponding to positions N4, E7, A67, T70, E172, and N214 of SEQ ID NO: 2. In some embodiments, the hydrolase comprises an amino acid substitution at positions corresponding to positions QUO and N214 of SEQ ID NO: 2 (e.g., Q110R and N214C amino acid substitutions relative to the sequence of SEQ ID NO: 2).

[0045] In some embodiments, the hydrolase comprises an amino acid substitution at a position corresponding to position E172 of SEQ ID NO: 2. In some embodiments, the amino acid substitution comprises a substitution of position E172 with an amino acid having a hydrophobic side chain (e.g., E172A, E172I, E172L, E172M, or E172V amino acid substitutions relative to the sequence of SEQ ID NO: 2). In some embodiments, the aminoacid substitution is E172L relative to the sequence of SEQ ID NO: 2. Without wishing to be bound by any theory, based on structural analyses, the improved thermotolerance conferred by E172L variants may be attributable to enhanced substrate binding due to increased hydrophobicity near the substrate binding pocket, which was further supported by the observation that a known PETase (LCC; SEQ ID NO: 5) contains a hydrophobic isoleucine at the corresponding position. In some embodiments, the hydrolase comprises an amino acid substitution at a position corresponding to position E172 and at one or more positions corresponding to positions N4, E7, A67, T70, QI 10, and N214 of SEQ ID NO: 2. In some embodiments, the hydrolase comprises an amino acid substitution at positions corresponding to positions E172 and N214 of SEQ ID NO: 2 (e.g., E172L and N214C amino acid substitutions relative to the sequence of SEQ ID NO: 2).

[0046] In some embodiments, the hydrolase comprises an amino acid substitution at a position corresponding to position N4 or E7 of SEQ ID NO: 2. In some embodiments, the amino acid substitution is N4C or E7C relative to the sequence of SEQ ID NO: 2. In some embodiments, the hydrolase comprises an amino acid substitution at positions corresponding to positions N4 and E7 of SEQ ID NO: 2 (e.g., N4C and E7C relative to the sequence of SEQ ID NO: 2). As described in Example 3, first and second rounds of screening identified several hydrolase variants having N4C and E7C amino acid substitutions relative to the sequence of SEQ ID NO: 2 which exhibited improved thermotolerance relative to the parent enzyme. Without wishing to be bound by any theory, based on structural analyses, the improved thermotolerance of these variants may be attributable to increased stability provided by disulfide bonding.

[0047] Thus, aspects of the disclosure relate to hydrolase variants (e.g., CARLase variants) with improved performance relative to a control hydrolase. In some embodiments, improved performance refers to improved thermo tolerance. As used herein, “improved thermotolerance” of a variant hydrolase relative to a control hydrolase refers to the ability of the hydrolase to be more enzymatically active than the control hydrolase at a specified temperature. In some embodiments, the specified temperature is a temperature that is not optimal for the control hydrolase. For example, in Example 3, the CARLase enzyme was found to be most active at 30 °C, with a gradual drop-off of activity at higher temperatures. Thermotolerant variants were identified that had higher activity than the wildtype CARLase enzyme at 37 °C. It should be appreciated that thermotolerance can be measured at temperatures other than 37 °C.

[0048] In some embodiments, variant hydrolases associated with the disclosure exhibit improved performance (e.g., thermotolerance) relative to a control hydrolase (e.g., CARLase). In some embodiments, variant hydrolases associated with the disclosure exhibit at least 1-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 5-fold, at least 7-fold, or at least 10-fold improved performance (e.g., thermotolerance) relative to a control hydrolase (e.g., CARLase). In some embodiments, the control hydrolase comprises the amino acid sequence of SEQ ID NO: 1 or 2.

[0049] It should be appreciated that while the amino acid positions of amino acid substitutions for hydrolase variants described herein are described relative to the sequence of SEQ ID NO: 2, corresponding amino acid substitutions could also be made relative to other base sequences, as would be understood by one of ordinary skill in the art. For example, SEQ ID NO: 1 corresponds to the full-length wildtype sequence of CARLase, containing the endogenous signal sequence. By aligning the sequences of SEQ ID NOs: 1 and 2, one of ordinary skill in the art would be able to identify the corresponding residues between these two sequences and would be able to make amino acid substitutions in the amino acid sequence of SEQ ID NO: 1 that correspond to amino acid substitutions described herein relative to the sequence of SEQ ID NO: 2. Similarly, if the sequence of SEQ ID NO: 2 were modified, such as by inserting a signal sequence or sequence tag, one of ordinary skill in the art would be able to identify which amino acids in the modified sequence correspond to the positions in SEQ ID NO: 2 described herein by aligning the sequences.

[0050] In some aspects, the disclosure provides a host cell that comprises a heterologous polynucleotide encoding a hydrolase (e.g., CARLase) or a hydrolase variant described herein. In some embodiments, the heterologous polynucleotide encodes a hydrolase comprising a sequence that is at least 60% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 60-80%, 70-90%, or 80-99%) identical to SEQ ID NO: 1 or 2. In some embodiments, the heterologous polynucleotide encodes a hydrolase comprising an amino acid substitution at one or more positions corresponding to positions N4, E7, A67, T70, QUO, E172, and N214 of SEQ ID NO: 2.

[0051] In some embodiments, a host cell is any cell capable of replicating, transcribing, and / or translating a heterologous polynucleotide for producing a recombinant hydrolase described herein. In some embodiments, the host cell is a prokaryotic cell (e.g., Escherichia coli, Bacillus subtilis, Salmonella typhimurium, a species of Streptomyces, or any prokaryotic cell known in the art to be suitable for expressing and / or isolating a recombinant protein, such as a hydrolase of the disclosure). In some embodiments, the host cell is a eukaryotic cell(e.g., yeast cell, mammalian cell, plant cell, avian cell, amphibian cell, plant cell, fish cell, or insect cell, or any eukaryotic cell known in the art to be suitable for expressing and / or isolating a recombinant protein, such as a hydrolase of the disclosure).

[0052] In some embodiments, the host cell is a species of Pichia. In some embodiments, the host cell is Pichia pastoris, Pichia membranifaciens, Pichia deserticola, Pichia cephalocereana, Pichia eremophila, Pichia myanmarensis, Pichia anomala, Pichia nakasei, Pichia siamensis, Pichia heedii, Pichia barkeri, Pichia norvegensis, Pichia thermomethanolica, Pichia stipites, Pichia subpelliculosa, Pichia exigua, Pichia occidentalis, or Pichia cactophila. In some embodiments, the host cell is a filamentous fungal cell. In some embodiments, the filamentous fungal cell is a species of Acremonium, Agaricus, Aspergillus, Aureobasidium, Chrysosporium, Coprinus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mortierella, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Panerochaete, Pleurotus, Schizophyllum, Talaromyces, Rasamsonia, Thermoascus, Thielavia, Tolypocladium, or Trichoderma.

[0053] In some embodiments, a host cell has been or is transformed or transfected with, or otherwise contained or contains, a heterologous polynucleotide, vector, or expression cassette that encodes a hydrolase or a hydrolase variant described herein. In some embodiments, the heterologous polynucleotide is introduced into a host cell using any method known in the art, including transformation, transfection, and transduction. It should be appreciated that any heterologous polynucleotide associated with the disclosure can be expressed transiently in a host cell or can be integrated into the genome of a host cell.

[0054] In some aspects, the disclosure provides a nucleic acid encoding a hydrolase having a sequence that is at least 60% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 60-80%, 70-90%, or 80-99%) identical to SEQ ID NO: 1 or 2. In some embodiments, the nucleic acid encodes a hydrolase comprising an amino acid substitution at one or more positions corresponding to positions N4, E7, A67, T70, QUO, E172, and N214 of SEQ ID NO: 2. In some aspects, the disclosure provides a nucleic acid encoding a hydrolase having the sequence of SEQ ID NO: 1 or 2. In some embodiments, the nucleic acid comprises a nucleotide sequence of SEQ ID NO: 3 or 4. The nucleic acid can be naturally-occurring or non-naturally occurring. The hydrolase encoded by the nucleic acid can be naturally-occurring or non-naturally occurring.

[0055] In some aspects, the disclosure provides a vector comprising a nucleic acid encoding a hydrolase having a sequence that is at least 60% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 60-80%, 70-90%, or 80-99%) identical to SEQID NO: 1 or 2. In some embodiments, the nucleic acid encodes a hydrolase comprising an amino acid substitution at one or more positions corresponding to positions N4, E7, A67, T70, QUO, E172, and N214 of SEQ ID NO: 2. In some aspects, the disclosure provides a vector comprising a nucleic acid encoding a hydrolase having the sequence of SEQ ID NO: 1 or 2. In some embodiments, the nucleic acid comprises a nucleotide sequence of SEQ ID NO: 3 or 4. The nucleic acid can be naturally-occurring or non-naturally occurring.Examples of vectors suitable for use in accordance with the disclosure include, without limitation, plasmids, phagemids, phasmids, cosmids, viruses, artificial chromosomes, shuttle vectors, expression vectors, and the like. Any vector suitable for expression of polynucleotides described herein may be compatible with aspects of the disclosure.

[0056] In some aspects, the disclosure provides an expression cassette comprising a nucleic acid encoding a hydrolase having a sequence that is at least 60% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 60-80%, 70-90%, or 80-99%) identical to SEQ ID NO: 1 or 2. In some embodiments, the nucleic acid encodes a hydrolase comprising an amino acid substitution at one or more positions corresponding to positions N4, E7, A67, T70, QUO, E172, and N214 of SEQ ID NO: 2. In some aspects, the disclosure provides an expression cassette comprising a nucleic acid encoding a hydrolase having the sequence of SEQ ID NO: 1 or 2. In some embodiments, the nucleic acid comprises a nucleotide sequence of SEQ ID NO: 3 or 4. The nucleic acid can be naturally-occurring or non-naturally occurring. In some embodiments, the expression cassette comprises a vector of the disclosure in operable linkage with a nucleic acid of the disclosure. In some embodiments, the expression cassette comprises one or more expression control elements known in the art, including, without limitation, enhancers, promoters, factor-specific binding sites, terminators, and / or ribosome binding sites.

[0057] In some aspects, the disclosure provides a composition comprising a hydrolase or a hydrolase variant described herein and one or more additional enzymes. In some embodiments, the composition comprises: a first hydrolase comprising a polypeptide having a sequence that is at least 60% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 60-80%, 70-90%, or 80-99%) identical to SEQ ID NO: 1 or 2, or comprises SEQ ID NO: 1 or 2; and a second hydrolase. In some embodiments, the first hydrolase comprises a polypeptide having an amino acid sequence that is at least 60% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 60-80%, 70- 90%, or 80-99%) identical to SEQ ID NO: 2, wherein the amino acid sequence comprises an amino acid substitution at one or more positions corresponding to positions N4, E7, A67,T70, QUO, E172, and N214 of SEQ ID NO: 2. In some embodiments, the first and second hydrolases share less than 90% sequence identity. For example, in some embodiments, the first and second hydrolases share less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, or less than 30% identity. In some embodiments, the second hydrolase is a mono(2-hydroxy ethyl) terephthalate hydrolase (MHETase). In some embodiments, the MHETase is from Ideonella sakaiensis. Such MHETase enzymes are known in the art (see, e.g., Palm, et al. Structure of the plastic-degrading Ideonella sakaiensis MHETase bound to a substrate. Nat Commun 10, 1717 (2019)).

[0058] In some aspects, the disclosure provides a kit comprising a hydrolase or a hydrolase variant described herein. In some embodiments, the kit comprises instructions for using the hydrolase or hydrolase variant in a method of degrading a polymer (e.g., a polyester- containing polymer as described herein). It should be appreciated that, in some embodiments, the kit can alternatively or additionally comprise a nucleic acid encoding the hydrolase or hydrolase variant (e.g., a naturally occurring or non-naturally occurring nucleic acid, such as a vector or expression cassette described herein). In some embodiments, the kit comprises one or more additional enzymes. In some embodiments, the kit comprises: a first hydrolase comprising a polypeptide having a sequence that is at least 60% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 60-80%, 70-90%, or 80- 99%) identical to SEQ ID NO: 1 or 2, or comprises SEQ ID NO: 1 or 2; and a second hydrolase. In some embodiments, the first hydrolase comprises a polypeptide having an amino acid sequence that is at least 60% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 60-80%, 70-90%, or 80-99%) identical to SEQ ID NO: 2, wherein the amino acid sequence comprises an amino acid substitution at one or more positions corresponding to positions N4, E7, A67, T70, QUO, E172, and N214 of SEQ ID NO: 2. In some embodiments, the first and second hydrolases share less than 90% sequence identity. For example, in some embodiments, the first and second hydrolases share less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, or less than 30% identity. In some embodiments, the second hydrolase is a mono(2-hydroxyethyl) terephthalate hydrolase (MHETase). In some embodiments, the MHETase is from Ideonella sakaiensis (see, e.g., Palm, et al. Structure of the plastic-degrading Ideonella sakaiensis MHETase bound to a substrate. Nat Commun 10, 1717 (2019)).

[0059] As described herein, in some embodiments, a hydrolase of the disclosure comprises a protein sequence that shares a percentage of sequence identity with SEQ ID NO: 1 or 2. For the purposes of comparing two or more protein sequences, the percentage of “sequenceidentity” between a first protein sequence and a second protein sequence refers to the percentage of amino acids that are identical at corresponding positions between the two protein sequences when the two protein sequences are aligned (optionally taking into account potential gaps in either sequence).

[0060] Percent identity between two protein sequences can be calculated by dividing the number of amino acid residues that are identical at the corresponding positions in the two protein sequences by the total number of amino acid residues in either of the two protein sequences and multiplying by 100. In some embodiments, when the two protein sequences are compared, percent identity is determined over the length of the shorter of the two sequences. In other embodiments, percent identity is determined over the length of the longer of the two sequences. In some embodiments, percent identity is determined over a fragment or specified region of a longer sequence. For example, in some embodiments, percent identity can be measured over a region that is at least about 25, 50, 75, or 100 amino acids in length, or over a region that is 100 to 150, 150 to 200, 100 to 200, or 200 or more, amino acids in length.

[0061] Percent identity can be calculated using algorithms known in the art and using standard settings, e.g., by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c; the algorithm of Needleman and Wunsch, J. Mol. Biol. (1970) 48:443; the method of Pearson and Lipman. Proc. Natl. Acad. Sci. USA (1998) 85:2444, and / or by using algorithms available as, e.g., Blast, Clustal Omega, or any other sequence alignment algorithms known in the art.Methods of Degradation

[0062] In some aspects, the disclosure provides methods of degrading a polyester-containing polymer. In some embodiments, a method of degrading a polyester-containing polymer comprises contacting the polyester-containing polymer with a hydrolase under conditions to degrade the polyester-containing polymer. In some embodiments, the hydrolase comprises a sequence that is at least 60% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 60-80%, 70-90%, or 80-99%) identical to SEQ ID NO: 1 or 2. In some embodiments, the hydrolase comprises the sequence of SEQ ID NO: 1 or 2. In some embodiments, the hydrolase is a hydrolase as described herein.

[0063] As used herein, a polyester-containing polymer refers to a polymer comprising one or more esters. In some embodiments, a polyester-containing polymer comprises one or more esters of the same type or a mixture of esters of a different type. In some embodiments, apolyester-containing polymer comprises one or more polyesters. In some embodiments, a polyester-containing polymer comprises one or more polyesters of the same type or a mixture of polyesters of a different type. In some embodiments, a polyester-containing polymer comprises polyethylene terephthalate, polyester-polyurethane, or a combination thereof. In some embodiments, a polyester-containing polymer comprises polyethylene terephthalate. In some embodiments, a polyester-containing polymer comprises polyester-polyurethane.

[0064] In some embodiments, a polyester-containing polymer corresponds to or includes a plastic material comprising one or more polyesters. In some embodiments, the plastic material is in a crystalline or semi-crystalline form. In some embodiments, the plastic material is in an amorphous form. In some embodiments, the plastic material is plastic waste in a suitable form for a degradation reaction described herein. For example, in some embodiments, the plastic material is plastic waste that has been processed to a more suitable form for degradation (e.g., pellets, flakes, powder, discs, sheets).

[0065] In some embodiments, degradation of a polyester-containing polymer by a hydrolase of the disclosure produces one or more degradation products comprising monomers and / or oligomers. In some embodiments, the monomers and / or oligomers comprise subunits of the polymer that may be used in a polymerization reaction to produce a polymer. In some embodiments, a method of the disclosure includes recovering the one or more degradation products.

[0066] In some embodiments, the one or more degradation products comprise any products resulting from degradation of polyethylene terephthalate and / or polyester-polyurethane by an enzyme having hydrolase (e.g., esterase) activity. In some embodiments, the one or more degradation products comprise polyols, polycarboxylic acids, and / or polyamines.

[0067] In some embodiments, one or more degradation products of a method described herein comprise one or more polyols. As used herein, a polyol refers to a compound having at least two hydroxyl groups. In some embodiments, the polyol has a molecular weight of less than 1,000 g / mol (e.g., less than 800, less than 600, less than 500, or less than 300 g / mol). In some embodiments, the one or more polyols are selected from the group consisting of ethylene glycol, diethylene glycol, 1 ,4-butanediol, triethylene glycol, propylene glycol, 1,2-dipropylene glycol, neopentyl glycol, glycerol, 1,1,1 -trimethylolpropane, sucrose, sorbitol, and pentaerythritol.

[0068] In some embodiments, one or more degradation products of a method described herein comprise one or more polycarboxylic acids. As used herein, a poly carboxy lie acid refers to a compound having at least two carboxyl groups. In some embodiments, thepolycarboxylic acid has a molecular weight of less than 1,000 g / mol (e.g., less than 800, less than 600, less than 500, or less than 300 g / mol). In some embodiments, the one or more polycarboxylic acids are selected from the group consisting of terephthalic acid (TPA), 2- hydroxy ethyl terephthalic acid (MHET), mono(2-hydroxyethyl)-isophthalate (BHET), succinic acid, glutaric acid, adipic acid, phthalic acid, benzenetricarboxylic acid, oleic acid, and ricinoleic acid.

[0069] In some embodiments, one or more degradation products of a method described herein comprise one or more poly amines. As used herein, a poly amine refers to a compound having at least two amino groups. In some embodiments, the polyamine has a molecular weight of less than 1,000 g / mol (e.g., less than 800, less than 600, less than 500, or less than 300 g / mol). In some embodiments, the one or more poly amines are selected from the group consisting of methylene-4,4'-diamine, methylene-2,4'-diamine, methylene-2,2'-diamine, naphthylene- 1,4-diamine, naphthylene- 1,5-diamine, naphthylene- 1,6-diamine, tolylene-2,4- diamine, and tolylene-2,6-diamine.

[0070] In some embodiments, methods of the disclosure comprise contacting a polyester- containing polymer with a hydrolase, where the hydrolase is purified. In some embodiments, a purified hydrolase refers to a hydrolase purified from a natural source or a recombinant hydrolase purified following recombinant protein production. Protein purification methods are known in the art and include, for example, gel purification, affinity column purification, and column chromatography.

[0071] In some embodiments, methods of the disclosure comprise contacting a polyester- containing polymer with a host cell expressing and secreting a hydrolase of the disclosure. In some embodiments, the host cell is a host cell as described herein. In some embodiments, the hydrolase comprises a signal sequence such that, following its expression by the host cell, the hydrolase is secreted by the host cell. For example, in some embodiments, the host cell expresses and secretes a hydrolase comprising a sequence of SEQ ID NO: 1. It should be appreciated that any hydrolase described herein can be expressed and secreted by a host cell for the purposes of contacting in a method of the disclosure.

[0072] In some embodiments, methods of the disclosure are performed under degradation conditions. For example, in some embodiments, the methods comprise contacting a polyester-containing polymer with a hydrolase under conditions to degrade the polyester- containing polymer. In some embodiments, the conditions comprise an incubation temperature of between about 30 °C and about 80 °C (e.g., 30-70 °C, 30-60 °C, 30-50 °C, 40- 70 °C, 40-60 °C, 40-50 °C). In some embodiments, the conditions comprise an incubationtemperature of between about 60 °C and about 80 °C (e.g., 65-80 °C, 70-80 °C, 60-75 °C, OS- 75 °C). In some embodiments, the incubation temperature is about 70 °C. In some embodiments, the conditions comprise a pH of between about 3.5 and about 9.0 (e.g., 3.5-8.0,3.5-7.5, 4.0-9.0, 4.0-8.0, 4.0-7.0, 5.0-9.0, 5.0-8.0, about 6.0, about 7.0, or about 8.0). In some embodiments, the conditions comprise a pH of between about 3.5 and about 5.5 (e.g., 3.5-5.0,3.5-4.5, 4.0-5.5, 4.0-5.0, about 4.0, about 5.0, or about 4.5).

[0073] In some embodiments, methods of the disclosure comprise contacting a polyester- containing polymer with a hydrolase described herein and one or more additional degradation enzymes. In some embodiments, the polyester-containing polymer is contacted with a first hydrolase comprising a polypeptide having a sequence that is at least 60% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 60-80%, 70-90%, or 80- 99%) identical to SEQ ID NO: 1 or 2 or comprises SEQ ID NO: 1 or 2, and a second hydrolase, where the second hydrolase is different from the first. In some embodiments, the second hydrolase is a mono(2-hydroxy ethyl) terephthalate hydrolase (MHETase). For example, in some embodiments, the MHETase is from Ideonella sakaiensis.

[0074] In some embodiments, the polyester-containing polymer is contacted with the first and second hydrolases simultaneously (e.g., contacting with a single mixture comprising the first and second hydrolases, or contacting with different hydrolase compositions comprising the first and second hydrolases at approximately the same time). In some embodiments, the polyester-containing polymer is contacted with the first and second hydrolases sequentially (e.g., contacting with one hydrolase composition followed by contacting with another hydrolase composition).

[0075] The present disclosure is further illustrated by the following Examples, which should not be construed as limiting.EXAMPLESExample 1. Bioprospecting for Novel Putative PETase Enzymes

[0076] As part of an environmental discovery effort to identify enzymes that hydrolyze different types of plastic, 218 plastic samples were collected throughout the US Pacific Northwest. An example workflow for the screening process is shown in FIG. 1. Following enrichment of samples with plastic nanoparticles, cultures were tested using HPLC for production of PET monomers TPA, MHET, and BHET, and standard curves using analytical standards were used to quantify monomers present in samples. FIG. 2 shows the sum of monomers identified by HPLC in metagenome samples (sum of TPA+BHET+MHET areaunder the curve). Metagenomes of microbial samples enriched on PET were isolated and sequenced. Following metagenomic sequencing, metagenomes were assembled using Flye (1), then gene-calling and annotation was performed using Prokka (2) using the Bacannot Nextflow pipeline (3). A previously described Hidden Markov Model for PETases (4) was used to identify putative PETases from the samples with e-values lower than 0.01. Signal peptides were identified using Deepsig (5) to identify secreted enzymes for use in subsequent bioinformatic analysis. FIG. 3 shows taxonomic distribution and abundance of microbes by taxonomic class in metagenome samples selected for next-generation sequencing.

[0077] Out of the 218 plastic samples tested, 64 samples produced monomer above the HPLC limit of detection. Sample “SAM 163” was selected for further analysis because it produced the highest sum of monomers (FIG. 2).

[0078] Fist of Cited References:1. Kolmogorov, M., Yuan, J., Fin, Y. et al. Assembly of long, error-prone reads using repeat graphs. Nat Biotechnol 37, 540-546 (2019).2. Seemann, T. Prokka: rapid prokaryotic genome annotation, Bioinformatics, Volume 30, Issue 14, July 2014.3. Felipe Marques de Almeida, & Georgios Joannis Pappas Jr. (2022). fmalmeida / bacannot: A generic but comprehensive bacterial annotation pipeline (v3.2). Zenodo.4. Erickson, E., Gado, J.E., Avilan, E. et al. Sourcing thermotolerant poly(ethylene terephthalate) hydrolase scaffolds from natural diversity. Nat Commun 13, 7850 (2022).5. Castrense Savojardo, Pier Euigi Martelli, Piero Fariselli, Rita Casadio, DeepSig: deep learning improves signal peptide detection in proteins, Bioinformatics, Volume 34, Issue 10, May 2018.Example 2. Discovery of PET Hydrolase for Enzymatic Digestion of Polyester-Containing Material

[0079] The screen described in Example 1 identified a novel PET hydrolase from the genus Aeromicrobium, referred to herein as “CARLase” (JIIDDLBP_27442). The amino acid sequence of CARLase, with and without its endogenous signal peptide, is provided in Table 1 with corresponding nucleotide sequences.Table 1. PET Hydrolase Sequences

[0080] The percent identity and percent similarity of CARLase to previously-identified leafbranch compost cutinase (LCC) and Ideonella sakaiensis PETase ( / .sPETasc) was evaluated. The amino acid sequences of LCC (UniProtKB Accession No.: G9BY57, incorporated herein as SEQ ID NO: 5) and / .sPETasc (UniProtKB Accession No.: A0A0K8P6T7, incorporated herein as SEQ ID NO: 6) are provided in Table 2. FIG. 4A shows a percent identity matrix of CARLase (SEQ ID NO: 2) relative to LCC and / .sPETasc. FIG. 4B shows a percent similarity matrix of CARLase relative to LCC and / .sPETasc. Surprisingly, the newly- identified hydrolase enzyme CARLase is only -45% identical to / .sPETasc and -51% identical to LCC.Table 2. LCC and / .sPETasc Sequences

[0081] FIG. 4C shows a predicted structure of CARLase (AlphaFold), with an arrow indicating the active site (shown as sticks). FIG. 4D shows an overlay of CARLase (dark shading) and LCC (light shading). FIG. 4E shows an overlay of CARLase (dark shading) and / .sPETasc (light shading). Taken together, the results shown in FIGs. 4A-4E reveal that the newly-identified CARLase is similar in structure but different in sequence relative to known PETase enzymes.

[0082] Cloning, Expression, and Purification

[0083] CARLase was cloned into a pet29b bacterial expression vector with a C-terminal 6X- His tag and co-expressed in E. coli with pGro7 in Shuffle T7 competent cells. Post growth and expression, cells were lysed, and CARLase was purified by Nickel column chromatography. FIG. 5A depicts a protein gel showing purified CARLase obtained following expression (Lane 1: Protein Ladder (BIO-RAD® #1610374) with 25 kDa (lower band) and 37 kDa (upper band) shown; Lane 2: Secretome of cells post-induction (media sample); Lane 3: Soluble fraction post-lysis of cells; Lane 4: Insoluble fraction post- lysis of cells; Lane 5: 250 mM imidazole elution post-Nickel column; Lane 6: 500 mM imidazole elution post-Nickel column). In all cases, 25 pL of sample was added to 5 pL of 5X SDS loading dye. Samples were run on an ExpressPlus™ PAGE Gel (GENSCRIPT® #76510- 660), stained and destained with standard coomassie protocol.

[0084] Agar plate assays

[0085] The activity of CARLase on both polyethylene terephthalate (PET) and polyesterpolyurethane was characterized by qualitative clearance assays on agar plates.

[0086] PET agar methodology. PET was solubilized in DMSO and added dropwise to LB- agar, stirred vigorously, and sonicated to generate amorphous PET emulsified LB-agar plates. E. coli cells expressing CARLase were grown in a 96 deep well plate and lysed to harvestsoluble CARLase from whole cell lysates (WCLs). WCLs were plated onto PET LB plates and incubated separately at 37 °C and 50 °C for 18 hours.

[0087] Polyester-polyurethane agar methodology. Impranil® DLN-SD (water soluble polyester-polyurethane) was added dropwise to LB-agar, stirred vigorously, and sonicated to generate Impranil® emulsified LB-agar plates. Purified CARLase from EIG. 5A was plated onto the polyester-polyurethane agar plate and incubated at 37 °C for 18 hours.

[0088] Results. Enzymatic-mediated breakdown of PET or polyester-polyurethane resulted in a zone of clearing within the agar plate. EIG. 5B depicts zone of clearing on PET agar plate with CARLase whole cell lysates at 37 °C and 50 °C. Demonstration of “clearing” of amorphous PET was indicative of CARLase mediated PET breakdown. FIG. 5C depicts zone of clearing on polyester-polyurethane agar plate with purified CARLase at 37 °C. Demonstration of “clearing” of polyester-polyurethane was indicative of CARLase mediated polyester-polyurethane breakdown. The results demonstrated that CARLase is active on PET and polyester-polyurethane using a qualitative clearance assay.

[0089] High-performance liquid chromatography (HPLC) assays

[0090] Whole cell lysates from E. coli cells expressing CARLase and the chaperone GroEL / ES were incubated with 20 mg of amorphous PET discs (Polymershapes LLC) for 96 hours at 37 °C. Monomer production was monitored and quantified via high-performance liquid chromatography (HPLC). From this reaction, a total of 61.14 PPM (sum of monomers) was produced. As shown in FIG. 5D, PET depolymerization by CARLase was demonstrated by detection of monomer products, including terephthalic acid (TPA), 2- hydroxyethyl terephthalic acid (MHET), and mono(2-hydroxyethyl)-isophthalate (BHET).

[0091] These results demonstrated that CARLase hydrolyzes PET and polyester- polyurethane materials.Example 3. Engineered Variants of CARLase

[0092] CARLase was codon-optimized, synthesized by Integrated DNA Technologies (IDT™), and cloned into a pet-29b plasmid with a C-terminal hexa-histidine epitope tag. To increase the success rate of protein folding and stability, CARLase was co-transformed into SHuffle® T7 Express Competent E. Coli (NEW ENGLAND BIOLABS®) with pGro7, which expresses the chaperone pair groEL / groES (Takara Bio Inc.™). Transformants were grown overnight at 30 °C to saturation in a 400 pL LB seed culture in a 96DW plate, followed by overnight induction in minimal M9 media (25 °C, 22 hours) supplemented with 0.4% casamino acids, 100 pM IPTG (pet-29b) and 0.1% Arabinose (pGro7). Post induction,cells were lysed using NEB Express® E. coli Lysis Reagent following manufacturer instructions.

[0093] Whole cell lysates were incubated with aPET discs (15 mg) for 96 hours at 37 °C. Using purified enzyme, it was empirically determined that CARLase was most active at 30 °C, with a gradual drop-off of activity at higher temperatures. Therefore, 37 °C was selected for screening CARLase libraries to identify enzyme variants with thermotolerance improvements. Thermotolerant enzymes typically exhibit longer half-lives, which reduces the quantity of enzyme needed to achieve high yield plastic degradation and also reduces overall process cost. Such thermostable enzyme variants allow for degradation reactions to be carried out at higher temperatures closer to the glass transition temperature of PET, which is expected to increase PET degradation rates due to increased polymer mobility, thus providing a more efficient bio-enzymatic degradation processes.

[0094] Two types of libraries were designed for CARLase: site saturation mutagenesis (SSM / NNK) and introduction of disulfide bonds (DSB). SSM libraries with NNK (NN(T / G)) degenerate codons were chosen to generate diversity for recombination. DDGun was used to predict all single mutation folding energy changes (Montanucci, et al. BMC Bioinformatics 20 (Suppl 14), 335 (2019)). Positions in the assumed binding pocket were excluded from mutation. A multiple sequence alignment (MSA) containing proteins with at least 50% sequence identity to the wildtype was constructed to identify mutable positions. Positions with more predicted beneficial mutations (via DDGun) and more alternative residues observed in the MSA were weighted higher for SSM selection. Ten of the most promising positions were selected as sites to introduce NNK degenerate codon sequences for a total of 200 mutants. Disulfide bond information was analyzed and used to generate 120 paired cysteine designs per input structure. Several disulfide bond pairs were constructed with PyMOL for visual validation of designs. The total number of mutants screened was approximately 320.

[0095] These libraries were screened using a 96DW plate workflow where enzymes variants were expressed via IPTG induction. Post induction, cells were lysed, and whole cell lysates were incubated with aPET film (15 mg) for 96 hours at 37 °C.

[0096] After successfully screening CARLase NNK and DSB libraries, 5 variants were identified (3 distinct NNK mutations and 2 distinct DSB pairs) with improved activity at 37 °C. A variant with improved activity was defined as any variant with a >1.5 Fold Improvement Over Parent (FIOP) after at least four biological replicates were performed. Favorable amino acid substitutions were recombined for a second round of screening ofvariant enzymes, identifying variants that exhibited up to 10-fold performance improvements at 37 °C. Results showing performance improvements relative to the parent enzyme (SEQ ID NO: 2) are depicted in FIG. 6 and shown below in Table 3.Table 3. Engineered CARLase VariantsExample 4. Industrial Process of Bio-enzymatic Degradation

[0097] FIG. 7 is a flowchart showing an example process for bio-enzymatic degradation of PET. In this process, plastic samples are granulated and combined in a stirred-tank chemical reactor with hydrolase in aqueous solution at 70 °C. Non-target solids are removed by filtration and hydrolysate is passed through an activated carbon column to remove plastic additives and colorants prior to sequential recovery and purification of TPA and EG.EQUIVALENTS AND SCOPE

[0098] In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one,more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0099] Furthermore, the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is / are referred to as comprising particular elements and / or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and / or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein.

[0100] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, z.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0101] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion ofexactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0102] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0103] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0104] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g., “comprising”) are also contemplated, in alternative embodiments, as “consisting of’ and “consisting essentially of’ the feature described by the open-ended transitional phrase. For example, if the application describes “a composition comprising A and B,” the 1application also contemplates the alternative embodiments “a composition consisting of A and B” and “a composition consisting essentially of A and B.”

[0105] Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0106] This application may refer to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

[0107] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.

[0108] The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

Claims

CLAIMSWhat is claimed is:

1. A hydrolase having an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 2, wherein the amino acid sequence of the hydrolase comprises an amino acid substitution at one or more positions corresponding to positions N4, E7, A67, T70, QUO, E172, and N214 of SEQ ID NO: 2.

2. The hydrolase of claim 1, wherein the amino acid sequence of the hydrolase comprises an amino acid substitution at a position corresponding to position N214 of SEQ ID NO: 2.

3. The hydrolase of claim 1 or 2, wherein the amino acid sequence of the hydrolase comprises an amino acid substitution at a position corresponding to position QUO of SEQ ID NO: 2.

4. The hydrolase of any one of claims 1-3, wherein the amino acid sequence of the hydrolase comprises an amino acid substitution at a position corresponding to position E172 of SEQ ID NO: 2.

5. The hydrolase of any one of claims 1-4, wherein the amino acid sequence of the hydrolase comprises an amino acid substitution at two or more positions corresponding to positions N4, E7, A67, T70, QUO, E172, and N214 of SEQ ID NO: 2.

6. The hydrolase of claim 5, wherein the amino acid sequence of the hydrolase comprises an amino acid substitution at positions corresponding to positions T70 and N214 of SEQ ID NO: 2.

7. The hydrolase of claim 5 or 6, wherein the amino acid sequence of the hydrolase comprises an amino acid substitution at positions corresponding to positions QUO and N214 of SEQ ID NO: 2.

8. The hydrolase of any one of claims 5-7, wherein the amino acid sequence of the hydrolase comprises an amino acid substitution at positions corresponding to positions E172 and N214 of SEQ ID NO: 2.

9. The hydrolase of any one of claims 5-8, wherein the amino acid sequence of the hydrolase comprises an amino acid substitution at positions corresponding to positions N4 and E7 of SEQ ID NO: 2.

10. The hydrolase of any one of claims 1-9, wherein the amino acid substitution is selected from N4C, E7C, A67G, T70C, Q110R, E172L, and N214C relative to the sequence of SEQ ID NO: 2.

11. The hydrolase of any one of claims 1-10, wherein the amino acid sequence of the hydrolase comprises amino acid substitutions at positions corresponding to the following positions within SEQ ID NO: 2: i) N4 and E7; ii) T70 and N214; iii) Q110 and N214; iv) E172 and N214; v) A67, QUO, and N214; vi) N4, E7, A67, and El 72; vii) N4, E7, QUO, and N214; viii) A67, QUO, E172, and N214; ix) A67, T70, E172, and N214; x) A67, T70, QUO, and N214; xi) N4, E7, A67, QUO, and N214; xii) N4, E7, QUO, E172, and N214; xiii) A67, T70, QUO, E 172, and N214; or xiv) N4, E7, A67, QUO, E172, and N214.

12. The hydrolase of claim 11, wherein the amino acid substitutions comprise relative to the sequence of SEQ ID NO: 2: i) N4C and E7C; ii) T70C and N214C;iii) Q110R and N214C; iv) E172L and N214C; v) A67G, Q11OR, and N214C; vi) N4C, E7C, A67G, and E172L; vii) N4C, E7C, Q11OR, and N214C; viii) A67G, Q110R, E172L, and N214C; ix) A67G, T70C, E172L, and N214C; x) A67G, T70C, Q11OR, and N214C; xi) N4C, E7C, A67G, Q11OR, and N214C; xii) N4C, E7C, Q110R, E172L, and N214C; xiii) A67G, T70C, Q110R, E172L, and N214C; or xiv) N4C, E7C, A67G, Q11OR, E172L, and N214C.

13. The hydrolase of any one of claims 1-12, wherein the hydrolase exhibits increased depolymerization activity relative to a control hydrolase.

14. The hydrolase of claim 13, wherein the hydrolase exhibits at least 1.5-fold, at least 2- fold, at least 5-fold or at least 10-fold increased depolymerization activity relative to the control hydrolase.

15. The hydrolase of any one of claims 1-14, wherein the hydrolase exhibits improved thermotolerance relative to a control hydrolase.

16. The hydrolase of claim 15, wherein the hydrolase exhibits at least 1.5-fold, at least 2- fold, at least 5-fold or at least 10-fold improved thermotolerance relative to the control hydrolase.

17. The hydrolase of any one of claims 13-16, wherein the control hydrolase is a hydrolase that comprises the amino acid sequence of SEQ ID NO: 2.

18. A host cell that comprises a heterologous polynucleotide encoding the hydrolase of any one of claims 1-17.

19. A nucleic acid encoding the hydrolase of any one of claims 1-17.

20. A vector comprising the nucleic acid of claim 19.

21. An expression cassette comprising the nucleic acid of claim 19.

22. A host cell that comprises a heterologous polynucleotide encoding a hydrolase, wherein the hydrolase comprises a sequence that is at least 90% identical to SEQ ID NO: 1 or 2.

23. A non-naturally occurring nucleic acid encoding a hydrolase having a sequence that is at least 90% identical to SEQ ID NO: 1 or 2.

24. A vector comprising the non-naturally occurring nucleic acid of claim 23.

25. An expression cassette comprising the non-naturally occurring nucleic acid of claim 23.

26. A composition comprising: a first hydrolase comprising a polypeptide having a sequence that is at least 90% identical to SEQ ID NO: 1 or 2; and a second hydrolase.

27. The composition of claim 26, wherein the first hydrolase comprises the hydrolase of any one of claims 1-17.

28. The composition of claim 26, wherein the second hydrolase comprises the hydrolase of any one of claims 1-17.

29. The composition of claim 26 or 27, wherein the second hydrolase is a mono(2- hydroxy ethyl) terephthalate hydrolase (MHETase).

30. The composition of claim 29, wherein the MHETase is from Ideonella sakaiensis.

31. A method of degrading a polyester-containing polymer, the method comprising: contacting the polyester-containing polymer with a hydrolase under conditions to degrade the polyester-containing polymer, wherein the hydrolase comprises a sequence that is at least 90% identical to SEQ ID NO: 1 or 2.

32. The method of claim 31, wherein the polyester-containing polymer comprises polyethylene terephthalate, polyester-polyurethane, or a combination thereof.

33. The method of claim 31 or 32, wherein degradation of the polyester-containing polymer by the hydrolase produces one or more degradation products comprising monomers and / or oligomers.

34. The method of claim 33, further comprising: recovering the one or more degradation products.

35. The method of claim 33 or 34, wherein the one or more degradation products are selected from the group consisting of polyols, polycarboxylic acids, and polyamines.

36. The method of any one of claims 33-35, wherein the one or more degradation products comprise one or more polyols selected from the group consisting of ethylene glycol, diethylene glycol, 1,4-butanediol, triethylene glycol, propylene glycol, 1,2-dipropylene glycol, neopentyl glycol, glycerol, 1,1,1 -trimethylolpropane, sucrose, sorbitol, and pentaerythritol.

37. The method of any one of claims 33-36, wherein the one or more degradation products comprise one or more polycarboxylic acids selected from the group consisting of terephthalic acid (TPA), 2-hydroxyethyl terephthalic acid (MHET), mono(2-hydroxyethyl)- isophthalate (BHET), succinic acid, glutaric acid, adipic acid, phthalic acid, benzenetricarboxylic acid, oleic acid, and ricinoleic acid.

38. The method of any one of claims 33-37, wherein the one or more degradation products comprise one or more polyamines selected from the group consisting of methylene- 4,4'-diamine, methylene-2, 4 '-diamine, methylene-2, 2 '-diamine, naphthylene- 1,4-diamine,naphthylene- 1,5-diamine, naphthylene- 1,6-diamine, tolylene-2,4-diamine, and tolylene-2,6- diamine.

39. The method of any one of claims 31-38, wherein the hydrolase is purified.

40. The method of any one of claims 31-39, wherein the polyester-containing polymer is contacted with a host cell expressing and secreting the hydrolase.

41. The method of any one of claims 31-40, wherein the conditions comprise an incubation temperature of between about 30 °C and about 80 °C.

42. The method of any one of claims 31-41, wherein the conditions comprise an incubation temperature of about 70 °C.

43. The method of any one of claims 31-42, wherein the conditions comprise a pH of between about 3.5 and about 9.0.

44. The method of any one of claims 31-43, wherein the conditions comprise a pH of between about 4.0 and about 5.0.

45. The method of any one of claims 31-44, wherein the hydrolase comprises a sequence that is at least 90% identical to SEQ ID NO: 1.

46. A method of degrading a polyester-polyurethane, the method comprising: contacting the polyester-polyurethane with a hydrolase under conditions to degrade the polyester-polyurethane, wherein the hydrolase comprises a sequence that is at least 90% identical to SEQ ID NO: 1 or 2.

47. The method of any one of claims 31-46, wherein the hydrolase comprises the hydrolase of any one of claims 1-17.

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