Hydrolase variants and methods of use thereof

Abstract

Provided are hydrolase variants and methods of using hydrolase variants for degrading organic polymers, such as polyester-containing polymers, polyether-containing polymers, polypropylene-containing polymers, poly ethylene-containing polymers, and combinations thereof.

Description

Docket No.: B1704.70002WO00 HYDROLASE VARIANTS AND METHODS OF USE THEREOF CROSS-REFERENCE TORELATEDAPPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No.63 / 637,820, filed April 23, 2024, entitled “HYDROLASE VARIANTS AND METHODS OF USE THEREOF,” and U.S. Provisional Application No.63 / 721,137, filed November 15, 2024, entitled “HYDROLASE VARIANTS AND METHODS OF USE THEREOF,” the entire disclosure of each of which is hereby incorporated by reference in its entirety. BACKGROUND

[0002] 100 million tons of polyethylene terephthalate (PET) are used annually to manufacture plastic products including beverage containers and textiles. Several methods of PET recycling are currently in use or under development, though none enable a true economical, sustainable, and circular plastic economy. Two predominant methods for recycling PET are mechanical and chemical recycling. Mechanical recycling requires elaborate and costly sortation, melting, and extrusion to produce recycled resin, and typically produces lower quality plastic resin that is downcycled into lower value products. Chemical recycling technologies such as methanolysis and glycolysis typically require expensive chemical processing and high temperatures to achieve high yield of circular PET monomers, resulting in an energy-intensive process with harmful environmental impacts. Innovation is needed to improve the economics of plastic recycling, the quality of recycled resin, and bring plastics into the circular economy. Bio-enzymatic plastic recycling uses enzymes as “molecular scissors” to hydrolyze plastic, such as PET and other polyester-containing polymers, into monomers or oligomers that can be recovered and used as a drop-in replacement for plastic manufacturing. Engineered enzymes enable efficient hydrolysis of PET polymers with improved economics, sustainability, and circularity of plastic recycling. Bio-enzymatic plastic recycling at scale will require improved enzymes having increased activity, for example, at high temperature and low pH conditions. SUMMARY

[0003] The present disclosure, in some aspects, provides hydrolases and their uses, including engineered hydrolase variants for use in plastic recycling. In someDocket No.: B1704.70002WO00 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 provide means for recycling plastic products by degrading one polymer to produce monomers and / or oligomers that can be repolymerized to form another polymer.

[0004] In some aspects, the disclosure provides a hydrolase comprising 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, where the amino acid sequence of the hydrolase comprises an amino acid substitution at one or more positions corresponding to positions D13, R19, S20, S24, N29, S31, L33, S34, S36, T42, R47, N49, N50, Y52, A54, V55, I57, S58, G63, E65, A66, S67, K73, I75, I83, I88, L91, Q93, E99, A103, A104, N106, I109, R111, A112, S113, S114, T115, R117, I120, S122, S123, V127, M128, S137, S142, Q143, A150, T154, W156, L158, N159, N161, S163, S164, V165, R166, V167, D175, L176, T178, I179, V182, L183, T184, H185, F189, N191, P194, T195, S196, I197, S198, L204, D205, G206, A207, T208, F210, N213, I214, P215, K217, I218, V224, N233, T235, T238, L241, P243, P245, R246, E252, and E254 of SEQ ID NO: 1.

[0005] In some embodiments, the hydrolase comprises at least one additional disulfide bond relative to a hydrolase that comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the hydrolase comprises one additional disulfide bond relative to a hydrolase that comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the hydrolase comprises two or more (e.g., 2, 3, 4, 5, 2-10, 2-5, 3-5, 5-10) additional disulfide bonds relative to a hydrolase that comprises the amino acid sequence of SEQ ID NO: 1.

[0006] In some embodiments, the amino acid sequence of the hydrolase comprises amino acid substitutions at positions corresponding to positions D205 and E254 of SEQ ID NO: 1. In some embodiments, the amino acid substitutions comprise D205C and E254C relative to SEQ ID NO: 1.

[0007] In some embodiments, the amino acid sequence of the hydrolase comprises amino acid substitutions at positions corresponding to positions I109 and S122 of SEQ ID NO: 1. In some embodiments, the amino acid substitutions comprise I109C and S122C relative to SEQ ID NO: 1.

[0008] In some embodiments, the amino acid sequence of the hydrolase comprises amino acid substitutions at positions corresponding to the following positions of SEQ ID NO: 1: (i)Docket No.: B1704.70002WO00 S137; and (ii) V127 or A150. In some embodiments, the amino acid substitutions comprise relative to SEQ ID NO: 1: (i) S137C and V127C; or (ii) S137C and A150C.

[0009] In some embodiments, the amino acid sequence of the hydrolase comprises amino acid substitutions at positions corresponding to positions P194 and S196 of SEQ ID NO: 1. In some embodiments, the amino acid substitutions comprise P194C and S196C relative to SEQ ID NO: 1.

[0010] In some embodiments, the amino acid sequence of the hydrolase comprises amino acid substitutions at positions corresponding to positions F210 and T235 of SEQ ID NO: 1. In some embodiments, the amino acid substitutions comprise relative to SEQ ID NO: 1: (i) F210G, F210I, F210K, F210N, F210R, or F210T; and (ii) T235D, T235F, T235G, T235I, T235K, T235L, T235Q, T235V, or T235Y. In some embodiments, the amino acid substitutions comprise relative to SEQ ID NO: 1: (i) F210I, F210R, or F210T; and (ii) T235D, T235F, T235G, T235I, T235K, T235L, T235Q, T235V, or T235Y. In some embodiments, the amino acid substitutions comprise relative to SEQ ID NO: 1: (i) F210R; and (ii) T235D, T235F, T235K, or T235Y.

[0011] In some embodiments, the amino acid sequence of the hydrolase comprises an amino acid substitution at one or more positions corresponding to positions A54, S58, and E65 of SEQ ID NO: 1. In some embodiments, the amino acid substitution is selected from A54G, S58C, E65C, E65K, E65Q, E65R, and E65V relative to SEQ ID NO: 1.

[0012] In some embodiments, the amino acid sequence of the hydrolase comprises an amino acid substitution at one or more positions corresponding to positions R47, S114, D175, H185, D205, F210, T235, and E254 of SEQ ID NO: 1. In some embodiments, the amino acid substitution is selected from R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C relative to SEQ ID NO: 1. In some embodiments, the amino acid substitution is D175Q relative to SEQ ID NO: 1. In some embodiments, the amino acid substitution is H185Y relative to SEQ ID NO: 1. In some embodiments, the amino acid substitution is F210R relative to SEQ ID NO: 1.

[0013] In some embodiments, the amino acid sequence of the hydrolase comprises amino acid substitutions at positions corresponding to positions R47, S114, D175, H185, D205, F210, T235, and E254 of SEQ ID NO: 1. In some embodiments, the amino acid substitutions comprise R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C relative to SEQ ID NO: 1.

[0014] In some embodiments, the amino acid sequence of the hydrolase comprises one or more amino acid substitutions selected from R19I, R19L, R19F, R19K, R19V, S24L, S24R,Docket No.: B1704.70002WO00 N29A, N29K, N29V, S34A, S34E, S34K, S36K, S36N, S36R, T42A, T42M, R47E, R47G, R47I, R47L, R47T, N49P, N49T, N50H, N50T, Y52F, A54G, V55I, I57L, I57M, I57V, S58C, G63L, G63S, G63C, G63N, E65C, E65K, E65Q, E65R, E65V, A66D, A66E, A66G, A66L, S67E, K73D, K73P, K73Q, I75L, I75T, I75V, I83L, I88H, I88N, L91I, L91R, L91V, Q93N, Q93S, A104S, I109C, A112S, S113P, S113H, S114D, S114G, S114N, S114R, I120V, S122C, S123A, S123N, V127C, M128T, S137C, S142E, A150C, T154S, N161D, S163F, S163Q, S163D, S163K, S163N, S163A, D175A, D175E, D175G, D175I, D175N, D175Q, D175R, D175S, D175T, L176A, L176I, L176R, L176T, L176V, T178G, T178Q, T178V, I179I, I179V, L183S, L183V, L183K, L183I, L183A, T184A, T184H, T184A, T184K, T184Q, H185Y, F189I, N191K, N191Q, N191A, P194C, T195E, T195M, T195P, S196C, S196P, S196T, S198A, S198E, S198N, S198T, L204G, D205C, G206R, A207V, T208G, T208S, F210G, F210I, F210K, F210N, F210R, F210T, I214V, I214K, I214M, P215V, I218T, V224L, N233V, T235E, T235G, T235I, T235L, T235Q, T235D, T235F, T235K, T235Y, T238N, L241M, P243G, P243R, P245K, R246L, E252A, and E254C relative to SEQ ID NO: 1.

[0015] 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: 1: R47, D205, and E254; R47, N50, D205, and E254; R47, K73, D205, and E254; I179, D205, and E254; R47, D205, and E254; D205, F210, and E254; T184, F189, D205, F210, and E254; D205, F210, A211, and E254; D205, F210, T235, and E254; D205, F210, P215, and E254; D205, F210, N233, P243, and E254; D205, F210, P215, T235, and E254; N49, K73, L91, D205, and E254; K73, D205, T235, P243, and E254; D205, F210, N233, T235, P243, and E254; D205, F210, N233, T235, and E254; S24, N29, D205, and E254; L183, D205, I218, and E254; R47, E65, S114, H185, D205, F210, T235, and E254; R47, S114, H185, D205, F210, T235, and E254; R47, K73, S114, I179, D205, F210, T235, and E254; R47, V55, G63, S114, H185, D205, F210, T235, and E254; R47, S114, H185, I179, D205, F210, T235, and E254; R47, I57, I83, S114, H185, D205, F210, T235, and E254; R47, I57, S114, H185, D205, F210, T235, and E254; R47, S114, D175, L176, H185, D205, F210, T235, and E254; R47, S114, N161, H185, D205, F210, T235, and E254; R47, H185, D205, F210, T235, and E254; R47, S114, H185, L204, D205, F210, T235, and E254; R47, S114, D175, H185, D205, F210, T235, and E254; R47, I75, S114, H185, D205, F210, T235, and E254; R47, S114, S123, H185, D205, F210, T235, and E254; R47, S114, D175, L176, D205, H185, F210, T235, and E254; R47, S114, L176, T178, H185, D205, F210, T235, and E254; R47, I57, I75, S114, H185, D205, F210, T235, and E254; I75, S114, H185, D205, F210, T235, and E254; R47, S113, S114, S123, H185, D205, F210, T235, and E254; R47, S114, H185, N191,Docket No.: B1704.70002WO00 D205, F210, T235, and E254; R47, S114, T178, H185, D205, F210, T235, and E254; S34, Y52, S114, H185, D205, F210, T235, and E254; R47, A112, S114, S123, H185, D205, F210, T235, and E254; R47, S114, I120, H185, D205, F210, T235, and E254; R47, S114, H185, S198, D205, F210, T235, and E254; R47, S114, H185, T195, D205, F210, T235, and E254; R47, S114, H185, D205, G206, F210, T235, and E254; R19, S36, N50, Y52, I57, I75, S114, H185, F210, and T235; R19, N50, Y52, I75, S114, H185, F210, and T235; R19, S34, R47, N50, Y52, I57, I75, S114, H185, F210, and T235; S34, N50, I57, I75, S114, S142, H185, F210, and T235; S34, Y52, I57, I75, S114, H185, F210, and T235; S36, N50, Y52, I57, S114, H185, F210, and T235; R47, S114, D175, H185, D205, F210, T235, P245, R246, and E254; R47, S114, H185, D205, F210, T235, T238, and E254; R19, R47, S114, D175, H185, D205, F210, T235, and E254; R19, R47, S114, D175, H185, D205, F210, T235, P245, R246, and E254; R19, S34, S36, R47, N50, Y52, D175, H185, F210, and T235; R47, D175, H185, D205, F210, T235, and E254; R47, I57, D175, H185, D205, F210, T235, and E254; R47, N50, Y52, I57, S114, D175, H185, D205, F210, T235, and E254; S34, S114, D175, H185, D205, F210, T235, P245, R246, and E254; S34, Y52, S114, D175, H185, D205, F210, T235, and E254; T42, R47, S114, D175, H185, D205, F210, T235, and E254; T42, R47, S114, D175, H185, D205, F210, V224, T235, and E254; R19, T42, R47, S114, D175, H185, D205, F210, T235, and E254; R47, I57, I75, S114, D175, H185, D205, F210, T235, and E254; R47, Y52, S114, D175, H185, D205, F210, L224, T235, and E254; S34, S36, R47, D175, H185, D205, F210, T235, and E254; S34, S36, R47, S114, D175, H185, D205, F210, T235, and E254; S34, S36, R47, S114, H185, D205, F210, T235, and E254; S34, D175, H185, D205, F210, T235, and E254; S34, R47, I75, S114, D175, H185, D205, F210, T235, and E254; S34, R47, S114, D175, H185, D205, F210, T235, and E254; R47, I57, I75, S114, H185, D205, T208, F210, T235, and E254; R47, I57, I75, S114, D175, H185, D205, A207, F210, T235, and E254; R47, I75, S114, D175, H185, D205, F210, I214, T235, and E254; R47, S114, M128, D175, H185, D205, F210, T235, and E254; S34, Y52, A104, S114, D175, H185, D205, F210, T235, and E254; R47, I57, G63, A66, S114, D175, H185, D205, F210, T235, and E254; R47, Y52, G63, A66, S114, D175, H185, D205, F210, T235, and E254; R47, I57, A66, S114, D175, H185, D205, F210, T235, and E254; R47, G63, A66, S114, D175, H185, D205, F210, T235, and E254; R47, E65, S67, S114, D175, H185, D205, F210, T235, and E254; R47, S114, S163, D175, H185, D205, F210, T235, and E254; R47, S114, D175, H185, D205, F210, T235, E252, and E254; R47, S114, D175, H185, D205, F210, T235, L241, and E254; R47, S114, D175, H185, D205, T208, F210, T235, and E254; R47, S114, T154, D175, T184, H185, D205, F210, T235, and E254; R47, I57, L91, Q93, S114, D175, H185, D205,Docket No.: B1704.70002WO00 F210, T235, and E254; R47, I88, S114, D175, H185, D205, F210, T235, and E254; R47, Q93, S114, D175, H185, D205, F210, T235, and E254; N29, S31, S36, R47, N49, S114, D175, H185, D205, F210, T235, and E254; R19, S20, T42, R47, S114, D175, H185, D205, F210, T235, and E254; R47, A103, N106, R111, S114, T115, D175, H185, D205, F210, T235, and E254; R47, A66, I75, S114, D175, H185, D205, F210, T235, and E254; R47, L91, N106, S114, D175, T184, H185, D205, F210, K217, T235, and E254; S34, Y52, G63, S114, D175, H185, D205, F210, T235, and E254; R47, R111, S114, D175, H185, D205, F210, T235, and E254; S31, R47, S114, D175, H185, D205, F210, T235, and E254; S34, Y52, L91, S113, S114, D175, H185, D205, F210, T235, and E254; R47, I57, G63, A66, S114, D175, H185, D205, T208, F210, T235, and E254; S34, G63, A66, S114, D175, H185, D205, F210, T235, and E254; R47, S114, W156, L158, D175, H185, S196, D205, F210, I214, T235, and E254; R47, S114, L158, R166, D175, H185, D205, F210, T235, and E254; R47, S114, L158, V167, D175, H185, N191, D205, F210, T235, and E254; R47, S114, D175, V182, H185, D205, F210, T235, and E254; R47, S114, S163, R166, D175, H185, N191, D205, F210, T235, and E254; R47, S114, L158, D175, H185, S196, D205, F210, T235, and E254; R47, S114, S163, S164, D175, H185, D205, F210, I214, T235, and E254; R47, S114, S164, D175, L183, H185, T195, D205, F210, T235, and E254; R47, S114, N159, D175, H185, D205, F210, T235, and E254; R47, S114, D175, S198, D205, F210, T235, and E254; R47, S114, V165, D175, L183, H185, I197, S198, D205, F210, T235, and E254; R47, S114, S142, T154, R166, D175, H185, S198, D205, F210, T235, and E254; R47, S114, Q143, S163, S164, D175, H185, T195, D205, F210, T235, and E254; R47, S114, L158, S163, S164, D175, H185, S198, D205, F210, N213, T235, and E254; R47, Y52, I57, G63, A66, I75, S114, D175, H185, D205, F210, T235, and E254; D13, R47, N50, I88, E99, S114, D175, H185, D205, F210, K217, T235, and E254; Y52, I57, G63, A66, I75, S114, D175, H185, D205, F210, T235, and E254; R47, G63, S114, R117, D175, L183, H185, D205, F210, I214, T235, and E254; R19, R47, G63, S114, R166, D175, L183, H185, D205, F210, T235, and E254; R47, S114, D175, L183, H185, D205, F210, T235, and E254; R47, S114, L158, S163, D175, T184, H185, D205, F210, T235, and E254; R47, Q93, S114, D175, H185, D205, T208, F210, T235, and E254; S34, R47, S114, L158, D175, H185, D205, F210, T235, and E254; or L33, R47, S114, R117, R166, D175, L183, H185, D205, F210, T235, and E254.

[0016] In some embodiments, the amino acid substitutions comprise relative to the sequence of SEQ ID NO: 1: R47V, D205C, and E254C; R47L, D205C, and E254C; R47E, N50G, D205C, and E254C; R47V, K73Q, D205C, and E254C; I179V, D205C, and E254C; R47T, D205C, and E254C; R47G, D205C, and E254C; D205C, F210G, and E254C; D205C,Docket No.: B1704.70002WO00 F210K, and E254C; T184H, F189I, D205C, F210K, and E254C; D205C, F210N, and E254C; D205C, F210K, A211G, and E254C; D205C, F210I, T235L, and E254C; D205C, F210I, T235G, and E254C; D205C, F210T, T235G, and E254C; D205C, F210R, P215V, and E254C; D205C, F210R, N233V, P243G, and E254C; D205C, F210R, P215V, T235V, and E254C; D205C, F210T, T235D, and E254C; D205C, F210R, T235L, and E254C; D205C, F210R, T235Y, and E254C; D205C, F210R, T235F, and E254C; D205C, F210R, T235Q, and E254C; D205C, F210R, T235K, and E254C; D205C, F210R, T235I, and E254C; D205C, F210I, T235D, and E254C; N49P, K73P, L91R, D205C, and E254C; K73D, D205C, T235E, P243R, and E254C; D205C, F210R, N233H, T235D, P243G, and E254C; D205C, F210R, N233H, T235G, and E254C; S24R, N29A, D205C, and E254C; S24L, N29K, D205C, and E254C; L183S, D205C, I218T, and E254C; R47E, E65K, S114N, H185Y, D205C, F210R, T235I, and E254C; R47E, S114D, H185Y, D205C, F210R, T235Y, and E254C; R47G, S114G, H185Y, D205C, F210R, T235F, and E254C; R47L, K73Q, S114D, I179V, D205C, F210R, T235Y, and E254C; R47E, V55I, G63S, S114D, H185Y, D205C, F210R, T235Y, and E254C; R47G, S114G, H185Y, I179I, D205C, F210R, T235Y, and E254C; R47G, I57V, I83L, S114D, H185Y, D205C, F210R, T235Y, and E254C; R47E, I57V, S114D, H185Y, D205C, F210R, T235Y, and E254C; R47E, S114D, D175G, L176A, H185Y, D205C, F210R, T235Y, and E254C; R47G, S114G, N161D, H185Y, D205C, F210R, T235Y, and E254C; R47E, S114G, D175S, L176A, H185Y, D205C, F210R, T235Y, and E254C; R47G, S114G, D175G, L176A, H185Y, D205C, F210R, T235Y, and E254C; R47I, H185Y, D205C, F210R, T235F, and E254C; R47G, S114G, H185Y, L204G, D205C, F210R, T235Y, and E254C; R47G, S114G, D175S, H185Y, D205C, F210R, T235Y, and E254C; R47E, I75L, S114D, H185Y, D205C, F210R, T235Y, and E254C; R47E, S114G, N161D, H185Y, D205C, F210R, T235Y, and E254C; R47G, S114D, H185Y, D205C, F210R, T235Y, and E254C; R47G, S114D, S123A, H185Y, D205C, F210R, T235Y, and E254C; R47E, S114D, D175R, L176V, H185Y, D205C, F210R, T235Y, and E254C; R47G, S114G, D175R, L176A, D205C, H185Y, F210R, T235Y, and E254C; R47G, S114G, L176R, T178Q, H185Y, D205C, F210R, T235Y, and E254C; R47G, I75V, S114G, H185Y, D205C, F210R, T235Y, and E254C; R47G, S114D, D175T, L176V, H185Y, D205C, F210R, T235Y, and E254C; R47E, I57M, I75V, S114D, H185Y, D205C, F210R, T235Y, and E254C; I75T, S114D, H185Y, D205C, F210R, T235I, and E254C; R47E, S114D, L176R, T178V, H185Y, D205C, F210R, T235Y, and E254C; R47E, I57V, I75V, S114D, H185Y, D205C, F210R, T235Y, and E254C; R47G, S114D, D175E, L176A, H185Y, D205C, F210R, T235Y, and E254C; R47E, I57V, I75L, S114D, H185Y, D205C, F210R, T235Y, and E254C; R47E, S113P, S114D, S123A, H185Y, D205C,Docket No.: B1704.70002WO00 F210R, T235Y, and E254C; R47G, S114G, D175S, L176I, H185Y, D205C, F210R, T235F, and E254C; R47E, S114D, H185Y, N191K, D205C, F210R, T235Y, and E254C; R47E, S114D, T178G, H185Y, D205C, F210R, T235Y, and E254C; S34E, Y52F, S114D, H185Y, D205C, F210R, T235I, and E254C; R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, S114G, D175I, L176A, H185Y, D205C, F210R, T235Y, and E254C; R47G, S114G, D175Q, L176I, H185Y, D205C, F210R, T235Y, and E254C; R47E, S114G, D175Q, L176T, H185Y, D205C, F210R, T235Y, and E254C; R47E, A112S, S114G, S123N, H185Y, D205C, F210R, T235Y, and E254C; R47E, S114G, I120V, H185Y, D205C, F210R, T235Y, and E254C; R47E, S114D, H185Y, S198A, D205C, F210R, T235Y, and E254C; R47G, S114G, H185Y, T195E, D205C, F210R, T235Y, and E254C; R47E, S114G, H185Y, D205C, G206R, F210R, T235Y, and E254C; R19I, S36R, N50T, Y52F, I57V, I75V, S114D, H185Y, F210R, and T235Y; R19L, N50T, Y52F, I75V, S114G, H185Y, F210R, and T235Y; R19L, S34E, R47E, N50T, Y52F, I57M, I75T, S114D, H185Y, F210R, and T235Y; S34A, N50T, I57M, I75V, S114D, S142E, H185Y, F210R, and T235Y; S34E, Y52F, I57V, I75V, S114D, H185Y, F210R, and T235Y; S36N, N50H, Y52F, I57M, S114G, H185Y, F210R, and T235Y; R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, P245K, R246L, and E254C; R47G, S114D, H185Y, D205C, F210R, T235Y, T238N, and E254C; R47G, S114G, H185Y, D205C, F210R, T235Y, T238N, and E254C; R19L, R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R19L, R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, P245K, R246L, and E254C; R19L, S34A, S36K, R47E, N50H, Y52F, D175Q, H185Y, F210R, and T235Y; R47E, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, I57V, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, N50H, Y52F, I57V, S114D, D175Q, H185Y, D205C, F210R, T235F, and E254C; R47E, S114G, D175Q, H185Y, D205C, F210R, T235Y, and E254C; S34E, S114D, D175Q, H185Y, D205C, F210R, T235Y, P245K, R246L, and E254C; S34E, Y52F, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; S34E, Y52F, S114G, D175Q, H185Y, D205C, F210R, T235Y, and E254C; T42A, R47E, S114D, D175Q, H185Y, D205C, F210R, T235F, and E254C; T42A, R47E, S114D, D175Q, H185Y, D205C, F210R, V224L, T235Y, and E254C; R19L, T42A, R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, I57V, I75L, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, Y52F, S114D, D175Q, H185Y, D205C, F210R, L224V, T235Y, and E254C; S34A, S36K, R47E, D175Q, H185Y, D205C, F210R, T235Y, and E254C; S34A, S36K, R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; S34A, S36K, R47E, S114G, H185Y, D205C, F210R, T235Y, and E254C; S34E, D175Q, H185Y, D205C, F210R, T235Y, and E254C; S34E, R47E, I75L, S114G, D175Q,Docket No.: B1704.70002WO00 H185Y, D205C, F210R, T235Y, and E254C; S34E, R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, S114D, D175A, H185Y, D205C, F210R, T235Y, and E254C; R47E, I57V, I75L, S114D, D175N, H185Y, D205C, F210R, T235Y, and E254C; R47E, I57V, I75L, S114D, H185Y, D205C, T208G, F210R, T235Y, and E254C; R47E, I57V, I75L, S114D, D175Q, H185Y, D205C, A207V, F210R, T235Y, and E254C; R47E, I75L, S114D, D175Q, H185Y, D205C, F210R, I214V, T235Y, and E254C; R47E, S114D, M128T, D175Q, H185Y, D205C, F210R, T235F, and E254C; S34E, Y52F, A104S, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, I57L, G63L, A66E, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, Y52F, G63L, A66D, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, I57L, A66G, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, G63L, A66L, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, E65C, S67E, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, S114G, S163F, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, E252A, and E254C; R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, L241M, and E254C; R47E, S114D, D175Q, H185Y, D205C, T208S, F210R, T235Y, and E254C; R47E, S114D, T154S, D175Q, T184A, H185Y, D205C, F210R, T235Y, and E254C; R47E, I57V, L91I, Q93N, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, I88H, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, Q93S, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; N29V, S31Q, S36K, R47E, N49T, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R19V, S20D, T42M, R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, A103Q, N106D, R111Y, S114R, T115P, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R19F, S20D, T42M, R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, A66E, I75V, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, L91I, N106T, S114D, D175Q, T184Q, H185Y, D205C, F210R, K217R, T235Y, and E254C; S34E, Y52F, G63N, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, R111E, S114G, D175Q, H185Y, D205C, F210R, T235Y, and E254C; S31K, R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; S34E, Y52F, L91V, S113H, S114G, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, I57L, G63L, A66E, S114D, D175Q, H185Y, D205C, T208G, F210R, T235Y, and E254C; R47E, I57L, G63L, A66E, S114G, D175Q, H185Y, D205C, F210R, T235Y, and E254C; S34E, G63L, A66E, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, I57V, L91I, Q93N, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, S114D, W156F, L158T, D175Q, H185Y, S196T, D205C, F210R, I214M, T235Y, and E254C;Docket No.: B1704.70002WO00 R47E, S114D, L158I, R166K, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, S114D, L158I, V167I, D175Q, H185Y, N191Q, D205C, F210R, T235Y, and E254C; R47E, S114D, D175Q, V182I, H185Y, D205C, F210R, T235Y, and E254C; R47E, S114D, S163A, R166I, D175Q, H185Y, N191A, D205C, F210R, T235Y, and E254C; R47E, S114D, L158T, D175Q, H185Y, S196P, D205C, F210R, T235Y, and E254C; R47E, S114D, S163A, S164T, D175Q, H185Y, D205C, F210R, I214M, T235Y, and E254C; R47E, S114D, S164N, D175Q, L183A, H185Y, T195M, D205C, F210R, T235Y, and E254C; R47E, S114D, N159A, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, S114D, D175Q, S198T, D205C, F210R, T235Y, and E254C; R47E, S114D, V165I, D175Q, L183V, H185Y, I197V, S198E, D205C, F210R, T235Y, and E254C; R47E, S114D, S142E, T154S, R166T, D175Q, H185Y, S198A, D205C, F210R, T235Y, and E254C; R47E, S114D, Q143K, S163A, S164T, D175Q, H185Y, T195P, D205C, F210R, T235Y, and E254C; R47E, S114D, L158V, S163N, S164T, D175Q, H185Y, S198N, D205C, F210R, N213T, T235Y, and E254C; R47E, Y52F, I57L, G63C, A66E, I75V, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; D13A, R47E, N50G, I88N, E99D, S114G, D175Q, H185Y, D205C, F210R, K217P, T235Y, and E254C; Y52F, I57L, G63C, A66E, I75V, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, G63L, S114D, R117K, D175Q, L183K, H185Y, D205C, F210R, I214K, T235Y, and E254C; R19K, R47E, G63L, S114D, R166T, D175Q, L183K, H185Y, D205C, F210R, T235Y, and E254C; R47E, S114D, D175Q, L183I, H185Y, D205C, F210R, T235Y, and E254C; R47E, S114D, L158N, S163K, D175Q, T184K, H185Y, D205C, F210R, T235Y, and E254C; R47E, S114D, L158T, S163Q, D175Q, T184K, H185Y, D205C, F210R, T235Y, and E254C; R47E, Q93S, S114D, D175Q, H185Y, D205C, T208S, F210R, T235Y, and E254C; S34K, R47E, S114D, L158T, D175Q, H185Y, D205C, F210R, T235Y, and E254C; L33K, R47E, S114D, R117K, R166T, D175Q, L183K, H185Y, D205C, F210R, T235Y, and E254C; or R47E, S114D, L158T, S163D, D175Q, T184K, H185Y, D205C, F210R, T235Y, and E254C.

[0017] In some embodiments, a hydrolase of the disclosure comprises an amino acid sequence of any one of SEQ ID NOs: 5-183. In some embodiments, a hydrolase of the disclosure comprises 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 any one of SEQ ID NOs: 5-183. In some embodiments, a hydrolase of the disclosure comprises an amino acid sequence that is a conservatively substituted version of any one of SEQ ID NOs: 5-183.

[0018] In some embodiments, a hydrolase of the disclosure comprises an amino acid sequence of any one of SEQ ID NOs: 85, 100, 103, 104, 114-117, 119, 123, 127, 128, 131-Docket No.: B1704.70002WO00 135, 137-145, 147-149, 152, 154, 157-166, 168-172, and 175-183. In some embodiments, a hydrolase of the disclosure comprises 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 any one of SEQ ID NOs: 85, 100, 103, 104, 114-117, 119, 123, 127, 128, 131-135, 137-145, 147-149, 152, 154, 157-166, 168-172, and 175-183. In some embodiments, a hydrolase of the disclosure comprises an amino acid sequence that is a conservatively substituted version of any one of SEQ ID NOs: 85, 100, 103, 104, 114-117, 119, 123, 127, 128, 131-135, 137-145, 147-149, 152, 154, 157-166, 168-172, and 175-183.

[0019] 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 depolymerization activity is determined at a temperature of more than 50 °C, between about 50 °C and about 80 °C, or about 70 °C. In some embodiments, the depolymerization activity is determined at a pH of below 7.0, between about 4.5 and about 6.5, or about 5.0. In some embodiments, the depolymerization activity is determined at a pH of above 7.0, between about 7.5 and about 8.5, or about 8.0. 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: 1.

[0020] In some aspects, the disclosure provides a host cell that comprises a heterologous polynucleotide encoding a hydrolase described herein. In some embodiments, the hydrolase comprises 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. In some embodiments, the amino acid sequence of the hydrolase comprises an amino acid substitution at one or more positions corresponding to positions of SEQ ID NO: 1 as described herein. In some embodiments, the hydrolase comprises 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%, or 100%) identical to any one of SEQ ID NOs: 5-183. In some embodiments, the hydrolase comprises 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%, or 100%) identical to any one of SEQ ID NOs: 85, 100, 103, 104, 114-117, 119, 123, 127, 128, 131-135, 137-145, 147-149, 152, 154, 157-166, 168-172, and 175-183.

[0021] 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, theDocket No.: B1704.70002WO00 hydrolase comprises 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. In some embodiments, the amino acid sequence of the hydrolase comprises an amino acid substitution at one or more positions corresponding to positions of SEQ ID NO: 1 as described herein. In some embodiments, the hydrolase comprises 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%, or 100%) identical to any one of SEQ ID NOs: 5-183. In some embodiments, the hydrolase comprises 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%, or 100%) identical to any one of SEQ ID NOs: 85, 100, 103, 104, 114-117, 119, 123, 127, 128, 131-135, 137-145, 147-149, 152, 154, 157-166, 168-172, and 175-183.

[0022] 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.

[0023] In some aspects, the disclosure provides a composition comprising a hydrolase described herein. In some embodiments, the composition comprises: a first hydrolase comprising a hydrolase described herein; and a second hydrolase, where the first and second hydrolases are different. In some embodiments, the first hydrolase comprises 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. In some embodiments, the amino acid sequence of the first hydrolase comprises an amino acid substitution at one or more positions corresponding to positions of SEQ ID NO: 1 as described herein. In some embodiments, the first hydrolase comprises 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%, or 100%) identical to any one of SEQ ID NOs: 5-183. In some embodiments, the first hydrolase comprises 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%, or 100%) identical to any one of SEQ ID NOs: 85, 100, 103, 104, 114-117, 119, 123, 127, 128, 131- 135, 137-145, 147-149, 152, 154, 157-166, 168-172, and 175-183. 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 composition further comprises a third hydrolase, where the third hydrolase is different from the first and second hydrolases. In some embodiments the third hydrolase is a PETase, a polyamidase, a polyurethanase, or a cellulase. In some embodiments, the composition comprises three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more) hydrolases.Docket No.: B1704.70002WO00

[0024] In some aspects, the disclosure provides a kit comprising a first hydrolase described herein and optionally one or more additional hydrolases. In some embodiments, the kit comprises instructions for using the hydrolase in a method of degrading an organic polymer (e.g., a polyether-containing polymer, a polyester-containing polymer, a polypropylene- containing polymer, a polyethylene-containing polymer, or a combination thereof). In some embodiments, the hydrolase comprises 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%, at least 98%, 60-80%, 70-90%, 80-99%) identical to SEQ ID NO: 1, where the amino acid sequence comprises an amino acid substitution at one or more positions corresponding to positions of SEQ ID NO: 1 as described herein. In some embodiments, the hydrolase comprises 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%, or 100%) identical to any one of SEQ ID NOs: 5-183. In some embodiments, the hydrolase comprises 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%, or 100%) identical to any one of SEQ ID NOs: 85, 100, 103, 104, 114-117, 119, 123, 127, 128, 131-135, 137-145, 147-149, 152, 154, 157-166, 168-172, and 175-183. In some embodiments, the kit comprises a first hydrolase possessing the characteristics described above and a second hydrolase, where the first hydrolase and the second hydrolase are different. In some embodiments, the second hydrolase comprises a MHETase, a PETase, a polyamidase, a polyurethanase, or a cellulase.

[0025] In some aspects, the disclosure provides methods of degrading an organic polymer. In some embodiments, a method of degrading an organic polymer comprises contacting the organic polymer with a hydrolase of the disclosure under conditions to degrade the organic polymer.

[0026] In some embodiments, the organic polymer comprises a polyether-containing polymer, a polyester-containing polymer, a polypropylene-containing polymer, a polyethylene-containing polymer, or a combination thereof. In some embodiments, the organic polymer comprises polyethylene glycol, polyethylene terephthalate, polyester- polyurethane, polytrimethylene terephthalate, polybutylene terephthalate, polyethylene isosorbide terephthalate, polylactic acid, polyhydroxyalkanoate, polybutylene succinate, polybutylene succinate adipate, polybutylene adipate terephthalate, polyethylene furanoate, polycaprolactone, poly(ethylene adipate), polyethylene naphthalate, or a combination thereof.

[0027] In some embodiments, degradation of the organic 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 degradationDocket No.: B1704.70002WO00 products. In some embodiments, the one or more degradation products include hydrolyzed polyols, hydrolyzed polycarboxylic acids, and / or hydrolyzed polyamines.

[0028] In some embodiments, the organic polymer comprises polyester-polyurethane. Accordingly, in some aspects, the disclosure provides methods of degrading a polyester- polyurethane. In some embodiments, a method of degrading a polyester-polyurethane comprises contacting the polyester-polyurethane with a hydrolase of the disclosure under conditions to degrade the polyester-polyurethane.

[0029] In some embodiments, a method of the disclosure comprises contacting an organic polymer with a hydrolase of the disclosure under conditions to degrade the organic polymer. In some embodiments, the hydrolase is a purified hydrolase (e.g., a purified recombinant hydrolase). For example, in some embodiments, the organic 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 a hydrolase of the disclosure. In some embodiments, the conditions comprise an incubation temperature of between about 30 °C and about 80 °C (e.g., about 70 °C). In some embodiments, the conditions comprise a pH of between about 3.5 and about 9.0 (e.g., between about 4.5 and about 5.5, between about 7.5 and about 8.5).

[0030] 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

[0031] 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.

[0032] FIG.1 shows PET depolymerization assay results evaluating thermotolerance with an engineered hydrolase, referred to herein as “BB212” (corresponding to SEQ ID NO: 1 (without a C-terminal polyhistidine tag) or SEQ ID NO: 2 (with a C-terminal polyhistidine tag)), and a predicted disulfide bond hydrolase variant of BB212 (D205C, E254C relative to the sequence of SEQ ID NO: 1), referred to herein as “DSB127” (corresponding to SEQ ID NO: 5 (without a C-terminal polyhistidine tag) or SEQ ID NO: 6 (with a C-terminal polyhistidine tag)) (standard deviation of replicates shown).

[0033] FIG.2 shows PET depolymerization assay results evaluating low pH activity with DSB127 and three variants of DSB127 within a larger library of engineered protein variants.Docket No.: B1704.70002WO00

[0034] FIG.3 shows three different perspectives of a BB212 Alpha Fold predicted structure of BB212 shown with docked PET trimer molecule (shown as sticks) and shaded at the residues where mutations were identified in improved variants (Table 4).

[0035] FIG.4 is a bar chart showing the average of 3 replicate assays evaluating the enzymatic hydrolysis of polyester-based thermoplastic polyurethane (TPU) by DSB127 (error bars: standard deviation of the replicates).

[0036] FIG.5 shows PET depolymerization assay results for DSB127 and five variants of DSB127.

[0037] FIGs.6A-6C show PET degradation results for a representative BB212 variant of the disclosure in pH-controlled stirred reactor tank scales. FIG.6A shows results from two replicates (n1: solid line; n2: dashed line) of a 50-liter scale reaction over time. FIG.6B shows the consistency of enzyme performance between 50-liter scale reaction (dashed line) and 0.1-liter (top plot, solid line), 2-liter (middle plot, solid line), and 10-liter (bottom plot, solid line) scale reactions. FIG.6C shows the percent yield of two replicates at different scale reactions (error bars represent replicate range; middle bar is mean of two replicates).

[0038] FIGs.7A-7B show results demonstrating enzyme specificity for PET over non-PET plastic contaminants. FIG.7A shows a mixture of PET and polypropylene plastics illustrating the relative amounts quantified, as reported in Table 6, before (left) and after (right) an enzymatic hydrolysis reaction with a representative BB212 variant of the disclosure. FIG.7B shows a mixture of PET and high-density polyethylene plastics illustrating the relative amounts quantified, as reported in Table 7, before (left) and after (right) an enzymatic hydrolysis reaction with the representative BB212 variant. DETAILEDDESCRIPTION

[0039] 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 an organic polymer (e.g., a polyester-containing polymer). In some embodiments, the methods comprise degrading an organic polymer to produce monomers and / or oligomers, which can be recycled into polymers for use in new plastic materials. HydrolasesDocket No.: B1704.70002WO00

[0040] Aspects of the disclosure relate to the identification of new hydrolase variants for use in bio-enzymatic degradation of plastic materials. As described in Example 1, a dienelactone hydrolase family protein from Thermobifida fusca corresponding to SEQ ID NO: 3 was modified, and the modified enzyme was used as a parent for further engineering to identify improved hydrolase variants. The modified enzyme, referred to as “BB212” comprises the amino acid sequence of SEQ ID NO: 1 (without a C-terminal polyhistidine tag) or the amino acid sequence of SEQ ID NO: 2 (including a C-terminal polyhistidine tag). These amino acid sequences are provided in Table 1. Table 1. Hydrolase Sequences

[0041] 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.”

[0042] In some aspects, the disclosure provides a hydrolase comprising an amino acid sequence of SEQ ID NO: 1 or 2. In some embodiments, a hydrolase of the disclosure 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: 1 or 2. In some embodiments, the hydrolase comprises an amino acid sequence that is 60-70%, 70-80%, 80-90%, 90-95%, 92-99%, 94-Docket No.: B1704.70002WO00 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. In some embodiments, the disclosure provides a hydrolase comprising an amino acid sequence that is a conservatively substituted version of SEQ ID NO: 1 or 2.

[0043] In some embodiments, a hydrolase of the disclosure 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: 1. 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%, 60-100%, 70-100%, 80- 100%, 90-100%, 92-100%, 94-100%, 95-100%, 96-100%, or 100% identical to SEQ ID NO: 1.

[0044] In some embodiments, a hydrolase of the disclosure 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. 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%, 60-100%, 70-100%, 80- 100%, 90-100%, 92-100%, 94-100%, 95-100%, 96-100%, or 100% identical to SEQ ID NO: 2.

[0045] 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: 1, corresponding amino acid substitutions could also be made relative to other similar sequences, as would be understood by one of ordinary skill in the art. For example, SEQ ID NO: 3 corresponds to the full-length wildtype sequence of Thermobifida fusca hydrolase, containing a predicted endogenous signal sequence. By aligning the sequences of SEQ ID NOs: 1 and 3, 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: 3 that correspond to amino acid substitutions described herein relative to the sequence of SEQ ID NO: 1. Similarly, if the sequence of SEQ ID NO: 1 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: 1 described herein by aligning the sequences. As discussed above, SEQ ID NO: 2 corresponds to a version of SEQDocket No.: B1704.70002WO00 ID NO: 1 that includes a C-terminal polyhistidine tag. 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: 2 that correspond to amino acid substitutions described herein relative to the sequence of SEQ ID NO: 1. One of ordinary skill in the art would also be able to identify homologous sequences to any one of SEQ ID NOs: 1-3 and identify corresponding residues in the homologous sequences.

[0046] As described in Example 1, library screening was performed to identify variants of the BB212 hydrolase 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 850-fold improvements in depolymerization activity under high-temperature conditions relative to the parent enzyme, which comprised the amino acid sequence of SEQ ID NO: 1 and 2 (Table 1). 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 in combination at position(s) corresponding to positions selected from: D13, R19, S20, S24, N29, S31, L33, S34, S36, T42, R47, N49, N50, Y52, A54, V55, I57, S58, G63, E65, A66, S67, K73, I75, I83, I88, L91, Q93, E99, A103, A104, N106, I109, R111, A112, S113, S114, T115, R117, I120, S122, S123, V127, M128, S137, S142, Q143, A150, T154, W156, L158, N159, N161, S163, S164, V165, R166, V167, D175, L176, T178, I179, V182, L183, T184, H185, F189, N191, P194, T195, S196, I197, S198, L204, D205, G206, A207, T208, F210, N213, I214, P215, K217, I218, V224, N233, T235, T238, L241, P243, P245, R246, E252, and E254 of SEQ ID NO: 1.

[0047] As used herein, a hydrolase “variant” 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 hydrolase variant 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.

[0048] In some aspects, the disclosure provides a hydrolase comprising 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: 1 or 2, where the amino acid sequence of the hydrolase comprises an amino acid substitution at one or more positions corresponding to positionsDocket No.: B1704.70002WO00 D13, R19, S20, S24, N29, S31, L33, S34, S36, T42, R47, N49, N50, Y52, A54, V55, I57, S58, G63, E65, A66, S67, K73, I75, I83, I88, L91, Q93, E99, A103, A104, N106, I109, R111, A112, S113, S114, T115, R117, I120, S122, S123, V127, M128, S137, S142, Q143, A150, T154, W156, L158, N159, N161, S163, S164, V165, R166, V167, D175, L176, T178, I179, V182, L183, T184, H185, F189, N191, P194, T195, S196, I197, S198, L204, D205, G206, A207, T208, F210, N213, I214, P215, K217, I218, V224, N233, T235, T238, L241, P243, P245, R246, E252, and E254 of SEQ ID NO: 1. 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 D13, R19, S20, S24, N29, S31, L33, S34, S36, T42, R47, N49, N50, Y52, A54, V55, I57, S58, G63, E65, A66, S67, K73, I75, I83, I88, L91, Q93, E99, A103, A104, N106, I109, R111, A112, S113, S114, T115, R117, I120, S122, S123, V127, M128, S137, S142, Q143, A150, T154, W156, L158, N159, N161, S163, S164, V165, R166, V167, D175, L176, T178, I179, V182, L183, T184, H185, F189, N191, P194, T195, S196, I197, S198, L204, D205, G206, A207, T208, F210, N213, I214, P215, K217, I218, V224, N233, T235, T238, L241, P243, P245, R246, E252, and E254 of SEQ ID NO: 1.

[0049] In some embodiments, the amino acid sequence of the hydrolase comprises one or more amino acid substitutions selected from R19I, R19L, R19F, R19K, R19V, S24L, S24R, N29A, N29K, N29V, S34A, S34E, S34K, S36K, S36N, S36R, T42A, T42M, R47E, R47G, R47I, R47L, R47T, N49P, N49T, N50H, N50T, Y52F, A54G, V55I, I57L, I57M, I57V, S58C, G63L, G63S, G63C, G63N, E65C, E65K, E65Q, E65R, E65V, A66D, A66E, A66G, A66L, S67E, K73D, K73P, K73Q, I75L, I75T, I75V, I83L, I88H, I88N, L91I, L91R, L91V, Q93N, Q93S, A104S, I109C, A112S, S113P, S113H, S114D, S114G, S114N, S114R, I120V, S122C, S123A, S123N, V127C, M128T, S137C, S142E, A150C, T154S, N161D, S163F, S163Q, S163D, S163K, S163N, S163A, D175A, D175E, D175G, D175I, D175N, D175Q, D175R, D175S, D175T, L176A, L176I, L176R, L176T, L176V, T178G, T178Q, T178V, I179I, I179V, L183S, L183V, L183K, L183I, L183A, T184A, T184H, T184A, T184K, T184Q, H185Y, F189I, N191K, N191Q, N191A, P194C, T195E, T195M, T195P, S196C, S196P, S196T, S198A, S198E, S198N, S198T, L204G, D205C, G206R, A207V, T208G, T208S, F210G, F210I, F210K, F210N, F210R, F210T, I214V, I214K, I214M, P215V, I218T, V224L, N233V, T235E, T235G, T235I, T235L, T235Q, T235D, T235F, T235K, T235Y, T238N, L241M, P243G, P243R, P245K, R246L, E252A, and E254C relative to the sequence of SEQ ID NO: 1.Docket No.: B1704.70002WO00

[0050] 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: 1 or 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: 1, 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: 1, 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: 1, wherein the amino acid sequence of the hydrolase comprises an amino acid substitution at one or more positions described herein.

[0051] In some aspects, the disclosure relates to hydrolase variants having one or more substitutions engineered to introduce one or more additional disulfide bonds relative to a parent hydrolase. As described in Examples 1 and 7, and as shown in Table 4, 120 variants having different pairs of amino acid substitutions to cysteine were designed and evaluated for thermotolerance improvements as indicated by increased depolymerization activity under high-temperature conditions. These efforts resulted in the identification of five paired substitutions to cysteine showing improved thermotolerance relative to the parent enzyme, which comprised the amino acid sequence of SEQ ID NO: 1 and 2: D205C and E254C; I109C and S122C; S137C and V127C; S137C and A150C; and P194C and S196C relative to the sequence of SEQ ID NO: 1. 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 the introduction of new disulfide bonds relative to the parent enzyme comprising the amino acid sequence of SEQ ID NO: 1.

[0052] Accordingly, in some embodiments, a hydrolase of the disclosure comprises at least one additional disulfide bond relative to a hydrolase that comprises an amino acid sequence of SEQ ID NO: 1. In some embodiments, the hydrolase comprises one additional disulfide bond relative to a hydrolase that comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the hydrolase comprises two or more (e.g., 2, 3, 4, 5, 2-10, 2-5, 3-5, 5-Docket No.: B1704.70002WO00 10) additional disulfide bonds relative to a hydrolase that comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the hydrolase comprises an amino acid substitution to cysteine at one or more positions of SEQ ID NO: 1 having an amino acid other than cysteine. In some embodiments, the hydrolase comprises an amino acid substitution to cysteine at two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 2-20, 2-10, 3-5, 5-10, 5-20) positions of SEQ ID NO: 1 having an amino acid other than cysteine.

[0053] In some embodiments, the amino acid sequence of the hydrolase comprises an amino acid substitution at one or more positions corresponding to positions D205, E254, I109, S122, S137, V127, S137, A150, P194, and S196 of SEQ ID NO: 1. In some embodiments, the amino acid substitutions are selected from D205C, E254C, I109C, S122C, S137C, V127C, S137C, A150C, P194C, and S196C relative to SEQ ID NO: 1. In some embodiments, the amino acid sequence of the hydrolase comprises one or more (e.g., 1, 2, 3, 4, 5, 2-5, 2-4, 3-5) pairs of amino acid substitutions selected from: D205C and E254C; I109C and S122C; S137C and V127C; S137C and A150C; and P194C and S196C relative to SEQ ID NO: 1.

[0054] In some embodiments, the amino acid sequence of the hydrolase comprises amino acid substitutions at positions corresponding to positions I109 and S122 of SEQ ID NO: 1. In some embodiments, the amino acid substitutions comprise I109C and S122C relative to SEQ ID NO: 1. In some embodiments, the amino acid sequence of the hydrolase comprises amino acid substitutions at positions corresponding to positions S137 and V127 of SEQ ID NO: 1. In some embodiments, the amino acid substitutions comprise S137C and V127C relative to SEQ ID NO: 1. In some embodiments, the amino acid sequence of the hydrolase comprises amino acid substitutions at positions corresponding to positions S137 and A150 of SEQ ID NO: 1. In some embodiments, the amino acid substitutions comprise S137C and A150C relative to SEQ ID NO: 1. In some embodiments, the amino acid sequence of the hydrolase comprises amino acid substitutions at positions corresponding to positions P194 and S196 of SEQ ID NO: 1. In some embodiments, the amino acid substitutions comprise P194C and S196C relative to SEQ ID NO: 1.

[0055] In some embodiments, the amino acid sequence of the hydrolase comprises amino acid substitutions at positions corresponding to positions D205 and E254 of SEQ ID NO: 1. In some embodiments, the amino acid substitutions comprise D205C and E254C relative to SEQ ID NO: 1. As described in Example 1, a hydrolase variant having a predicted disulfide bond pair of D205C and E254C showed an approximately 50-fold increase in PET depolymerization activity under high-temperature conditions relative to the parent enzyme, which comprised the amino acid sequence of SEQ ID NO: 1 and 2 (Table 1). This disulfideDocket No.: B1704.70002WO00 bond variant, referred to herein as “DSB127,” was further shown to hydrolyze polyester- based thermoplastic polyurethane, indicating the ability to hydrolyze different types of ester bond-containing polymers in addition to PET (Example 5, FIG.4). DSB127 comprises the amino acid sequence of SEQ ID NO: 5 (without a C-terminal polyhistidine tag) or the amino acid sequence of SEQ ID NO: 6 (including a C-terminal polyhistidine tag). These amino acid sequences are provided in Table 2. Table 2. DSB127 Hydrolase Sequences

[0056] Accordingly, in some aspects, the disclosure provides a hydrolase comprising an amino acid sequence of SEQ ID NO: 5 or 6. In some embodiments, a hydrolase of the disclosure 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: 5 or 6. 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%, 60-100%, 70-100%, 80-100%, 90-100%, 92-100%, 94-100%, 95- 100%, 96-100%, or 100% identical to SEQ ID NO: 5 or 6. In some embodiments, the disclosure provides a hydrolase comprising an amino acid sequence that is a conservatively substituted version of SEQ ID NO: 5 or 6.

[0057] In some embodiments, a hydrolase of the disclosure 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: 5. 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%, 60-100%, 70-100%, 80- 100%, 90-100%, 92-100%, 94-100%, 95-100%, 96-100%, or 100% identical to SEQ ID NO:Docket No.: B1704.70002WO00 5. In some embodiments, a hydrolase of the disclosure 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: 6. 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%, 60-100%, 70-100%, 80-100%, 90-100%, 92-100%, 94-100%, 95-100%, 96-100%, or 100% identical to SEQ ID NO: 6.

[0058] In some aspects, the disclosure provides a hydrolase comprising 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: 5 or 6, where the amino acid sequence of the hydrolase comprises an amino acid substitution at one or more positions corresponding to positions D13, R19, S20, S24, N29, S31, L33, S34, S36, T42, R47, N49, N50, Y52, A54, V55, I57, S58, G63, E65, A66, S67, K73, I75, I83, I88, L91, Q93, E99, A103, A104, N106, I109, R111, A112, S113, S114, T115, R117, I120, S122, S123, V127, M128, S137, S142, Q143, A150, T154, W156, L158, N159, N161, S163, S164, V165, R166, V167, D175, L176, T178, I179, V182, L183, T184, H185, F189, N191, P194, T195, S196, I197, S198, L204, G206, A207, T208, F210, N213, I214, P215, K217, I218, V224, N233, T235, T238, L241, P243, P245, R246, and E252 of SEQ ID NO: 5. 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 D13, R19, S20, S24, N29, S31, L33, S34, S36, T42, R47, N49, N50, Y52, A54, V55, I57, S58, G63, E65, A66, S67, K73, I75, I83, I88, L91, Q93, E99, A103, A104, N106, I109, R111, A112, S113, S114, T115, R117, I120, S122, S123, V127, M128, S137, S142, Q143, A150, T154, W156, L158, N159, N161, S163, S164, V165, R166, V167, D175, L176, T178, I179, V182, L183, T184, H185, F189, N191, P194, T195, S196, I197, S198, L204, G206, A207, T208, F210, N213, I214, P215, K217, I218, V224, N233, T235, T238, L241, P243, P245, R246, and E252 of SEQ ID NO: 5.

[0059] As described in Example 1, five hydrolase variants comprising substitutions at positions F210 and T235 showed an approximately 164-fold or more increase in PET depolymerization activity under high-temperature conditions relative to the parent enzyme, which comprised the amino acid sequence of SEQ ID NO: 1 and 2 (Table 1). The five hydrolase variants, which also included the DSB127 substitutions (D205C and E254C), were shown to exhibit an approximately 2-fold increase in PET depolymerization activity relativeDocket No.: B1704.70002WO00 to a variant having only the DSB127 substitutions, suggesting that the 2-fold improvements were attributable to F210 and T235 substitutions (Example 6, FIG.5). Additional hydrolase variants comprising the DSB127 substitutions (D205C and E254C) in combination with substitutions at positions F210 and T235 showed up to 850-fold increase in PET depolymerization activity under high-temperature conditions relative to the parent enzyme, which comprised the amino acid sequence of SEQ ID NO: 1 and 2 (Table 1).

[0060] Accordingly, in some embodiments, the amino acid sequence of the hydrolase comprises amino acid substitutions at positions corresponding to positions F210 and T235 of SEQ ID NO: 1. In some embodiments, the amino acid substitutions comprise relative to SEQ ID NO: 1: (i) F210G, F210I, F210K, F210N, F210R, or F210T; and (ii) T235D, T235F, T235G, T235I, T235K, T235L, T235Q, T235V, or T235Y. In some embodiments, the amino acid substitutions comprise relative to SEQ ID NO: 1: (i) F210I, F210R, or F210T; and (ii) T235D, T235F, T235G, T235I, T235K, T235L, T235Q, T235V, or T235Y. In some embodiments, the amino acid substitutions comprise relative to SEQ ID NO: 1, F210R and T235D. In some embodiments, the amino acid substitutions comprise relative to SEQ ID NO: 1, F210R and T235F. In some embodiments, the amino acid substitutions comprise relative to SEQ ID NO: 1, F210R and T235I. In some embodiments, the amino acid substitutions comprise relative to SEQ ID NO: 1, F210R and T235K. In some embodiments, the amino acid substitutions comprise relative to SEQ ID NO: 1, F210R and T235Q. In some embodiments, the amino acid substitutions comprise relative to SEQ ID NO: 1, F210R and T235Y.

[0061] In some embodiments, a hydrolase of the disclosure comprises, relative to SEQ ID NO: 1, one or more amino acid substitutions selected from Table 4. In some embodiments, the amino acid sequence of the hydrolase comprises an amino acid substitution at one or more positions corresponding to positions A54, S58, and E65 of SEQ ID NO: 1. In some embodiments, the amino acid substitution is selected from A54G, S58C, E65C, E65K, E65Q, E65R, and E65V relative to SEQ ID NO: 1.

[0062] In some embodiments, the amino acid sequence of the hydrolase comprises an amino acid substitution at one or more positions corresponding to positions R47, S114, D175, H185, D205, F210, T235, and E254 of SEQ ID NO: 1. In some embodiments, the amino acid substitution is selected from R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C relative to SEQ ID NO: 1. In some embodiments, the amino acid substitution is D175Q relative to SEQ ID NO: 1. In some embodiments, the amino acid substitution is H185Y relative to SEQ ID NO: 1. In some embodiments, the amino acid substitution isDocket No.: B1704.70002WO00 F210R relative to SEQ ID NO: 1. In some embodiments, the amino acid sequence of the hydrolase comprises 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) amino acid substitutions selected from R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C relative to SEQ ID NO: 1. 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: 1: R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C.

[0063] In some embodiments, the amino acid sequence of the hydrolase comprises an amino acid substitution selected from R19I, R19L, S24L, S24R, N29A, N29K, S34A, S34E, S36K, S36N, S36R, T42A, R47E, R47G, R47I, R47L, R47T, N49P, N50H, N50T, Y52F, V55I, I57L, I57M, I57V, G63L, G63S, A66D, A66E, A66G, A66L, S67E, K73D, K73P, K73Q, I75L, I75T, I75V, I83L, I88H, L91I, L91R, Q93N, Q93S, A104S, I109C, A112S, S113P, S114D, S114G, S114N, I120V, S122C, S123A, S123N, V127C, M128T, S137C, S142E, A150C, T154S, N161D, S163F, D175A, D175E, D175G, D175I, D175N, D175Q, D175R, D175S, D175T, L176A, L176I, L176R, L176T, L176V, T178G, T178Q, T178V, I179I, I179V, L183S, T184A, T184H, H185Y, F189I, N191K, P194C, T195E, S196C, S198A, L204G, D205C, G206R, A207V, T208G, T208S, F210G, F210I, F210K, F210N, F210R, F210T, I214V, P215V, I218T, V224L, N233V, T235E, T235G, T235I, T235L, T235Q, T235D, T235F, T235K, T235Y, T238N, L241M, P243G, P243R, P245K, R246L, E252A, and E254C relative to SEQ ID NO: 1. In some embodiments, the amino acid sequence of the hydrolase comprises 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) amino acid substitutions selected from R19I, R19L, S24L, S24R, N29A, N29K, S34A, S34E, S36K, S36N, S36R, T42A, R47E, R47G, R47I, R47L, R47T, N49P, N50H, N50T, Y52F, V55I, I57L, I57M, I57V, G63L, G63S, A66D, A66E, A66G, A66L, S67E, K73D, K73P, K73Q, I75L, I75T, I75V, I83L, I88H, L91I, L91R, Q93N, Q93S, A104S, I109C, A112S, S113P, S114D, S114G, S114N, I120V, S122C, S123A, S123N, V127C, M128T, S137C, S142E, A150C, T154S, N161D, S163F, D175A, D175E, D175G, D175I, D175N, D175Q, D175R, D175S, D175T, L176A, L176I, L176R, L176T, L176V, T178G, T178Q, T178V, I179I, I179V, L183S, T184A, T184H, H185Y, F189I, N191K, P194C, T195E, S196C, S198A, L204G, D205C, G206R, A207V, T208G, T208S, F210G, F210I, F210K, F210N, F210R, F210T, I214V, P215V, I218T, V224L, N233V, T235E, T235G, T235I, T235L, T235Q, T235D,Docket No.: B1704.70002WO00 T235F, T235K, T235Y, T238N, L241M, P243G, P243R, P245K, R246L, E252A, and E254C relative to SEQ ID NO: 1.

[0064] 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: 1: R47, D205, and E254; R47, N50, D205, and E254; R47, K73, D205, and E254; I179, D205, and E254; R47, D205, and E254; D205, F210, and E254; T184, F189, D205, F210, and E254; D205, F210, A211, and E254; D205, F210, T235, and E254; D205, F210, P215, and E254; D205, F210, N233, P243, and E254; D205, F210, P215, T235, and E254; N49, K73, L91, D205, and E254; K73, D205, T235, P243, and E254; D205, F210, N233, T235, P243, and E254; D205, F210, N233, T235, and E254; S24, N29, D205, and E254; L183, D205, I218, and E254; R47, E65, S114, H185, D205, F210, T235, and E254; R47, S114, H185, D205, F210, T235, and E254; R47, K73, S114, I179, D205, F210, T235, and E254; R47, V55, G63, S114, H185, D205, F210, T235, and E254; R47, S114, H185, I179, D205, F210, T235, and E254; R47, I57, I83, S114, H185, D205, F210, T235, and E254; R47, I57, S114, H185, D205, F210, T235, and E254; R47, S114, D175, L176, H185, D205, F210, T235, and E254; R47, S114, N161, H185, D205, F210, T235, and E254; R47, H185, D205, F210, T235, and E254; R47, S114, H185, L204, D205, F210, T235, and E254; R47, S114, D175, H185, D205, F210, T235, and E254; R47, I75, S114, H185, D205, F210, T235, and E254; R47, S114, S123, H185, D205, F210, T235, and E254; R47, S114, D175, L176, D205, H185, F210, T235, and E254; R47, S114, L176, T178, H185, D205, F210, T235, and E254; R47, I57, I75, S114, H185, D205, F210, T235, and E254; I75, S114, H185, D205, F210, T235, and E254; R47, S113, S114, S123, H185, D205, F210, T235, and E254; R47, S114, H185, N191, D205, F210, T235, and E254; R47, S114, T178, H185, D205, F210, T235, and E254; S34, Y52, S114, H185, D205, F210, T235, and E254; R47, A112, S114, S123, H185, D205, F210, T235, and E254; R47, S114, I120, H185, D205, F210, T235, and E254; R47, S114, H185, S198, D205, F210, T235, and E254; R47, S114, H185, T195, D205, F210, T235, and E254; R47, S114, H185, D205, G206, F210, T235, and E254; R19, S36, N50, Y52, I57, I75, S114, H185, F210, and T235; R19, N50, Y52, I75, S114, H185, F210, and T235; R19, S34, R47, N50, Y52, I57, I75, S114, H185, F210, and T235; S34, N50, I57, I75, S114, S142, H185, F210, and T235; S34, Y52, I57, I75, S114, H185, F210, and T235; S36, N50, Y52, I57, S114, H185, F210, and T235; R47, S114, D175, H185, D205, F210, T235, P245, R246, and E254; R47, S114, H185, D205, F210, T235, T238, and E254; R19, R47, S114, D175, H185, D205, F210, T235, and E254; R19, R47, S114, D175, H185, D205, F210, T235, P245, R246, and E254; R19, S34, S36, R47, N50, Y52, D175, H185, F210, and T235; R47, D175, H185,Docket No.: B1704.70002WO00 D205, F210, T235, and E254; R47, I57, D175, H185, D205, F210, T235, and E254; R47, N50, Y52, I57, S114, D175, H185, D205, F210, T235, and E254; S34, S114, D175, H185, D205, F210, T235, P245, R246, and E254; S34, Y52, S114, D175, H185, D205, F210, T235, and E254; T42, R47, S114, D175, H185, D205, F210, T235, and E254; T42, R47, S114, D175, H185, D205, F210, V224, T235, and E254; R19, T42, R47, S114, D175, H185, D205, F210, T235, and E254; R47, I57, I75, S114, D175, H185, D205, F210, T235, and E254; R47, Y52, S114, D175, H185, D205, F210, L224, T235, and E254; S34, S36, R47, D175, H185, D205, F210, T235, and E254; S34, S36, R47, S114, D175, H185, D205, F210, T235, and E254; S34, S36, R47, S114, H185, D205, F210, T235, and E254; S34, D175, H185, D205, F210, T235, and E254; S34, R47, I75, S114, D175, H185, D205, F210, T235, and E254; S34, R47, S114, D175, H185, D205, F210, T235, and E254; R47, I57, I75, S114, H185, D205, T208, F210, T235, and E254; R47, I57, I75, S114, D175, H185, D205, A207, F210, T235, and E254; R47, I75, S114, D175, H185, D205, F210, I214, T235, and E254; R47, S114, M128, D175, H185, D205, F210, T235, and E254; S34, Y52, A104, S114, D175, H185, D205, F210, T235, and E254; R47, I57, G63, A66, S114, D175, H185, D205, F210, T235, and E254; R47, Y52, G63, A66, S114, D175, H185, D205, F210, T235, and E254; R47, I57, A66, S114, D175, H185, D205, F210, T235, and E254; R47, G63, A66, S114, D175, H185, D205, F210, T235, and E254; R47, E65, S67, S114, D175, H185, D205, F210, T235, and E254; R47, S114, S163, D175, H185, D205, F210, T235, and E254; R47, S114, D175, H185, D205, F210, T235, E252, and E254; R47, S114, D175, H185, D205, F210, T235, L241, and E254; R47, S114, D175, H185, D205, T208, F210, T235, and E254; R47, S114, T154, D175, T184, H185, D205, F210, T235, and E254; R47, I57, L91, Q93, S114, D175, H185, D205, F210, T235, and E254; R47, I88, S114, D175, H185, D205, F210, T235, and E254; R47, Q93, S114, D175, H185, D205, F210, T235, and E254; N29, S31, S36, R47, N49, S114, D175, H185, D205, F210, T235, and E254; R19, S20, T42, R47, S114, D175, H185, D205, F210, T235, and E254; R47, A103, N106, R111, S114, T115, D175, H185, D205, F210, T235, and E254; R47, A66, I75, S114, D175, H185, D205, F210, T235, and E254; R47, L91, N106, S114, D175, T184, H185, D205, F210, K217, T235, and E254; S34, Y52, G63, S114, D175, H185, D205, F210, T235, and E254; R47, R111, S114, D175, H185, D205, F210, T235, and E254; S31, R47, S114, D175, H185, D205, F210, T235, and E254; S34, Y52, L91, S113, S114, D175, H185, D205, F210, T235, and E254; R47, I57, G63, A66, S114, D175, H185, D205, T208, F210, T235, and E254; S34, G63, A66, S114, D175, H185, D205, F210, T235, and E254; R47, S114, W156, L158, D175, H185, S196, D205, F210, I214, T235, and E254; R47, S114, L158, R166, D175, H185, D205, F210, T235, and E254; R47, S114, L158,Docket No.: B1704.70002WO00 V167, D175, H185, N191, D205, F210, T235, and E254; R47, S114, D175, V182, H185, D205, F210, T235, and E254; R47, S114, S163, R166, D175, H185, N191, D205, F210, T235, and E254; R47, S114, L158, D175, H185, S196, D205, F210, T235, and E254; R47, S114, S163, S164, D175, H185, D205, F210, I214, T235, and E254; R47, S114, S164, D175, L183, H185, T195, D205, F210, T235, and E254; R47, S114, N159, D175, H185, D205, F210, T235, and E254; R47, S114, D175, S198, D205, F210, T235, and E254; R47, S114, V165, D175, L183, H185, I197, S198, D205, F210, T235, and E254; R47, S114, S142, T154, R166, D175, H185, S198, D205, F210, T235, and E254; R47, S114, Q143, S163, S164, D175, H185, T195, D205, F210, T235, and E254; R47, S114, L158, S163, S164, D175, H185, S198, D205, F210, N213, T235, and E254; R47, Y52, I57, G63, A66, I75, S114, D175, H185, D205, F210, T235, and E254; D13, R47, N50, I88, E99, S114, D175, H185, D205, F210, K217, T235, and E254; Y52, I57, G63, A66, I75, S114, D175, H185, D205, F210, T235, and E254; R47, G63, S114, R117, D175, L183, H185, D205, F210, I214, T235, and E254; R19, R47, G63, S114, R166, D175, L183, H185, D205, F210, T235, and E254; R47, S114, D175, L183, H185, D205, F210, T235, and E254; R47, S114, L158, S163, D175, T184, H185, D205, F210, T235, and E254; R47, Q93, S114, D175, H185, D205, T208, F210, T235, and E254; S34, R47, S114, L158, D175, H185, D205, F210, T235, and E254; or L33, R47, S114, R117, R166, D175, L183, H185, D205, F210, T235, and E254. In some embodiments, the amino acid substitutions comprise relative to the sequence of SEQ ID NO: 1: R47V, D205C, and E254C; R47L, D205C, and E254C; R47E, N50G, D205C, and E254C; R47V, K73Q, D205C, and E254C; I179V, D205C, and E254C; R47T, D205C, and E254C; R47G, D205C, and E254C; D205C, F210G, and E254C; D205C, F210K, and E254C; T184H, F189I, D205C, F210K, and E254C; D205C, F210N, and E254C; D205C, F210K, A211G, and E254C; D205C, F210I, T235L, and E254C; D205C, F210I, T235G, and E254C; D205C, F210T, T235G, and E254C; D205C, F210R, P215V, and E254C; D205C, F210R, N233V, P243G, and E254C; D205C, F210R, P215V, T235V, and E254C; D205C, F210T, T235D, and E254C; D205C, F210R, T235L, and E254C; D205C, F210R, T235Y, and E254C; D205C, F210R, T235F, and E254C; D205C, F210R, T235Q, and E254C; D205C, F210R, T235K, and E254C; D205C, F210R, T235I, and E254C; D205C, F210I, T235D, and E254C; N49P, K73P, L91R, D205C, and E254C; K73D, D205C, T235E, P243R, and E254C; D205C, F210R, N233H, T235D, P243G, and E254C; D205C, F210R, N233H, T235G, and E254C; S24R, N29A, D205C, and E254C; S24L, N29K, D205C, and E254C; L183S, D205C, I218T, and E254C; R47E, E65K, S114N, H185Y, D205C, F210R, T235I, and E254C; R47E, S114D, H185Y, D205C, F210R, T235Y, and E254C; R47G, S114G, H185Y,Docket No.: B1704.70002WO00 D205C, F210R, T235F, and E254C; R47L, K73Q, S114D, I179V, D205C, F210R, T235Y, and E254C; R47E, V55I, G63S, S114D, H185Y, D205C, F210R, T235Y, and E254C; R47G, S114G, H185Y, I179I, D205C, F210R, T235Y, and E254C; R47G, I57V, I83L, S114D, H185Y, D205C, F210R, T235Y, and E254C; R47E, I57V, S114D, H185Y, D205C, F210R, T235Y, and E254C; R47E, S114D, D175G, L176A, H185Y, D205C, F210R, T235Y, and E254C; R47G, S114G, N161D, H185Y, D205C, F210R, T235Y, and E254C; R47E, S114G, D175S, L176A, H185Y, D205C, F210R, T235Y, and E254C; R47G, S114G, D175G, L176A, H185Y, D205C, F210R, T235Y, and E254C; R47I, H185Y, D205C, F210R, T235F, and E254C; R47G, S114G, H185Y, L204G, D205C, F210R, T235Y, and E254C; R47G, S114G, D175S, H185Y, D205C, F210R, T235Y, and E254C; R47E, I75L, S114D, H185Y, D205C, F210R, T235Y, and E254C; R47E, S114G, N161D, H185Y, D205C, F210R, T235Y, and E254C; R47G, S114D, H185Y, D205C, F210R, T235Y, and E254C; R47G, S114D, S123A, H185Y, D205C, F210R, T235Y, and E254C; R47E, S114D, D175R, L176V, H185Y, D205C, F210R, T235Y, and E254C; R47G, S114G, D175R, L176A, D205C, H185Y, F210R, T235Y, and E254C; R47G, S114G, L176R, T178Q, H185Y, D205C, F210R, T235Y, and E254C; R47G, I75V, S114G, H185Y, D205C, F210R, T235Y, and E254C; R47G, S114D, D175T, L176V, H185Y, D205C, F210R, T235Y, and E254C; R47E, I57M, I75V, S114D, H185Y, D205C, F210R, T235Y, and E254C; I75T, S114D, H185Y, D205C, F210R, T235I, and E254C; R47E, S114D, L176R, T178V, H185Y, D205C, F210R, T235Y, and E254C; R47E, I57V, I75V, S114D, H185Y, D205C, F210R, T235Y, and E254C; R47G, S114D, D175E, L176A, H185Y, D205C, F210R, T235Y, and E254C; R47E, I57V, I75L, S114D, H185Y, D205C, F210R, T235Y, and E254C; R47E, S113P, S114D, S123A, H185Y, D205C, F210R, T235Y, and E254C; R47G, S114G, D175S, L176I, H185Y, D205C, F210R, T235F, and E254C; R47E, S114D, H185Y, N191K, D205C, F210R, T235Y, and E254C; R47E, S114D, T178G, H185Y, D205C, F210R, T235Y, and E254C; S34E, Y52F, S114D, H185Y, D205C, F210R, T235I, and E254C; R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, S114G, D175I, L176A, H185Y, D205C, F210R, T235Y, and E254C; R47G, S114G, D175Q, L176I, H185Y, D205C, F210R, T235Y, and E254C; R47E, S114G, D175Q, L176T, H185Y, D205C, F210R, T235Y, and E254C; R47E, A112S, S114G, S123N, H185Y, D205C, F210R, T235Y, and E254C; R47E, S114G, I120V, H185Y, D205C, F210R, T235Y, and E254C; R47E, S114D, H185Y, S198A, D205C, F210R, T235Y, and E254C; R47G, S114G, H185Y, T195E, D205C, F210R, T235Y, and E254C; R47E, S114G, H185Y, D205C, G206R, F210R, T235Y, and E254C; R19I, S36R, N50T, Y52F, I57V, I75V, S114D, H185Y, F210R, and T235Y; R19L, N50T, Y52F, I75V, S114G, H185Y, F210R, and T235Y; R19L,Docket No.: B1704.70002WO00 S34E, R47E, N50T, Y52F, I57M, I75T, S114D, H185Y, F210R, and T235Y; S34A, N50T, I57M, I75V, S114D, S142E, H185Y, F210R, and T235Y; S34E, Y52F, I57V, I75V, S114D, H185Y, F210R, and T235Y; S36N, N50H, Y52F, I57M, S114G, H185Y, F210R, and T235Y; R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, P245K, R246L, and E254C; R47G, S114D, H185Y, D205C, F210R, T235Y, T238N, and E254C; R47G, S114G, H185Y, D205C, F210R, T235Y, T238N, and E254C; R19L, R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R19L, R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, P245K, R246L, and E254C; R19L, S34A, S36K, R47E, N50H, Y52F, D175Q, H185Y, F210R, and T235Y; R47E, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, I57V, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, N50H, Y52F, I57V, S114D, D175Q, H185Y, D205C, F210R, T235F, and E254C; R47E, S114G, D175Q, H185Y, D205C, F210R, T235Y, and E254C; S34E, S114D, D175Q, H185Y, D205C, F210R, T235Y, P245K, R246L, and E254C; S34E, Y52F, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; S34E, Y52F, S114G, D175Q, H185Y, D205C, F210R, T235Y, and E254C; T42A, R47E, S114D, D175Q, H185Y, D205C, F210R, T235F, and E254C; T42A, R47E, S114D, D175Q, H185Y, D205C, F210R, V224L, T235Y, and E254C; R19L, T42A, R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, I57V, I75L, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, Y52F, S114D, D175Q, H185Y, D205C, F210R, L224V, T235Y, and E254C; S34A, S36K, R47E, D175Q, H185Y, D205C, F210R, T235Y, and E254C; S34A, S36K, R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; S34A, S36K, R47E, S114G, H185Y, D205C, F210R, T235Y, and E254C; S34E, D175Q, H185Y, D205C, F210R, T235Y, and E254C; S34E, R47E, I75L, S114G, D175Q, H185Y, D205C, F210R, T235Y, and E254C; S34E, R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, S114D, D175A, H185Y, D205C, F210R, T235Y, and E254C; R47E, I57V, I75L, S114D, D175N, H185Y, D205C, F210R, T235Y, and E254C; R47E, I57V, I75L, S114D, H185Y, D205C, T208G, F210R, T235Y, and E254C; R47E, I57V, I75L, S114D, D175Q, H185Y, D205C, A207V, F210R, T235Y, and E254C; R47E, I75L, S114D, D175Q, H185Y, D205C, F210R, I214V, T235Y, and E254C; R47E, S114D, M128T, D175Q, H185Y, D205C, F210R, T235F, and E254C; S34E, Y52F, A104S, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, I57L, G63L, A66E, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, Y52F, G63L, A66D, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, I57L, A66G, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, G63L, A66L, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, E65C, S67E, S114D, D175Q, H185Y, D205C, F210R, T235Y,Docket No.: B1704.70002WO00 and E254C; R47E, S114G, S163F, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, E252A, and E254C; R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, L241M, and E254C; R47E, S114D, D175Q, H185Y, D205C, T208S, F210R, T235Y, and E254C; R47E, S114D, T154S, D175Q, T184A, H185Y, D205C, F210R, T235Y, and E254C; R47E, I57V, L91I, Q93N, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, I88H, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, Q93S, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; N29V, S31Q, S36K, R47E, N49T, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R19V, S20D, T42M, R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, A103Q, N106D, R111Y, S114R, T115P, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R19F, S20D, T42M, R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, A66E, I75V, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, L91I, N106T, S114D, D175Q, T184Q, H185Y, D205C, F210R, K217R, T235Y, and E254C; S34E, Y52F, G63N, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, R111E, S114G, D175Q, H185Y, D205C, F210R, T235Y, and E254C; S31K, R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; S34E, Y52F, L91V, S113H, S114G, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, I57L, G63L, A66E, S114D, D175Q, H185Y, D205C, T208G, F210R, T235Y, and E254C; R47E, I57L, G63L, A66E, S114G, D175Q, H185Y, D205C, F210R, T235Y, and E254C; S34E, G63L, A66E, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, I57V, L91I, Q93N, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, S114D, W156F, L158T, D175Q, H185Y, S196T, D205C, F210R, I214M, T235Y, and E254C; R47E, S114D, L158I, R166K, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, S114D, L158I, V167I, D175Q, H185Y, N191Q, D205C, F210R, T235Y, and E254C; R47E, S114D, D175Q, V182I, H185Y, D205C, F210R, T235Y, and E254C; R47E, S114D, S163A, R166I, D175Q, H185Y, N191A, D205C, F210R, T235Y, and E254C; R47E, S114D, L158T, D175Q, H185Y, S196P, D205C, F210R, T235Y, and E254C; R47E, S114D, S163A, S164T, D175Q, H185Y, D205C, F210R, I214M, T235Y, and E254C; R47E, S114D, S164N, D175Q, L183A, H185Y, T195M, D205C, F210R, T235Y, and E254C; R47E, S114D, N159A, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, S114D, D175Q, S198T, D205C, F210R, T235Y, and E254C; R47E, S114D, V165I, D175Q, L183V, H185Y, I197V, S198E, D205C, F210R, T235Y, and E254C; R47E, S114D, S142E, T154S, R166T, D175Q, H185Y, S198A, D205C, F210R, T235Y, and E254C; R47E, S114D, Q143K, S163A, S164T, D175Q, H185Y, T195P, D205C, F210R, T235Y, and E254C; R47E, S114D, L158V, S163N, S164T, D175Q, H185Y,Docket No.: B1704.70002WO00 S198N, D205C, F210R, N213T, T235Y, and E254C; R47E, Y52F, I57L, G63C, A66E, I75V, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; D13A, R47E, N50G, I88N, E99D, S114G, D175Q, H185Y, D205C, F210R, K217P, T235Y, and E254C; Y52F, I57L, G63C, A66E, I75V, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; R47E, G63L, S114D, R117K, D175Q, L183K, H185Y, D205C, F210R, I214K, T235Y, and E254C; R19K, R47E, G63L, S114D, R166T, D175Q, L183K, H185Y, D205C, F210R, T235Y, and E254C; R47E, S114D, D175Q, L183I, H185Y, D205C, F210R, T235Y, and E254C; R47E, S114D, L158N, S163K, D175Q, T184K, H185Y, D205C, F210R, T235Y, and E254C; R47E, S114D, L158T, S163Q, D175Q, T184K, H185Y, D205C, F210R, T235Y, and E254C; R47E, Q93S, S114D, D175Q, H185Y, D205C, T208S, F210R, T235Y, and E254C; S34K, R47E, S114D, L158T, D175Q, H185Y, D205C, F210R, T235Y, and E254C; L33K, R47E, S114D, R117K, R166T, D175Q, L183K, H185Y, D205C, F210R, T235Y, and E254C; or R47E, S114D, L158T, S163D, D175Q, T184K, H185Y, D205C, F210R, T235Y, and E254C.

[0065] In some embodiments, a hydrolase of the disclosure comprises an amino acid sequence of any one of SEQ ID NOs: 5-183. In some embodiments, a hydrolase of the disclosure 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 any one of SEQ ID NOs: 5-183. 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%, 60-100%, 70-100%, 80-100%, 90-100%, 92-100%, 94-100%, 95-100%, 96-100%, or 100% identical to any one of SEQ ID NOs: 5-183. In some embodiments, the disclosure provides a hydrolase comprising an amino acid sequence that is a conservatively substituted version of any one of SEQ ID NOs: 5-183.

[0066] In some embodiments, a hydrolase of the disclosure comprises an amino acid sequence of any one of SEQ ID NOs: 85, 100, 103, 104, 114-117, 119, 123, 127, 128, 131- 135, 137-145, 147-149, 152, 154, 157-166, 168-172, and 175-183. In some embodiments, a hydrolase of the disclosure 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 any one of SEQ ID NOs: 85, 100, 103, 104, 114-117, 119, 123, 127, 128, 131-135, 137-145, 147-149, 152, 154, 157-166, 168-172, and 175-183. 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%, 60-100%, 70-100%, 80-Docket No.: B1704.70002WO00 100%, 90-100%, 92-100%, 94-100%, 95-100%, 96-100%, or 100% identical to any one of SEQ ID NOs: 85, 100, 103, 104, 114-117, 119, 123, 127, 128, 131-135, 137-145, 147-149, 152, 154, 157-166, 168-172, and 175-183. In some embodiments, the disclosure provides a hydrolase comprising an amino acid sequence that is a conservatively substituted version of any one of SEQ ID NOs: 85, 100, 103, 104, 114-117, 119, 123, 127, 128, 131-135, 137-145, 147-149, 152, 154, 157-166, 168-172, and 175-183.

[0067] In some aspects, the disclosure relates to hydrolase variants with improved performance relative to a control hydrolase. In some embodiments, improved performance refers to increased depolymerization activity relative to a control hydrolase. For example, as described in Example 1, library screening identified hydrolase variants that exhibited up to 850-fold increased depolymerization relative to the parent enzyme, which comprised the amino acid sequence of SEQ ID NO: 1 and 2 (Table 1). Accordingly, in some embodiments, a hydrolase described herein exhibits 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, at least 10-fold, at least 20-fold, at least 30-fold, at least 50-fold, at least 75-fold, at least 100-fold, at least 125-fold, at least 150-fold, at least 175-fold, at least 200-fold, at least 225-fold, at least 240-fold, at least 250-fold, at least 300-fold, at least 350-fold, at least 400-fold, at least 500-fold, at least 550-fold, at least 600-fold, at least 650-fold, at least 700-fold, at least 750-fold, or at least 800-fold increased depolymerization activity relative to a control hydrolase. In some embodiments, the hydrolase exhibits 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, at least 10-fold, at least 20-fold, at least 30-fold, at least 50-fold, at least 75-fold, at least 100-fold, at least 125-fold, at least 150-fold, at least 175- fold, at least 200-fold, at least 225-fold, at least 240-fold, at least 250-fold, at least 300-fold, at least 350-fold, at least 400-fold, at least 500-fold, at least 550-fold, at least 600-fold, at least 650-fold, at least 700-fold, at least 750-fold, or at least 800-fold increased depolymerization of PET relative to a control hydrolase. In some embodiments, the hydrolase exhibits at least 250-fold, at least 275-fold, at least 300-fold, at least 325-fold, at least 350-fold, at least 375-fold, at least 400-fold, at least 500-fold, at least 550-fold, at least 600-fold, at least 650-fold, at least 700-fold, at least 750-fold, or at least 800-fold increased depolymerization activity relative to a control hydrolase. In some embodiments, the hydrolase exhibits at least 250-fold, at least 275-fold, at least 300-fold, at least 325-fold, at least 350-fold, at least 375-fold, at least 400-fold, at least 500-fold, at least 550-fold, at least 600-fold, at least 650-fold, at least 700-fold, at least 750-fold, or at least 800-fold increased depolymerization of PET relative to a control hydrolase.Docket No.: B1704.70002WO00

[0068] In some embodiments, improved performance refers to improved thermotolerance. As used herein, “improved thermotolerance” of a hydrolase variant relative to a control hydrolase refers to the ability of the hydrolase variant to be more enzymatically active than the control hydrolase at a specified temperature (e.g., more than 50 °C, more than 60 °C, more than 70 °C, between about 50 °C and about 80 °C, or about 70 °C). In some embodiments, the specified temperature is a temperature that is not optimal for the control hydrolase. For example, as described in Example 1, library screening identified hydrolase variants that exhibited thermotolerance improvements as indicated by increased depolymerization activity at 70 ºC relative to the parent enzyme, which comprised the amino acid sequence of SEQ ID NO: 1 and 2 (Table 1).

[0069] In some embodiments, hydrolase variants associated with the disclosure exhibit improved thermotolerance relative to a control hydrolase. In some embodiments, hydrolase variants associated with the disclosure exhibit 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, at least 10-fold, at least 20-fold, at least 30- fold, at least 50-fold, at least 75-fold, at least 100-fold, at least 125-fold, at least 150-fold, at least 175-fold, at least 200-fold, at least 225-fold, or at least 240-fold improved thermotolerance relative to a control hydrolase. In some embodiments, the control hydrolase comprises the amino acid sequence of SEQ ID NO: 1 and / or 2.

[0070] In some embodiments, improved performance refers to improved acid tolerance. As used herein, “improved acid tolerance” of a hydrolase variant relative to a control hydrolase refers to the ability of the hydrolase variant to be more enzymatically active than the control hydrolase at a specified pH below about 7.0 (e.g., below about 6.5, below about 6.0, between about 4.5 and about 6.5, or about 5.0). In some embodiments, the specified pH is a pH that is not optimal for the control hydrolase. For example, as described in Example 3, and as shown in FIG.2, hydrolase variants of the disclosure exhibited improved acid tolerance as indicated by increased depolymerization activity at pH 5 relative to the parent enzyme. As also shown in Example 3 and FIG.2, the hydrolase variants further exhibited increased depolymerization activity at pH 8 relative to the parent enzyme. Thus, it should be appreciated that hydrolase variants described herein can be more enzymatically active than a control hydrolase at a specified pH above about 7.0 (e.g., above about 7.5, above about 8.0, between about 7.5 and about 8.5, or about 8.0).

[0071] In some embodiments, hydrolase variants associated with the disclosure exhibit improved acid tolerance relative to a control hydrolase. In some embodiments, hydrolase variants associated with the disclosure exhibit at least 1.5-fold, at least 2-fold, at least 2.5-Docket No.: B1704.70002WO00 fold, at least 3-fold, at least 5-fold, at least 7-fold, at least 10-fold, at least 20-fold, at least 30- fold, or at least 50-fold improved acid tolerance relative to a control hydrolase. In some embodiments, the control hydrolase comprises the amino acid sequence of SEQ ID NO: 1 and / or 2.

[0072] In some aspects, the disclosure provides a host cell that comprises a heterologous polynucleotide encoding a hydrolase or a hydrolase variant described herein. In some embodiments, the heterologous polynucleotide encodes a hydrolase comprising 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%, at least 98%, 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 D13, R19, S20, S24, N29, S31, L33, S34, S36, T42, R47, N49, N50, Y52, A54, V55, I57, S58, G63, E65, A66, S67, K73, I75, I83, I88, L91, Q93, E99, A103, A104, N106, I109, R111, A112, S113, S114, T115, R117, I120, S122, S123, V127, M128, S137, S142, Q143, A150, T154, W156, L158, N159, N161, S163, S164, V165, R166, V167, D175, L176, T178, I179, V182, L183, T184, H185, F189, N191, P194, T195, S196, I197, S198, L204, D205, G206, A207, T208, F210, N213, I214, P215, K217, I218, V224, N233, T235, T238, L241, P243, P245, R246, E252, and E254 of SEQ ID NO: 1. In some embodiments, the heterologous polynucleotide encodes a hydrolase comprising 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%, at least 98%, 60-80%, 70-90%, 80-99%, or 100%) identical to any one of SEQ ID NOs: 5-183. In some embodiments, the heterologous polynucleotide encodes a hydrolase comprising 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%, at least 98%, 60-80%, 70-90%, 80-99%, or 100%) identical to any one of SEQ ID NOs: 85, 100, 103, 104, 114-117, 119, 123, 127, 128, 131- 135, 137-145, 147-149, 152, 154, 157-166, 168-172, and 175-183. In some embodiments, the heterologous polynucleotide encodes a hydrolase having an amino acid sequence of SEQ ID NO: 1 or 2. In some embodiments, the heterologous polynucleotide comprises a nucleotide sequence of SEQ ID NO: 4.

[0073] 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, suchDocket No.: B1704.70002WO00 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).

[0074] 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.

[0075] 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.

[0076] In some aspects, the disclosure provides a nucleic acid encoding a hydrolase or a hydrolase variant described herein. In some embodiments, the nucleic acid encodes a hydrolase comprising 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%, at least 98%, 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 D13, R19, S20, S24, N29, S31, L33, S34, S36, T42, R47, N49, N50, Y52, A54, V55, I57, S58, G63, E65, A66, S67, K73, I75, I83, I88, L91, Q93, E99, A103, A104, N106, I109, R111, A112, S113, S114, T115, R117, I120, S122, S123, V127, M128, S137, S142, Q143, A150, T154, W156, L158, N159, N161, S163, S164, V165, R166, V167, D175, L176, T178, I179, V182, L183, T184, H185, F189, N191, P194, T195, S196, I197, S198, L204, D205, G206, A207, T208, F210, N213, I214, P215, K217, I218, V224, N233, T235, T238,Docket No.: B1704.70002WO00 L241, P243, P245, R246, E252, and E254 of SEQ ID NO: 1. In some embodiments, the nucleic acid encodes a hydrolase comprising 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%, at least 98%, 60-80%, 70-90%, 80-99%, or 100%) identical to any one of SEQ ID NOs: 5-183. In some embodiments, the nucleic acid encodes a hydrolase comprising 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%, at least 98%, 60-80%, 70-90%, 80-99%, or 100%) identical to any one of SEQ ID NOs: 85, 100, 103, 104, 114-117, 119, 123, 127, 128, 131-135, 137-145, 147-149, 152, 154, 157-166, 168-172, and 175-183. In some embodiments, the nucleic acid encodes a hydrolase having an amino acid sequence of SEQ ID NO: 1 or 2. In some embodiments, the nucleic acid comprises a nucleotide sequence of SEQ ID NO: 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.

[0077] In some aspects, the disclosure provides a vector comprising a nucleic acid encoding a hydrolase comprising 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%, at least 98%, 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 D13, R19, S20, S24, N29, S31, L33, S34, S36, T42, R47, N49, N50, Y52, A54, V55, I57, S58, G63, E65, A66, S67, K73, I75, I83, I88, L91, Q93, E99, A103, A104, N106, I109, R111, A112, S113, S114, T115, R117, I120, S122, S123, V127, M128, S137, S142, Q143, A150, T154, W156, L158, N159, N161, S163, S164, V165, R166, V167, D175, L176, T178, I179, V182, L183, T184, H185, F189, N191, P194, T195, S196, I197, S198, L204, D205, G206, A207, T208, F210, N213, I214, P215, K217, I218, V224, N233, T235, T238, L241, P243, P245, R246, E252, and E254 of SEQ ID NO: 1. In some embodiments, the vector comprises a nucleic acid encoding a hydrolase comprising 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%, at least 98%, 60-80%, 70-90%, 80-99%, or 100%) identical to any one of SEQ ID NOs: 5-183. In some embodiments, the vector comprises a nucleic acid encoding a hydrolase comprising 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%, at least 98%, 60-80%, 70-90%, 80-99%, or 100%) identical to any one of SEQ ID NOs: 85, 100, 103, 104, 114-117, 119, 123, 127, 128, 131-135, 137-145, 147-149, 152, 154, 157-166, 168-172, and 175-183. In some embodiments, the vector comprises a nucleic acid encoding a hydrolase having an amino acidDocket No.: B1704.70002WO00 sequence of SEQ ID NO: 1 or 2. In some embodiments, the nucleic acid comprises a nucleotide sequence of SEQ ID NO: 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.

[0078] In some aspects, the disclosure provides an expression cassette comprising a nucleic acid encoding a hydrolase comprising 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%, at least 98%, 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 D13, R19, S20, S24, N29, S31, L33, S34, S36, T42, R47, N49, N50, Y52, A54, V55, I57, S58, G63, E65, A66, S67, K73, I75, I83, I88, L91, Q93, E99, A103, A104, N106, I109, R111, A112, S113, S114, T115, R117, I120, S122, S123, V127, M128, S137, S142, Q143, A150, T154, W156, L158, N159, N161, S163, S164, V165, R166, V167, D175, L176, T178, I179, V182, L183, T184, H185, F189, N191, P194, T195, S196, I197, S198, L204, D205, G206, A207, T208, F210, N213, I214, P215, K217, I218, V224, N233, T235, T238, L241, P243, P245, R246, E252, and E254 of SEQ ID NO: 1. In some embodiments, the disclosure provides an expression cassette comprising a nucleic acid encoding a hydrolase comprising 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%, at least 98%, 60- 80%, 70-90%, 80-99%, or 100%) identical to any one of SEQ ID NOs: 5-183. In some embodiments, the disclosure provides an expression cassette comprising a nucleic acid encoding a hydrolase comprising 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%, at least 98%, 60- 80%, 70-90%, 80-99%, or 100%) identical to any one of SEQ ID NOs: 85, 100, 103, 104, 114-117, 119, 123, 127, 128, 131-135, 137-145, 147-149, 152, 154, 157-166, 168-172, and 175-183. 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: 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 moreDocket No.: B1704.70002WO00 expression control elements known in the art, including, without limitation, enhancers, promoters, factor-specific binding sites, terminators, and / or ribosome binding sites.

[0079] 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 comprising 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: 1 or 2, or comprises SEQ ID NO: 1 or 2; and a second hydrolase. In some embodiments, the first hydrolase comprises a polypeptide comprising 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: 1, where the amino acid sequence comprises an amino acid substitution at one or more positions corresponding to positions D13, R19, S20, S24, N29, S31, L33, S34, S36, T42, R47, N49, N50, Y52, A54, V55, I57, S58, G63, E65, A66, S67, K73, I75, I83, I88, L91, Q93, E99, A103, A104, N106, I109, R111, A112, S113, S114, T115, R117, I120, S122, S123, V127, M128, S137, S142, Q143, A150, T154, W156, L158, N159, N161, S163, S164, V165, R166, V167, D175, L176, T178, I179, V182, L183, T184, H185, F189, N191, P194, T195, S196, I197, S198, L204, D205, G206, A207, T208, F210, N213, I214, P215, K217, I218, V224, N233, T235, T238, L241, P243, P245, R246, E252, and E254 of SEQ ID NO: 1. In some embodiments, the first hydrolase comprises a polypeptide comprising 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%, 80-99%, or 100%) identical to any one of SEQ ID NOs: 5-183. In some embodiments, the first hydrolase comprises a polypeptide comprising 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%, 80-99%, or 100%) identical to any one of SEQ ID NOs: 85, 100, 103, 104, 114-117, 119, 123, 127, 128, 131-135, 137-145, 147-149, 152, 154, 157-166, 168-172, and 175-183.

[0080] 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. 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, 1717Docket No.: B1704.70002WO00 (2019)). In some embodiments, the second hydrolase is a PETase (e.g., a crystalline PET- degrading enzyme, an acid tolerant PET-degrading enzyme). In some embodiments, the second hydrolase is a polyamidase (e.g., a nylon-degrading enzyme). In some embodiments, the second hydrolase is a polyurethanase (e.g., a polyurethane-degrading enzyme). In some embodiments, the second hydrolase is a cellulase (e.g., a cellulose-degrading enzyme).

[0081] In some embodiments, the composition comprises three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 3-10, 5-10, or more) hydrolases. For example, in some embodiments, the composition comprises the first hydrolase, the second hydrolase, and a third hydrolase. In some embodiments, the second and third hydrolases are selected from a MHETase, a PETase (e.g., a crystalline PET-degrading enzyme, an acid tolerant PET-degrading enzyme), a polyamidase (e.g., a nylon-degrading enzyme), a polyurethanase (e.g., a polyurethane-degrading enzyme), and a cellulase (e.g., a cellulose-degrading enzyme). In some embodiments, the second hydrolase is a MHETase, and the third hydrolase is a PETase, a polyamidase, a polyurethanase, or a cellulase.

[0082] 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). It should be appreciated that, in some embodiments, the kit can alternatively or additionally comprise a nucleic acid encoding the hydrolase (e.g., a naturally occurring or non-naturally occurring nucleic acid, such as a vector or expression cassette described herein).

[0083] In some embodiments, the kit comprises a hydrolase comprising 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%, at least 98%, 60-80%, 70-90%, 80-99%) identical to SEQ ID NO: 1, where the amino acid sequence comprises an amino acid substitution at one or more positions corresponding to positions of SEQ ID NO: 1 as described herein. In some embodiments, the kit comprises a hydrolase comprising 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%, or 100%) identical to any one of SEQ ID NOs: 5-183. In some embodiments, the kit comprises a hydrolase comprising 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%, or 100%) identical to any one of SEQ ID NOs: 85, 100, 103, 104, 114-117, 119, 123, 127, 128, 131-135, 137-145, 147-149, 152, 154, 157-166, 168-172, and 175-183.

[0084] In some embodiments, the kit comprises one or more enzymes in addition to the hydrolase. For example, in some embodiments, the kit comprises a first hydrolase (e.g., aDocket No.: B1704.70002WO00 hydrolase possessing the characteristics described in

[0083] or elsewhere herein) and a second hydrolase, where the first hydrolase and the second hydrolase are different. In some embodiments, the first hydrolase and the second hydrolase share less than 90% sequence identity (e.g., 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)). In some embodiments, the second hydrolase is a PETase. In some embodiments, the second hydrolase is a polyamidase. In some embodiments, the second hydrolase is a polyurethanase. In some embodiments, the second hydrolase is a cellulase.

[0085] 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 or with any other reference sequence described herein. For the purposes of comparing two or more protein sequences, the percentage of “sequence identity” 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).

[0086] 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.

[0087] 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 byDocket No.: B1704.70002WO00 using algorithms available as, e.g., Blast, Clustal Omega, or any other sequence alignment algorithms known in the art. Methods of Degradation

[0088] In some aspects, the disclosure provides methods of degrading an organic polymer. In some embodiments, a method of degrading an organic polymer comprises contacting the organic polymer with a hydrolase under conditions to degrade the organic polymer. In some embodiments, the hydrolase comprises 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%, at least 98%, 60-80%, 70-90%, or 80-99%) identical to SEQ ID NO: 1 or 2. In some embodiments, the hydrolase comprises an amino acid sequence of SEQ ID NO: 1 or 2 or a conservatively substituted version thereof. In some embodiments, the hydrolase comprises an amino acid substitution at one or more positions corresponding to positions D13, R19, S20, S24, N29, S31, L33, S34, S36, T42, R47, N49, N50, Y52, A54, V55, I57, S58, G63, E65, A66, S67, K73, I75, I83, I88, L91, Q93, E99, A103, A104, N106, I109, R111, A112, S113, S114, T115, R117, I120, S122, S123, V127, M128, S137, S142, Q143, A150, T154, W156, L158, N159, N161, S163, S164, V165, R166, V167, D175, L176, T178, I179, V182, L183, T184, H185, F189, N191, P194, T195, S196, I197, S198, L204, D205, G206, A207, T208, F210, N213, I214, P215, K217, I218, V224, N233, T235, T238, L241, P243, P245, R246, E252, and E254 of SEQ ID NO: 1. In some embodiments, the hydrolase comprises 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 any one of SEQ ID NOs: 5-183. In some embodiments, the hydrolase comprises an amino acid sequence of any one of SEQ ID NOs: 5-183 or a conservatively substituted version thereof. In some embodiments, the hydrolase comprises 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 any one of SEQ ID NOs: 85, 100, 103, 104, 114-117, 119, 123, 127, 128, 131- 135, 137-145, 147-149, 152, 154, 157-166, 168-172, and 175-183. In some embodiments, the hydrolase comprises an amino acid sequence of any one of SEQ ID NOs: 85, 100, 103, 104, 114-117, 119, 123, 127, 128, 131-135, 137-145, 147-149, 152, 154, 157-166, 168-172, and 175-183 or a conservatively substituted version thereof. In some embodiments, the hydrolase is a hydrolase as described herein.

[0089] As used herein, an organic polymer refers to a compound or mixture of compounds comprising monomers that are linked by covalent chemical bonds and comprise one or more carbon atoms (e.g., one or more carbon-containing groups). Within the context of theDocket No.: B1704.70002WO00 disclosure, an organic polymer includes natural or synthetic polymers, which can comprise a single type of monomer (e.g., homopolymers) or a mixture of different monomers (e.g., copolymers or heteropolymers).

[0090] In some embodiments, an organic polymer comprises polyester (e.g., polyethylene terephthalate), polyamide (e.g., nylon), polycarbonate, low-density polyethylene (e.g., polyethylene having a density of up to 930 kg / m3, such as 917-930 kg / m3), linear low-density polyethylene (e.g., linear polyethylene having a density of up to 930 kg / m3, such as 917-930 kg / m3), high-density polyethylene (e.g., polyethylene having a density of at least 930 kg / m3, such as 930-970 kg / m3), polypropylene, polystyrene, polyvinyl chloride, acrylic or polymethyl methacrylate, polyurethane, polycaprolactone, polylactic acid (PLA), polyglycolide (PGA), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), polyanhydride, polyurea, poly(lactic-co-glycolic acid) (PLGA), polydioxanone (PDS), elastane (e.g., spandex, lycra), or a combination thereof.

[0091] In some embodiments, an organic polymer comprises a polyester-containing polymer, a polyether-containing polymer, a polypropylene-containing polymer, a polyethylene- containing polymer, or a combination thereof. In some embodiments, an organic polymer comprises a polyester-containing polymer (e.g., a polymer comprising one or more esters or polyesters, which can be one or more esters or polyesters of the same type or a mixture of esters or polyesters of a different type). In some embodiments, an organic polymer comprises polyethylene glycol, polyethylene terephthalate, polyester-polyurethane, polytrimethylene terephthalate, polybutylene terephthalate, polyethylene isosorbide terephthalate, polylactic acid, polyhydroxyalkanoate, polybutylene succinate, polybutylene succinate adipate, polybutylene adipate terephthalate, polyethylene furanoate, polycaprolactone, poly(ethylene adipate), polyethylene naphthalate, or a combination thereof. In some embodiments, an organic polymer comprises polyethylene terephthalate.

[0092] In some embodiments, an organic polymer comprises a polyester-containing polymer. 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, a polyester- 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. InDocket No.: B1704.70002WO00 some embodiments, a polyester-containing polymer comprises polyethylene terephthalate. In some embodiments, a polyester-containing polymer comprises polyester-polyurethane.

[0093] In some embodiments, an organic polymer corresponds to or includes a plastic material comprising one or more polyesters, one or more polyethers, one or more polypropylenes, or a combination thereof. 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).

[0094] In some embodiments, degradation of an organic 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.

[0095] In some embodiments, the one or more degradation products comprise any products resulting from degradation of the organic polymer (e.g., polyethylene terephthalate, polyester-polyurethane) by an enzyme having hydrolase (e.g., esterase) activity. In some embodiments, the one or more degradation products comprise hydrolyzed polyols, hydrolyzed polycarboxylic acids, and / or hydrolyzed polyamines.

[0096] In some embodiments, one or more degradation products of a method described herein comprise one or more hydrolyzed polyols. As used herein, a hydrolyzed polyol refers to a compound having at least two hydroxyl groups. In some embodiments, the hydrolyzed 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 hydrolyzed 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.

[0097] In some embodiments, one or more degradation products of a method described herein comprise one or more hydrolyzed polycarboxylic acids. As used herein, a hydrolyzed polycarboxylic acid refers to a compound having at least two carboxyl groups. In some embodiments, the hydrolyzed polycarboxylic 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 someDocket No.: B1704.70002WO00 embodiments, the one or more hydrolyzed polycarboxylic acids are 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.

[0098] In some embodiments, one or more degradation products of a method described herein comprise one or more hydrolyzed polyamines. As used herein, a hydrolyzed polyamine refers to a compound having at least two amino groups. In some embodiments, the hydrolyzed 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 hydrolyzed polyamines 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.

[0099] In some embodiments, methods of the disclosure comprise contacting an organic 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.

[0100] In some embodiments, methods of the disclosure comprise contacting an organic 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. 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.

[0101] In some embodiments, methods of the disclosure are performed under degradation conditions. For example, in some embodiments, the methods comprise contacting an organic polymer with a hydrolase under conditions to degrade the organic 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 incubation temperature of between about 60 °C and about 80 °C (e.g., 65-80 °C, 70-80 °C, 60-75 °C, 65-75 °C). In some embodiments, the incubation temperature is about 70 °C. In some embodiments, the conditions comprise a pHDocket No.: B1704.70002WO00 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, 7.5-8.5, 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, 4.5- 5.5, about 4.0, about 5.0, or about 4.5).

[0102] In some embodiments, methods of the disclosure comprise contacting an organic polymer with a hydrolase described herein and one or more additional degradation enzymes. In some embodiments, the organic polymer is contacted with two or more hydrolases, including at least a first hydrolase and a second hydrolase. In some embodiments, the organic polymer is contacted with a first hydrolase comprising a polypeptide comprising 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: 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 first hydrolase comprises an amino acid substitution at one or more positions corresponding to positions D13, R19, S20, S24, N29, S31, L33, S34, S36, T42, R47, N49, N50, Y52, A54, V55, I57, S58, G63, E65, A66, S67, K73, I75, I83, I88, L91, Q93, E99, A103, A104, N106, I109, R111, A112, S113, S114, T115, R117, I120, S122, S123, V127, M128, S137, S142, Q143, A150, T154, W156, L158, N159, N161, S163, S164, V165, R166, V167, D175, L176, T178, I179, V182, L183, T184, H185, F189, N191, P194, T195, S196, I197, S198, L204, D205, G206, A207, T208, F210, N213, I214, P215, K217, I218, V224, N233, T235, T238, L241, P243, P245, R246, E252, and E254 of SEQ ID NO: 1. In some embodiments, the second hydrolase is a mono(2-hydroxyethyl) terephthalate hydrolase (MHETase). For example, in some embodiments, the MHETase is from Ideonella sakaiensis.

[0103] In some embodiments, the organic polymer is contacted with the two or more hydrolases simultaneously (e.g., contacting with a single mixture comprising at least the first and second hydrolases, or contacting with different hydrolase compositions comprising at least the first and second hydrolases at approximately the same time). In some embodiments, the organic polymer is contacted with the two or more hydrolases sequentially (e.g., contacting with one hydrolase composition followed by contacting with another hydrolase composition).

[0104] In some embodiments, a method of degrading an organic polymer comprises: (i) contacting a first polymer composition comprising the organic polymer with a first hydrolase composition under first degradation condition(s); and (ii) contacting a second polymer composition comprising the organic polymer with a second hydrolase composition underDocket No.: B1704.70002WO00 second degradation condition(s). In some embodiments, the method further comprises, after (i) and before (ii), recovering one or more degradation products from the first polymer composition. In some embodiments, the method further comprises, after (ii), recovering one or more degradation products from the second polymer composition. In some embodiments, the first and second hydrolase compositions are different. In some embodiments, at least one of the first and second hydrolase compositions comprise one or more hydrolases described herein. In some embodiments, the first and second degradation condition(s) are different. In some embodiments, at least one of the first and second degradation condition(s) comprise degradation condition(s) described herein.

[0105] The present disclosure is further illustrated by the following Examples, which should not be construed as limiting. EXAMPLESExample 1. Engineered Improved Hydrolase Variants

[0106] A dienelactone hydrolase family protein from Thermobifida fusca (NCBI Ref.: WP_104613137.1) was modified to exclude a predicted signal peptide and introduce a leading methionine and a C-terminal 6X polyhistidine tag. The modified enzyme, referred to herein as “BB212,” was used as a parent enzyme for further engineering to identify improved variants for increased thermostability, acid tolerance, and activity on PET and other polyester-containing polymers. The amino acid sequence of the hydrolase from Thermobifida fusca is provided in Table 3, along with the amino acid sequence of BB212 and its corresponding nucleotide sequence. Table 3. Hydrolase SequencesDocket No.: B1704.70002WO00

[0107] BB212 enzyme variant libraries were designed and evaluated in a 96-well plate using a HPLC-based PET degradation assay as described in Example 7. The initial variant screening focused on identifying mutations that retained or improved PET depolymerization activity closer to the glass transition temperature of PET (68-70 ºC), where PET is typically easier to hydrolyze by enzymes due to improved polymer mobility.

[0108] A total of 178 variants with improved activity compared to BB212 were identified. These variants are shown in Table 4 with values for the fold improvement over parent (FIOP) measurements of enzyme activity on amorphous PET (aPET) discs in the described 96-well plate HPLC-based PET degradation assay. Across the 178 variants, mutations spanned 94 residue positions with a total of 201 unique single residue changes. The mutations were distributed across the protein sequence and across the structure as described in Example 4. The FIOP results for all variants are shown in Table 4, with select variants further evaluated as described in Examples 2-3 and 5-9. Table 4. Engineered BB212 VariantsDocket No.: B1704.70002WO00Docket No.: B1704.70002WO00Docket No.: B1704.70002WO00Docket No.: B1704.70002WO00Docket No.: B1704.70002WO00Docket No.: B1704.70002WO00Docket No.: B1704.70002WO00Docket No.: B1704.70002WO00Docket No.: B1704.70002WO00Example 2. Engineered Disulfide Bond Variant with Increased Thermotolerance

[0109] As described in Example 7, 120 paired cysteines were designed and tested for improved thermotolerance as indicated by increased activity in a 96-well plate HPLC-based PET degradation assay at 70 ºC. One engineered disulfide bond pair of a variant referred to herein as “DSB127” (BB212 with mutations D205C, E254C) generated the largest increase in overall activity compared to the parent enzyme BB212, relative to the other members of the disulfide bond variant library (Table 4). The thermotolerance effects of the DSB127 variant were further evaluated in this example.

[0110] The parent BB212 enzyme and the DSB127 variant were purified and evaluated in PET depolymerization assays carried out at 50 ºC and 70 ºC. In these assays, enzymes were incubated with either amorphous PET (aPET) or crystalline PET (cPow) powder at 0.7 mg enzyme / g PET loading. Biological duplicates were conducted at 50 ºC or 70 ºC for 96 hours in 50 mM KH2PO4,100 mM NaCl pH 8.0. Analyte analysis of TPA and MHET was performed on an Infinity II 1290 ultra-high-performance liquid chromatography (UHPLC) system (Agilent Technologies). TPA and MHET concentrations were calculated by preparation of standard curves.

[0111] FIG.1 shows bar charts depicting the results from PET depolymerization assays with purified BB212 (“BB212 WT”) and DSB127 (“BB212 D205C / E254C”) (standard deviation of replicates shown). As shown, DSB127 exhibited significantly increased activity at 70 ºC. The improvements in activity at 70 ºC relative to 50 ºC indicated that the two mutations ofDocket No.: B1704.70002WO00 DSB127 provided increased thermotolerance. The DSB127 variant was used for further engineering and served as a parent enzyme in further screens (Table 4, Example 6). Example 3. Engineered Variants with Increased Activity in Low pH Conditions

[0112] Breakdown of PET plastic is an acidogenic process in which the pH of the reaction environment decreases as more PET is depolymerized through the release of terephthalic acid (TPA) into solution. As such, engineered enzymes having improved activity at low pH would be beneficial for increasing total depolymerization in an industrial enzymatic plastic recycling process.

[0113] BB212 variants were tested in 96-well plate HPLC-based PET degradation assays conducted at standard (pH 8) and acidic (pH 5) reaction conditions via the addition of citrate buffer. Three variants of DSB127 (BB212 with mutations D205C, E254C) that exhibit increased activity in reaction conditions at both pH 8 and pH 5 were identified: (1) F210R, T235Y (BB212 with mutations D205C, F210R, T235Y, E254C); (2) F210R, T235K (BB212 with mutations D205C, F210R, T235K, E254C); and (3) F210R, T235F (BB212 with mutations D205C, F210R, T235F, E254C).

[0114] FIG.2 shows assay results for the three variants and the parent enzyme (DSB127). Values are the sum of monomer levels of TPA, MHET, and BHET as measured via HPLC. Enzymes were incubated with amorphous PET (aPET) for 24 hours at 70 ºC at either pH 8 or pH 5. As annotated in FIG.2, the three variants exhibited high activity in both standard pH conditions (pH 8) and low pH conditions (pH 5). Parent enzyme data points are colored in blue and represented as triangles. The dotted line is unity. Example 4. Structural Analysis of BB212 Positions Mutated in Improved Variants

[0115] FIG.3 shows three different perspectives of a BB212 Alpha Fold predicted structure of BB212 shown with docked PET trimer molecule (shown as sticks) and shaded at the residues where mutations were identified in improved variants (Table 4). As shown in FIG.3 and summarized in Table 5, the mutations are spread throughout the protein sequence and structure, including surface exposed residues, mutations within the protein core, and mutations contacting the PET substrate. Table 5. Structural Analysis of BB212 MutationsDocket No.: B1704.70002WO00Docket No.: B1704.70002WO00Docket No.: B1704.70002WO00Docket No.: B1704.70002WO00Docket No.: B1704.70002WO00Docket No.: B1704.70002WO00 Example 5. Polyurethanase Activity of BB212 Variant

[0116] PET and other commonly used synthetic polymers contain ester bonds. Therefore, engineered enzymes that are active across a spectrum of these polymers would be advantageous for different recycling processes.

[0117] The enzymatic hydrolysis of polyester-based thermoplastic polyurethane (TPU) by DSB127 (BB212 with mutations D205C, E254C) was assessed by measuring adipic acid production using high resolution LCMS. Adipic acid is a key monomer of the substrate of interest making up the soft segments of the TPU. In these assays, 0.125 µM of enzyme was incubated with ~35 mg of TPU in 10 mM ammonium bicarbonate pH 8.0 at 70 °C for 24 hours. A similar reaction containing only buffer was also incubated as a control. The reactions were quenched with 20:80 acetonitrile:0.1% FA in water and filtered. Samples were analyzed without column separation by ESI(-) LCMS with the abundance at 145.05 m / z correlating to adipic acid. The concentration of adipic acid was quantified using a standard curve from 0.5-20 µM adipic acid. Samples were diluted as needed to measure adipic acid concentration.

[0118] FIG.4 is a bar chart showing the average of 3 replicate assays evaluating the enzymatic hydrolysis of polyester-based TPU by DSB127 (error bars: standard deviation of the replicates). As shown, the results demonstrated depolymerization of polyester-based TPU by DSB127. This data indicated the ability of this enzyme, and potentially engineered variants thereof, to hydrolyze different polymer types, including PU, and suggested activity on other ester bond containing polymers. Example 6. Engineered Variants of DSB127

[0119] As described in Example 2, DSB127 (BB212 with mutations D205C, E254C) generated the largest increase in overall activity among the disulfide bond variant library as compared to the parent enzyme BB212. DSB127 was selected as the base protein variant for further protein engineering efforts, including the three methods for library design as described in Example 7.

[0120] PET depolymerization assays were conducted with purified DSB127 and five protein variants of DSB127 having different substitutions at residues F210 and T235: (1) F210R, T235I (BB212 with mutations D205C, F210R, T235I, E254C); (2) F210R, T235Y (BB212 with mutations D205C, F210R, T235Y, E254C); (3) F210R, T235K (BB212 with mutations D205C, F210R, T235K, E254C); (4) F210R, T235Q (BB212 with mutations D205C, F210R,Docket No.: B1704.70002WO00 T235Q, E254C); and (5) F210R, T235F (BB212 with mutations D205C, F210R, T235F, E254C).

[0121] In these PET depolymerization assays, 2.0% loading by mass of an amorphous PET film (Polymershapes) was incubated with 10 ug of purified enzyme in a 450 uL reaction (1 mg enzyme / g PET). Biological triplicates were conducted at 70 ºC for 96 hours in 100 mM potassium phosphate pH 8.0. After 96 hours, reactions were quenched by the addition of an equal volume of a cold methanol and 13 mM trifluoracetic acid solution. Analyte analysis of TPA and MHET was performed on an Infinity II 1290 ultra-high-performance liquid chromatography (UHPLC) system (Agilent Technologies). TPA and MHET concentrations were calculated by preparation of standard curves and the sum of monomers (PPM) was determined.

[0122] FIG.5 is a bar graph showing assay results for the parent enzyme (DSB127) and the five variants indicated above (graph shows mean value for the sum of monomers (PPM) with standard deviation). As shown, each of the five variants exhibited an approximately 2-fold increased depolymerization activity relative to the parent enzyme. Example 7. Methods associated with Examples 1-6

[0123] HPLC-based PET degradation assay

[0124] E. coli NEB T7 express transformants expressing secreted hydrolases were seeded as single colonies in 96 deep well plates, grown for 16 hours in 400 µl of LB (37 ºC) at 1,000 RPM and 80% humidity. This was followed by a 22-hour induction at 25 ºC by transferring 100 µl of seed into 900 µl of LB containing 250 µM IPTG at 1,000 RPM and 80% humidity. After induction, plates were centrifuged and 400 µl of supernatants were incubated in 96 deep well plates with amorphous PET discs for 24 hours at 50 ºC or 70 ºC. In some instances, pH adjustments were made to incubation plates by adding sodium citrate buffer. Samples were then quenched in an equal volume of a cold (-20 ºC) methanol and 13 mM trifluoracetic acid solution and diluted as needed into ddH2O before HPLC analysis. Four biological replicates were performed. Analyte analysis of TPA and MHET was performed on an Infinity II 1290 ultra-high-performance liquid chromatography (UHPLC) system (Agilent Technologies) using OpenLab CDS software. TPA, MHET, and BHET concentrations were calculated by preparation of standard curves.

[0125] Purified enzyme incubation with amorphous PET discs or crystalline PET powder (Example 3, Example 6)Docket No.: B1704.70002WO00

[0126] After purification of enzyme, between 0.7-2.0% loading by mass of an amorphous PET film (Polymershapes) or crystalline PET powder (Goodfellow) was incubated with 10 ug of purified enzyme in a 450 uL reaction (1 mg enzyme / g PET). Biological triplicates were conducted at 70 ºC for 96 hours in 100 mM potassium phosphate pH 8.0. After 96 hours, reactions were quenched by the addition of an equal volume of a cold (-20 ºC) methanol and 13 mM trifluoracetic acid solution. Analyte analysis of TPA and MHET was performed on an Infinity II 1290 ultra-high-performance liquid chromatography (UHPLC) system (Agilent Technologies). TPA and MHET concentrations were calculated by preparation of standard curves.

[0127] LCMS measurement of Adipic acid production (Example 5)

[0128] Enzymatic hydrolysis of a polyester-based thermoplastic polyurethane (TPU) by DSB127 was assessed by measuring adipic acid production using high resolution LCMS. 0.125 µM of enzyme was incubated with ~35 mg of TPU in 10 mM ammonium bicarbonate pH 8.0 at 70 °C for 24 hours. A similar reaction containing only buffer was also incubated as a control. The reactions were quenched with 20:80 acetonitrile:0.1% formic acid in water and filtered. Samples were analyzed without column separation by ESI(-) LCMS with the abundance at 145.05 m / z correlating to adipic acid. The concentration of adipic acid was quantified using a standard curve from 0.5-20 µM adipic acid. Samples were diluted as needed to measure adipic acid concentration.

[0129] Library design

[0130] Initially, two types of libraries were designed for BB212: (1) site saturation mutagenesis (SSM / NNK), and (2) introduction of disulfide bonds (DSB).

[0131] SSM libraries with NNK (NN(T / G)) degenerate codons were chosen to generate diversity for recombination at amino acid residues predicted to contribute to unfolding. DDGun (Montanucci, et al.) was used to predict all single mutation folding energy changes. 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 300 mutants.

[0132] Engineered disulfide bond protein variants were constructed using amino acid residues in close proximity that would enable disulfide bond formation across the full length of BB212, using PyMOL for visual validation of designs. Paired cysteines were introducedDocket No.: B1704.70002WO00 into the BB212 coding sequence using PCR and verified by DNA sequencing.120 paired cysteines were designed and tested for improved thermostability.

[0133] One engineered disulfide bond pair, DSB127 (D205C, E254C), generated the largest increase in overall activity compared to the parent enzyme BB212, relative to the other members of the disulfide bond variant library. DSB127 was selected as the base protein variant for further protein engineering efforts, including the following three methods for library design.

[0134] DSB127 library design methods (Example 6)

[0135] The first library design method applied an evolutionary couplings model (Hopf, et al.) to predict epistatic effects of single mutations. This model was used to predict the effects of all possible single mutations along the protein. Residues in the sequence that had more predicted beneficial mutations were selected for mutation and degenerate codons were chosen to incorporate all positively predicted mutations at those positions. Using this method, three libraries were constructed across the DSB127 sequence which contained single and double mutants. In total, the libraries contained 974 unique variants which were screened via the described 96 well plate assay.

[0136] The second library design method used a model (Gado, et al.) that predicts pH optimum based on protein sequence. The aim was to identify mutations in the DSB127 mutant (D205C, E254C) of BB212 primary amino acid sequence that would lower the pH optimum of this enzyme, enabling improved hydrolysis under low pH conditions. PET hydrolysis is highly acidogenic (due to release of terephthalic acid into solution), and higher enzyme activity under low pH conditions will lower process cost and process carbon intensity at industrial scale. The model was used to predict the pH optimum effect of every possible single mutation along the protein sequence. Residues in the BB212 sequence with mutations predicted to lower pH optimum were selected for mutation. Degenerate codons were selected to incorporate all mutations predicted to lower pH optimum at those positions. Three protein variant libraries were constructed in this manner containing one, two, or three amino acid substitutions. A total of 2,354 variants were constructed and screened via the described 96 well plate assay.

[0137] The third library design method focused on the predicted “binding pocket” residues of BB212 that are predicted to contact a PET polymer during catalysis. The predicted binding pocket residues were determined by overlaying and aligning the BB212 structure onto the known Ideonella sakaiensis PETase structure with a PET ligand (Han, et al.). Predicted binding pocket contact residues include Y61, T62, G63, Q93, H130, S131, M132, T154,Docket No.: B1704.70002WO00 P155, W156, H157, L158, N159, K160, N161, A174, D177, I179, A180, P181, T184, H185, P188, H209, F210, A211, C242, C260. Degenerate NNK codons were incorporated at various double site combinations, covering all listed sites in the binding pocket residues to create single and double variants. Two protein variant libraries were constructed in this manner and a total of 1,120 E. coli transformant colonies were constructed and screened via the described 96 well plate assay.

[0138] References

[0139] Montanucci, Ludovica, et al. “DDGun: an untrained predictor of protein stability changes upon amino acid variants.” Nucleic Acids Research 50.W1 (2022): W222-W227.

[0140] Hopf, Thomas A., et al. “Mutation effects predicted from sequence co-variation.” Nature Biotechnology 35.2 (2017): 128-135.

[0141] Gado, Japheth E., et al. “Deep learning prediction of enzyme optimum pH.” bioRxiv (2023): 2023-06.

[0142] Han, Xu, et al. “Structural insight into catalytic mechanism of PET hydrolase.” Nature Communications 8.1 (2017): 2106. Example 8. Performance of BB212 Variant in pH-Controlled Stirred Reactor Tank Scales

[0143] The scalability of enzyme performance was evaluated using a representative BB212 variant of Table 4 having substitutions including H185Y, D205C, and E254C. PET degradation yields over time were monitored in stirred tank reactors containing the representative BB212 variant, size-reduced PET, and buffer at 0.1-liter, 2-liter, 10-liter, and 50-liter scale reactions. The stirred tank reactors were pH-controlled with base at pH 8, and the reactions were carried out at 70 ºC.

[0144] The results from two replicates of a 50-liter scale reaction over time are shown in FIG.6A. FIG.6B shows the consistency of enzyme performance between 50-liter scale reaction (dashed line) and 0.1-liter (top plot, solid line), 2-liter (middle plot, solid line), and 10-liter (bottom plot, solid line) scale reactions. The percent yield of two replicates at each of the different scale reactions was determined and is shown in the plot of FIG.6C (error bars represent replicate range; middle bar is mean of two replicates). The results demonstrated consistent performance of PET hydrolysis by the representative BB212 variant across pH- controlled reactor scales from 0.1-liter up to 50-liter scale reactions. Example 9. Enzyme Specificity for PET over Non-PET PlasticsDocket No.: B1704.70002WO00

[0145] The enzyme specificity for PET over non-PET plastics was evaluated using a representative BB212 variant of Table 4 having substitutions including H185Y, D205C, and E254C. Hydrolysis reactions were carried out at 70 ºC for 72 hours in a stirred tank reactor containing the representative BB212 variant, PET plastic (post-consumer PET 4mm flake), and either (a) polypropylene plastic, or (b) high-density polyethylene plastic.

[0146] The results from the enzymatic hydrolysis reaction with PET and polypropylene plastics are shown in FIG.7A and below in Table 6. FIG.7A shows PET and polypropylene plastics illustrating the relative amounts quantified before (left) and after (right) the reaction with the representative BB212 variant. Table 6 provides the mass of PET and polypropylene as determined before and after the hydrolysis reaction. As shown, the enzyme variant was highly active toward PET plastic while being inert to the polypropylene plastic, with the hydrolysis reaction resulting in an approximate 98.7% reduction in mass of PET without any measured change in mass of polypropylene.

[0147] The results from the enzymatic hydrolysis reaction with PET and high-density polyethylene plastics are shown in FIG.7B and below in Table 7. FIG.7B shows PET and high-density polyethylene plastics illustrating the relative amounts quantified before (left) and after (right) the reaction with the representative BB212 variant. Table 7 provides the mass of PET and high-density polyethylene as determined before and after the hydrolysis reaction. As shown, the enzyme variant was again highly active toward PET plastic while being inert to the high-density polyethylene plastic, with the hydrolysis reaction resulting in an approximate 98.3% reduction in mass of PET without any measured change in mass of high-density polyethylene.

[0148] These results demonstrated that the representative BB212 variant was highly specific for PET in mixtures containing non-PET plastics. While the enzyme was inert toward the non-PET plastics in hydrolysis reactions, it remained highly active against PET, resulting in more than 98% degradation of PET without any measured degradation of non-PET plastics.

[0149] Non-PET contaminants are common in commercial bales of mixed plastic waste. The high specificity for PET plastic and inertness toward non-PET contaminants as demonstrated in this example, in addition to the scalability demonstrated in Example 8, are further indicative of the usefulness of the variants described herein for effective and efficient plastic recycling processes. Table 6. Hydrolysis Reaction with PET and Polypropylene (PP)Docket No.: B1704.70002WO00Table 7. Hydrolysis Reaction with PET and High-Density Polyethylene (HDPE)EQUIVALENTS AND SCOPE

[0150] 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.

[0151] 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.

[0152] 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, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. MultipleDocket No.: B1704.70002WO00 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.

[0153] 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 of exactly 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.

[0154] 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.Docket No.: B1704.70002WO00

[0155] 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.

[0156] 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 application also contemplates the alternative embodiments “a composition consisting of A and B” and “a composition consisting essentially of A and B.”

[0157] 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.

[0158] 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.

[0159] 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 artDocket No.: B1704.70002WO00 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.

[0160] 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

Docket No.: B1704.70002WO00 CLAIMS What is claimed is:

1. A hydrolase comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, wherein the amino acid sequence of the hydrolase comprises an amino acid substitution at one or more positions corresponding to positions D13, R19, S20, S24, N29, S31, L33, S34, S36, T42, R47, N49, N50, Y52, A54, V55, I57, S58, G63, E65, A66, S67, K73, I75, I83, I88, L91, Q93, E99, A103, A104, N106, I109, R111, A112, S113, S114, T115, R117, I120, S122, S123, V127, M128, S137, S142, Q143, A150, T154, W156, L158, N159, N161, S163, S164, V165, R166, V167, D175, L176, T178, I179, V182, L183, T184, H185, F189, N191, P194, T195, S196, I197, S198, L204, D205, G206, A207, T208, F210, N213, I214, P215, K217, I218, V224, N233, T235, T238, L241, P243, P245, R246, E252, and E254 of SEQ ID NO:

1.

2. The hydrolase of claim 1, wherein the hydrolase comprises at least one additional disulfide bond relative to a hydrolase that comprises the amino acid sequence of SEQ ID NO:

1.

3. The hydrolase of claim 1 or 2, wherein the amino acid sequence of the hydrolase comprises amino acid substitutions at positions corresponding to positions D205 and E254 of SEQ ID NO:

1.

4. The hydrolase of claim 3, wherein the amino acid substitutions comprise D205C and E254C relative to SEQ ID NO:

1.

5. The hydrolase of any one of claims 1-4, wherein the amino acid sequence of the hydrolase comprises amino acid substitutions at positions corresponding to positions I109 and S122 of SEQ ID NO:

1.

6. The hydrolase of claim 5, wherein the amino acid substitutions comprise I109C and S122C relative to SEQ ID NO: 1.Docket No.: B1704.70002WO00 7. The hydrolase of any one of claims 1-6, wherein the amino acid sequence of the hydrolase comprises amino acid substitutions at positions corresponding to the following positions of SEQ ID NO: 1: (i) S137; and (ii) V127 or A150.

8. The hydrolase of claim 7, wherein the amino acid substitutions comprise relative to SEQ ID NO: 1: (i) S137C and V127C; or (ii) S137C and A150C.

9. The hydrolase of any one of claims 1-8, wherein the amino acid sequence of the hydrolase comprises amino acid substitutions at positions corresponding to positions P194 and S196 of SEQ ID NO:

1.

10. The hydrolase of claim 9, wherein the amino acid substitutions comprise P194C and S196C relative to SEQ ID NO:

1.

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 positions F210 and T235 of SEQ ID NO:

1.

12. The hydrolase of claim 11, wherein the amino acid substitutions comprise relative to SEQ ID NO: 1: (i) F210G, F210I, F210K, F210N, F210R, or F210T; and (ii) T235D, T235F, T235G, T235I, T235K, T235L, T235Q, T235V, or T235Y.

13. The hydrolase of claim 11, wherein the amino acid substitutions comprise relative to SEQ ID NO: 1: (i) F210I, F210R, or F210T; and (ii) T235D, T235F, T235G, T235I, T235K, T235L, T235Q, T235V, or T235Y.

14. The hydrolase of any one of claims 11-13, wherein the amino acid substitutions comprise relative to SEQ ID NO: 1: (i) F210R; and (ii) T235D, T235F, T235K, or T235Y.

15. The hydrolase of any one of claims 1-14, wherein the amino acid sequence of the hydrolase comprises an amino acid substitution at one or more positions corresponding to positions A54, S58, and E65 of SEQ ID NO: 1.Docket No.: B1704.70002WO00 16. The hydrolase of claim 15, wherein the amino acid substitution is selected from A54G, S58C, E65C, E65K, E65Q, E65R, and E65V relative to SEQ ID NO:

1.

17. The hydrolase of any one of claims 1-16, wherein the amino acid sequence of the hydrolase comprises an amino acid substitution at one or more positions corresponding to positions R47, S114, D175, H185, D205, F210, T235, and E254 of SEQ ID NO:

1.

18. The hydrolase of claim 17, wherein the amino acid substitution is D175Q relative to SEQ ID NO:

1.

19. The hydrolase of claim 17 or 18, wherein the amino acid substitution is H185Y relative to SEQ ID NO:

1.

20. The hydrolase of any one of claims 17-19, wherein the amino acid substitution is F210R relative to SEQ ID NO:

1.

21. The hydrolase of any one of claims 1-20, wherein the amino acid sequence of the hydrolase comprises amino acid substitutions at positions corresponding to positions R47, S114, D175, H185, D205, F210, T235, and E254 of SEQ ID NO:

1.

22. The hydrolase of claim 21, wherein the amino acid substitutions comprise R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C relative to SEQ ID NO:

1.

23. The hydrolase of any one of claims 1-22, wherein the amino acid substitution is selected from R19I, R19L, R19F, R19K, R19V, S24L, S24R, N29A, N29K, N29V, S34A, S34E, S34K, S36K, S36N, S36R, T42A, T42M, R47E, R47G, R47I, R47L, R47T, N49P, N49T, N50H, N50T, Y52F, A54G, V55I, I57L, I57M, I57V, S58C, G63L, G63S, G63C, G63N, E65C, E65K, E65Q, E65R, E65V, A66D, A66E, A66G, A66L, S67E, K73D, K73P, K73Q, I75L, I75T, I75V, I83L, I88H, I88N, L91I, L91R, L91V, Q93N, Q93S, A104S, I109C, A112S, S113P, S113H, S114D, S114G, S114N, S114R, I120V, S122C, S123A, S123N, V127C, M128T, S137C, S142E, A150C, T154S, N161D, S163F, S163Q, S163D, S163K, S163N, S163A, D175A, D175E, D175G, D175I, D175N, D175Q, D175R, D175S, D175T, L176A, L176I, L176R, L176T, L176V, T178G, T178Q, T178V, I179I, I179V, L183S, L183V, L183K, L183I, L183A, T184A, T184H, T184A, T184K, T184Q, H185Y, F189I, N191K,Docket No.: B1704.70002WO00 N191Q, N191A, P194C, T195E, T195M, T195P, S196C, S196P, S196T, S198A, S198E, S198N, S198T, L204G, D205C, G206R, A207V, T208G, T208S, F210G, F210I, F210K, F210N, F210R, F210T, I214V, I214K, I214M, P215V, I218T, V224L, N233V, T235E, T235G, T235I, T235L, T235Q, T235D, T235F, T235K, T235Y, T238N, L241M, P243G, P243R, P245K, R246L, E252A, and E254C relative to SEQ ID NO:

1.

24. The hydrolase of any one of claims 1-23, wherein the amino acid sequence of the hydrolase comprises amino acid substitutions at positions corresponding to the following positions within SEQ ID NO: 1: i) R47, D205, and E254; ii) R47, N50, D205, and E254; iii) R47, K73, D205, and E254; iv) I179, D205, and E254; v) R47, D205, and E254; vi) D205, F210, and E254; vii) T184, F189, D205, F210, and E254; viii) D205, F210, A211, and E254; ix) D205, F210, T235, and E254; x) D205, F210, P215, and E254; xi) D205, F210, N233, P243, and E254; xii) D205, F210, P215, T235, and E254; xiii) N49, K73, L91, D205, and E254; xiv) K73, D205, T235, P243, and E254; xv) D205, F210, N233, T235, P243, and E254; xvi) D205, F210, N233, T235, and E254; xvii) S24, N29, D205, and E254; xviii) L183, D205, I218, and E254; xix) R47, E65, S114, H185, D205, F210, T235, and E254; xx) R47, S114, H185, D205, F210, T235, and E254; xxi) R47, K73, S114, I179, D205, F210, T235, and E254; xxii) R47, V55, G63, S114, H185, D205, F210, T235, and E254; xxiii) R47, S114, H185, I179, D205, F210, T235, and E254; xxiv) R47, I57, I83, S114, H185, D205, F210, T235, and E254; xxv) R47, I57, S114, H185, D205, F210, T235, and E254;Docket No.: B1704.70002WO00 xxvi) R47, S114, D175, L176, H185, D205, F210, T235, and E254; xxvii) R47, S114, N161, H185, D205, F210, T235, and E254; xxviii) R47, H185, D205, F210, T235, and E254; xxix) R47, S114, H185, L204, D205, F210, T235, and E254; xxx) R47, S114, D175, H185, D205, F210, T235, and E254; xxxi) R47, I75, S114, H185, D205, F210, T235, and E254; xxxii) R47, S114, S123, H185, D205, F210, T235, and E254; xxxiii) R47, S114, D175, L176, D205, H185, F210, T235, and E254; xxxiv) R47, S114, L176, T178, H185, D205, F210, T235, and E254; xxxv) R47, I57, I75, S114, H185, D205, F210, T235, and E254; xxxvi) I75, S114, H185, D205, F210, T235, and E254; xxxvii) R47, S113, S114, S123, H185, D205, F210, T235, and E254; xxxviii) R47, S114, H185, N191, D205, F210, T235, and E254; xxxix) R47, S114, T178, H185, D205, F210, T235, and E254; xl) S34, Y52, S114, H185, D205, F210, T235, and E254; xli) R47, A112, S114, S123, H185, D205, F210, T235, and E254; xlii) R47, S114, I120, H185, D205, F210, T235, and E254; xliii) R47, S114, H185, S198, D205, F210, T235, and E254; xliv) R47, S114, H185, T195, D205, F210, T235, and E254; xlv) R47, S114, H185, D205, G206, F210, T235, and E254; xlvi) R19, S36, N50, Y52, I57, I75, S114, H185, F210, and T235; xlvii) R19, N50, Y52, I75, S114, H185, F210, and T235; xlviii) R19, S34, R47, N50, Y52, I57, I75, S114, H185, F210, and T235; xlix) S34, N50, I57, I75, S114, S142, H185, F210, and T235; l) S34, Y52, I57, I75, S114, H185, F210, and T235; li) S36, N50, Y52, I57, S114, H185, F210, and T235; lii) R47, S114, D175, H185, D205, F210, T235, P245, R246, and E254; liii) R47, S114, H185, D205, F210, T235, T238, and E254; liv) R19, R47, S114, D175, H185, D205, F210, T235, and E254; lv) R19, R47, S114, D175, H185, D205, F210, T235, P245, R246, and E254; lvi) R19, S34, S36, R47, N50, Y52, D175, H185, F210, and T235; lvii) R47, D175, H185, D205, F210, T235, and E254; lviii) R47, I57, D175, H185, D205, F210, T235, and E254; lix) R47, N50, Y52, I57, S114, D175, H185, D205, F210, T235, and E254;Docket No.: B1704.70002WO00 lx) S34, S114, D175, H185, D205, F210, T235, P245, R246, and E254; lxi) S34, Y52, S114, D175, H185, D205, F210, T235, and E254; lxii) T42, R47, S114, D175, H185, D205, F210, T235, and E254; lxiii) T42, R47, S114, D175, H185, D205, F210, V224, T235, and E254; lxiv) R19, T42, R47, S114, D175, H185, D205, F210, T235, and E254; lxv) R47, I57, I75, S114, D175, H185, D205, F210, T235, and E254; lxvi) R47, Y52, S114, D175, H185, D205, F210, L224, T235, and E254; lxvii) S34, S36, R47, D175, H185, D205, F210, T235, and E254; lxviii) S34, S36, R47, S114, D175, H185, D205, F210, T235, and E254; lxix) S34, S36, R47, S114, H185, D205, F210, T235, and E254; lxx) S34, D175, H185, D205, F210, T235, and E254; lxxi) S34, R47, I75, S114, D175, H185, D205, F210, T235, and E254; lxxii) S34, R47, S114, D175, H185, D205, F210, T235, and E254; lxxiii) R47, I57, I75, S114, H185, D205, T208, F210, T235, and E254; lxxiv) R47, I57, I75, S114, D175, H185, D205, A207, F210, T235, and E254; lxxv) R47, I75, S114, D175, H185, D205, F210, I214, T235, and E254; lxxvi) R47, S114, M128, D175, H185, D205, F210, T235, and E254; lxxvii) S34, Y52, A104, S114, D175, H185, D205, F210, T235, and E254; lxxviii) R47, I57, G63, A66, S114, D175, H185, D205, F210, T235, and E254; lxxix) R47, Y52, G63, A66, S114, D175, H185, D205, F210, T235, and E254; lxxx) R47, I57, A66, S114, D175, H185, D205, F210, T235, and E254; lxxxi) R47, G63, A66, S114, D175, H185, D205, F210, T235, and E254; lxxxii) R47, E65, S67, S114, D175, H185, D205, F210, T235, and E254; lxxxiii) R47, S114, S163, D175, H185, D205, F210, T235, and E254; lxxxiv) R47, S114, D175, H185, D205, F210, T235, E252, and E254; lxxxv) R47, S114, D175, H185, D205, F210, T235, L241, and E254; lxxxvi) R47, S114, D175, H185, D205, T208, F210, T235, and E254; lxxxvii) R47, S114, T154, D175, T184, H185, D205, F210, T235, and E254; lxxxviii) R47, I57, L91, Q93, S114, D175, H185, D205, F210, T235, and E254; lxxxix) R47, I88, S114, D175, H185, D205, F210, T235, and E254; xc) R47, Q93, S114, D175, H185, D205, F210, T235, and E254; xci) N29, S31, S36, R47, N49, S114, D175, H185, D205, F210, T235, and E254; xcii) R19, S20, T42, R47, S114, D175, H185, D205, F210, T235, and E254; xciii) R47, A103, N106, R111, S114, T115, D175, H185, D205, F210, T235, and E254;Docket No.: B1704.70002WO00 xciv) R47, A66, I75, S114, D175, H185, D205, F210, T235, and E254; xcv) R47, L91, N106, S114, D175, T184, H185, D205, F210, K217, T235, and E254; xcvi) S34, Y52, G63, S114, D175, H185, D205, F210, T235, and E254; xcvii) R47, R111, S114, D175, H185, D205, F210, T235, and E254; xcviii) S31, R47, S114, D175, H185, D205, F210, T235, and E254; xcix) S34, Y52, L91, S113, S114, D175, H185, D205, F210, T235, and E254; c) R47, I57, G63, A66, S114, D175, H185, D205, T208, F210, T235, and E254; ci) S34, G63, A66, S114, D175, H185, D205, F210, T235, and E254; cii) R47, S114, W156, L158, D175, H185, S196, D205, F210, I214, T235, and E254; ciii) R47, S114, L158, R166, D175, H185, D205, F210, T235, and E254; civ) R47, S114, L158, V167, D175, H185, N191, D205, F210, T235, and E254; cv) R47, S114, D175, V182, H185, D205, F210, T235, and E254; cvi) R47, S114, S163, R166, D175, H185, N191, D205, F210, T235, and E254; cvii) R47, S114, L158, D175, H185, S196, D205, F210, T235, and E254; cviii) R47, S114, S163, S164, D175, H185, D205, F210, I214, T235, and E254; cix) R47, S114, S164, D175, L183, H185, T195, D205, F210, T235, and E254; cx) R47, S114, N159, D175, H185, D205, F210, T235, and E254; cxi) R47, S114, D175, S198, D205, F210, T235, and E254; cxii) R47, S114, V165, D175, L183, H185, I197, S198, D205, F210, T235, and E254; cxiii) R47, S114, S142, T154, R166, D175, H185, S198, D205, F210, T235, and E254; cxiv) R47, S114, Q143, S163, S164, D175, H185, T195, D205, F210, T235, and E254; cxv) R47, S114, L158, S163, S164, D175, H185, S198, D205, F210, N213, T235, and E254; cxvi) R47, Y52, I57, G63, A66, I75, S114, D175, H185, D205, F210, T235, and E254; cxvii) D13, R47, N50, I88, E99, S114, D175, H185, D205, F210, K217, T235, and E254; cxviii) Y52, I57, G63, A66, I75, S114, D175, H185, D205, F210, T235, and E254; cxix) R47, G63, S114, R117, D175, L183, H185, D205, F210, I214, T235, and E254; cxx) R19, R47, G63, S114, R166, D175, L183, H185, D205, F210, T235, and E254; cxxi) R47, S114, D175, L183, H185, D205, F210, T235, and E254; cxxii) R47, S114, L158, S163, D175, T184, H185, D205, F210, T235, and E254; cxxiii) R47, Q93, S114, D175, H185, D205, T208, F210, T235, and E254; cxxiv) S34, R47, S114, L158, D175, H185, D205, F210, T235, and E254; or cxxv) L33, R47, S114, R117, R166, D175, L183, H185, D205, F210, T235, and E254.Docket No.: B1704.70002WO00 25. The hydrolase of claim 24, wherein the amino acid substitutions comprise relative to the sequence of SEQ ID NO: 1: i) R47V, D205C, and E254C; ii) R47L, D205C, and E254C; iii) R47E, N50G, D205C, and E254C; iv) R47V, K73Q, D205C, and E254C; v) I179V, D205C, and E254C; vi) R47T, D205C, and E254C; vii) R47G, D205C, and E254C; viii) D205C, F210G, and E254C; ix) D205C, F210K, and E254C; x) T184H, F189I, D205C, F210K, and E254C; xi) D205C, F210N, and E254C; xii) D205C, F210K, A211G, and E254C; xiii) D205C, F210I, T235L, and E254C; xiv) D205C, F210I, T235G, and E254C; xv) D205C, F210T, T235G, and E254C; xvi) D205C, F210R, P215V, and E254C; xvii) D205C, F210R, N233V, P243G, and E254C; xviii) D205C, F210R, P215V, T235V, and E254C; xix) D205C, F210T, T235D, and E254C; xx) D205C, F210R, T235L, and E254C; xxi) D205C, F210R, T235Y, and E254C; xxii) D205C, F210R, T235F, and E254C; xxiii) D205C, F210R, T235Q, and E254C; xxiv) D205C, F210R, T235K, and E254C; xxv) D205C, F210R, T235I, and E254C; xxvi) D205C, F210I, T235D, and E254C; xxvii) N49P, K73P, L91R, D205C, and E254C; xxviii) K73D, D205C, T235E, P243R, and E254C; xxix) D205C, F210R, N233H, T235D, P243G, and E254C; xxx) D205C, F210R, N233H, T235G, and E254C; xxxi) S24R, N29A, D205C, and E254C;Docket No.: B1704.70002WO00 xxxii) S24L, N29K, D205C, and E254C; xxxiii) L183S, D205C, I218T, and E254C; xxxiv) R47E, E65K, S114N, H185Y, D205C, F210R, T235I, and E254C; xxxv) R47E, S114D, H185Y, D205C, F210R, T235Y, and E254C; xxxvi) R47G, S114G, H185Y, D205C, F210R, T235F, and E254C; xxxvii) R47L, K73Q, S114D, I179V, D205C, F210R, T235Y, and E254C; xxxviii) R47E, V55I, G63S, S114D, H185Y, D205C, F210R, T235Y, and E254C; xxxix) R47G, S114G, H185Y, I179I, D205C, F210R, T235Y, and E254C; xl) R47G, I57V, I83L, S114D, H185Y, D205C, F210R, T235Y, and E254C; xli) R47E, I57V, S114D, H185Y, D205C, F210R, T235Y, and E254C; xlii) R47E, S114D, D175G, L176A, H185Y, D205C, F210R, T235Y, and E254C; xliii) R47G, S114G, N161D, H185Y, D205C, F210R, T235Y, and E254C; xliv) R47E, S114G, D175S, L176A, H185Y, D205C, F210R, T235Y, and E254C; xlv) R47G, S114G, D175G, L176A, H185Y, D205C, F210R, T235Y, and E254C; xlvi) R47I, H185Y, D205C, F210R, T235F, and E254C; xlvii) R47G, S114G, H185Y, L204G, D205C, F210R, T235Y, and E254C; xlviii) R47G, S114G, D175S, H185Y, D205C, F210R, T235Y, and E254C; xlix) R47E, I75L, S114D, H185Y, D205C, F210R, T235Y, and E254C; l) R47E, S114G, N161D, H185Y, D205C, F210R, T235Y, and E254C; li) R47G, S114D, H185Y, D205C, F210R, T235Y, and E254C; lii) R47G, S114D, S123A, H185Y, D205C, F210R, T235Y, and E254C; liii) R47E, S114D, D175R, L176V, H185Y, D205C, F210R, T235Y, and E254C; liv) R47G, S114G, D175R, L176A, D205C, H185Y, F210R, T235Y, and E254C; lv) R47G, S114G, L176R, T178Q, H185Y, D205C, F210R, T235Y, and E254C; lvi) R47G, I75V, S114G, H185Y, D205C, F210R, T235Y, and E254C; lvii) R47G, S114D, D175T, L176V, H185Y, D205C, F210R, T235Y, and E254C; lviii) R47E, I57M, I75V, S114D, H185Y, D205C, F210R, T235Y, and E254C; lix) I75T, S114D, H185Y, D205C, F210R, T235I, and E254C; lx) R47E, S114D, L176R, T178V, H185Y, D205C, F210R, T235Y, and E254C; lxi) R47E, I57V, I75V, S114D, H185Y, D205C, F210R, T235Y, and E254C; lxii) R47G, S114D, D175E, L176A, H185Y, D205C, F210R, T235Y, and E254C; lxiii) R47E, I57V, I75L, S114D, H185Y, D205C, F210R, T235Y, and E254C; lxiv) R47E, S113P, S114D, S123A, H185Y, D205C, F210R, T235Y, and E254C; lxv) R47G, S114G, D175S, L176I, H185Y, D205C, F210R, T235F, and E254C;Docket No.: B1704.70002WO00 lxvi) R47E, S114D, H185Y, N191K, D205C, F210R, T235Y, and E254C; lxvii) R47E, S114D, T178G, H185Y, D205C, F210R, T235Y, and E254C; lxviii) S34E, Y52F, S114D, H185Y, D205C, F210R, T235I, and E254C; lxix) R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; lxx) R47E, S114G, D175I, L176A, H185Y, D205C, F210R, T235Y, and E254C; lxxi) R47G, S114G, D175Q, L176I, H185Y, D205C, F210R, T235Y, and E254C; lxxii) R47E, S114G, D175Q, L176T, H185Y, D205C, F210R, T235Y, and E254C; lxxiii) R47E, A112S, S114G, S123N, H185Y, D205C, F210R, T235Y, and E254C; lxxiv) R47E, S114G, I120V, H185Y, D205C, F210R, T235Y, and E254C; lxxv) R47E, S114D, H185Y, S198A, D205C, F210R, T235Y, and E254C; lxxvi) R47G, S114G, H185Y, T195E, D205C, F210R, T235Y, and E254C; lxxvii) R47E, S114G, H185Y, D205C, G206R, F210R, T235Y, and E254C; lxxviii) R19I, S36R, N50T, Y52F, I57V, I75V, S114D, H185Y, F210R, and T235Y; lxxix) R19L, N50T, Y52F, I75V, S114G, H185Y, F210R, and T235Y; lxxx) R19L, S34E, R47E, N50T, Y52F, I57M, I75T, S114D, H185Y, F210R, and T235Y; lxxxi) S34A, N50T, I57M, I75V, S114D, S142E, H185Y, F210R, and T235Y; lxxxii) S34E, Y52F, I57V, I75V, S114D, H185Y, F210R, and T235Y; lxxxiii) S36N, N50H, Y52F, I57M, S114G, H185Y, F210R, and T235Y; lxxxiv) R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, P245K, R246L, and E254C; lxxxv) R47G, S114D, H185Y, D205C, F210R, T235Y, T238N, and E254C; lxxxvi) R47G, S114G, H185Y, D205C, F210R, T235Y, T238N, and E254C; lxxxvii) R19L, R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; lxxxviii) R19L, R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, P245K, R246L, and E254C; lxxxix) R19L, S34A, S36K, R47E, N50H, Y52F, D175Q, H185Y, F210R, and T235Y; xc) R47E, D175Q, H185Y, D205C, F210R, T235Y, and E254C; xci) R47E, I57V, D175Q, H185Y, D205C, F210R, T235Y, and E254C; xcii) R47E, N50H, Y52F, I57V, S114D, D175Q, H185Y, D205C, F210R, T235F, and E254C; xciii) R47E, S114G, D175Q, H185Y, D205C, F210R, T235Y, and E254C; xciv) S34E, S114D, D175Q, H185Y, D205C, F210R, T235Y, P245K, R246L, and E254C;Docket No.: B1704.70002WO00 xcv) S34E, Y52F, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; xcvi) S34E, Y52F, S114G, D175Q, H185Y, D205C, F210R, T235Y, and E254C; xcvii) T42A, R47E, S114D, D175Q, H185Y, D205C, F210R, T235F, and E254C; xcviii) T42A, R47E, S114D, D175Q, H185Y, D205C, F210R, V224L, T235Y, and E254C; xcix) R19L, T42A, R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; c) R47E, I57V, I75L, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; ci) R47E, Y52F, S114D, D175Q, H185Y, D205C, F210R, L224V, T235Y, and E254C; cii) S34A, S36K, R47E, D175Q, H185Y, D205C, F210R, T235Y, and E254C; ciii) S34A, S36K, R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; civ) S34A, S36K, R47E, S114G, H185Y, D205C, F210R, T235Y, and E254C; cv) S34E, D175Q, H185Y, D205C, F210R, T235Y, and E254C; cvi) S34E, R47E, I75L, S114G, D175Q, H185Y, D205C, F210R, T235Y, and E254C; cvii) S34E, R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; cviii) R47E, S114D, D175A, H185Y, D205C, F210R, T235Y, and E254C; cix) R47E, I57V, I75L, S114D, D175N, H185Y, D205C, F210R, T235Y, and E254C; cx) R47E, I57V, I75L, S114D, H185Y, D205C, T208G, F210R, T235Y, and E254C; cxi) R47E, I57V, I75L, S114D, D175Q, H185Y, D205C, A207V, F210R, T235Y, and E254C; cxii) R47E, I75L, S114D, D175Q, H185Y, D205C, F210R, I214V, T235Y, and E254C; cxiii) R47E, S114D, M128T, D175Q, H185Y, D205C, F210R, T235F, and E254C; cxiv) S34E, Y52F, A104S, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; cxv) R47E, I57L, G63L, A66E, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; cxvi) R47E, Y52F, G63L, A66D, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; cxvii) R47E, I57L, A66G, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; cxviii) R47E, G63L, A66L, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; cxix) R47E, E65C, S67E, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; cxx) R47E, S114G, S163F, D175Q, H185Y, D205C, F210R, T235Y, and E254C; cxxi) R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, E252A, and E254C; cxxii) R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, L241M, and E254C; cxxiii) R47E, S114D, D175Q, H185Y, D205C, T208S, F210R, T235Y, and E254C;Docket No.: B1704.70002WO00 cxxiv) R47E, S114D, T154S, D175Q, T184A, H185Y, D205C, F210R, T235Y, and E254C; cxxv) R47E, I57V, L91I, Q93N, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; cxxvi) R47E, I88H, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; cxxvii) R47E, Q93S, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; cxxviii) N29V, S31Q, S36K, R47E, N49T, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; cxxix) R19V, S20D, T42M, R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; cxxx) R47E, A103Q, N106D, R111Y, S114R, T115P, D175Q, H185Y, D205C, F210R, T235Y, and E254C; cxxxi) R19F, S20D, T42M, R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; cxxxii) R47E, A66E, I75V, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; cxxxiii) R47E, L91I, N106T, S114D, D175Q, T184Q, H185Y, D205C, F210R, K217R, T235Y, and E254C; cxxxiv) S34E, Y52F, G63N, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; cxxxv) R47E, R111E, S114G, D175Q, H185Y, D205C, F210R, T235Y, and E254C; cxxxvi) S31K, R47E, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; cxxxvii) S34E, Y52F, L91V, S113H, S114G, D175Q, H185Y, D205C, F210R, T235Y, and E254C; cxxxviii) R47E, I57L, G63L, A66E, S114D, D175Q, H185Y, D205C, T208G, F210R, T235Y, and E254C; cxxxix) R47E, I57L, G63L, A66E, S114G, D175Q, H185Y, D205C, F210R, T235Y, and E254C; cxl) S34E, G63L, A66E, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; cxli) R47E, I57V, L91I, Q93N, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; cxlii) R47E, S114D, W156F, L158T, D175Q, H185Y, S196T, D205C, F210R, I214M, T235Y, and E254C; cxliii) R47E, S114D, L158I, R166K, D175Q, H185Y, D205C, F210R, T235Y, and E254C;Docket No.: B1704.70002WO00 cxliv) R47E, S114D, L158I, V167I, D175Q, H185Y, N191Q, D205C, F210R, T235Y, and E254C; cxlv) R47E, S114D, D175Q, V182I, H185Y, D205C, F210R, T235Y, and E254C; cxlvi) R47E, S114D, S163A, R166I, D175Q, H185Y, N191A, D205C, F210R, T235Y, and E254C; cxlvii) R47E, S114D, L158T, D175Q, H185Y, S196P, D205C, F210R, T235Y, and E254C; cxlviii) R47E, S114D, S163A, S164T, D175Q, H185Y, D205C, F210R, I214M, T235Y, and E254C; cxlix) R47E, S114D, S164N, D175Q, L183A, H185Y, T195M, D205C, F210R, T235Y, and E254C; cl) R47E, S114D, N159A, D175Q, H185Y, D205C, F210R, T235Y, and E254C; cli) R47E, S114D, D175Q, S198T, D205C, F210R, T235Y, and E254C; clii) R47E, S114D, V165I, D175Q, L183V, H185Y, I197V, S198E, D205C, F210R, T235Y, and E254C; cliii) R47E, S114D, S142E, T154S, R166T, D175Q, H185Y, S198A, D205C, F210R, T235Y, and E254C; cliv) R47E, S114D, Q143K, S163A, S164T, D175Q, H185Y, T195P, D205C, F210R, T235Y, and E254C; clv) R47E, S114D, L158V, S163N, S164T, D175Q, H185Y, S198N, D205C, F210R, N213T, T235Y, and E254C; clvi) R47E, Y52F, I57L, G63C, A66E, I75V, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; clvii) D13A, R47E, N50G, I88N, E99D, S114G, D175Q, H185Y, D205C, F210R, K217P, T235Y, and E254C; clviii) Y52F, I57L, G63C, A66E, I75V, S114D, D175Q, H185Y, D205C, F210R, T235Y, and E254C; clix) R47E, G63L, S114D, R117K, D175Q, L183K, H185Y, D205C, F210R, I214K, T235Y, and E254C; clx) R19K, R47E, G63L, S114D, R166T, D175Q, L183K, H185Y, D205C, F210R, T235Y, and E254C; clxi) R47E, S114D, D175Q, L183I, H185Y, D205C, F210R, T235Y, and E254C; clxii) R47E, S114D, L158N, S163K, D175Q, T184K, H185Y, D205C, F210R, T235Y, and E254C;Docket No.: B1704.70002WO00 clxiii) R47E, S114D, L158T, S163Q, D175Q, T184K, H185Y, D205C, F210R, T235Y, and E254C; clxiv) R47E, Q93S, S114D, D175Q, H185Y, D205C, T208S, F210R, T235Y, and E254C; clxv) S34K, R47E, S114D, L158T, D175Q, H185Y, D205C, F210R, T235Y, and E254C; clxvi) L33K, R47E, S114D, R117K, R166T, D175Q, L183K, H185Y, D205C, F210R, T235Y, and E254C; or clxvii) R47E, S114D, L158T, S163D, D175Q, T184K, H185Y, D205C, F210R, T235Y, and E254C.

26. The hydrolase of any one of claims 1-25, wherein the amino acid sequence of the hydrolase comprises any one of SEQ ID NOs: 5-183.

27. The hydrolase of any one of claims 1-26, wherein the amino acid sequence of the hydrolase comprises any one of SEQ ID NOs: 85, 100, 103, 104, 114-117, 119, 123, 127, 128, 131-135, 137-145, 147-149, 152, 154, 157-166, 168-172, and 175-183.

28. The hydrolase of any one of claims 1-27, wherein the hydrolase exhibits increased depolymerization activity relative to a control hydrolase.

29. The hydrolase of claim 28, 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.

30. The hydrolase of claim 28 or 29, wherein the depolymerization activity is determined at a temperature of more than 50 °C, between about 50 °C and about 80 °C, or about 70 °C.

31. The hydrolase of any one of claims 28-30, wherein the depolymerization activity is determined at a pH of below 7.0, between about 4.5 and about 6.5, or about 5.

0.

32. The hydrolase of any one of claims 28-30, wherein the depolymerization activity is determined at a pH of above 7.0, between about 7.5 and about 8.5, or about 8.0.Docket No.: B1704.70002WO00 33. The hydrolase of any one of claims 28-32, wherein the control hydrolase is a hydrolase that comprises the amino acid sequence of SEQ ID NO:

1.

34. A host cell that comprises a heterologous polynucleotide encoding the hydrolase of any one of claims 1-33.

35. A nucleic acid encoding the hydrolase of any one of claims 1-33.

36. A vector comprising the nucleic acid of claim 35.

37. An expression cassette comprising the nucleic acid of claim 35.

38. A composition comprising: a first hydrolase comprising the hydrolase of any one of claims 1-33; and a second hydrolase.

39. The composition of claim 38, wherein the second hydrolase is a mono(2- hydroxyethyl) terephthalate hydrolase (MHETase).

40. The composition of claim 39, wherein the MHETase is from Ideonella sakaiensis.

41. The composition of any one of claims 38-40, further comprising a third hydrolase.

42. The composition of claim 41, wherein the third hydrolase is a PETase, a polyamidase, a polyurethanase, or a cellulase.

43. A method of degrading an organic polymer, the method comprising: contacting the organic polymer with the hydrolase of any one of claims 1-33 under conditions to degrade the organic polymer.

44. The method of claim 43, wherein the organic polymer comprises a polyether- containing polymer, a polyester-containing polymer, a polypropylene-containing polymer, a polyethylene-containing polymer, or a combination thereof.Docket No.: B1704.70002WO00 45. The method of claim 43 or 44, wherein the organic polymer comprises polyethylene glycol, polyethylene terephthalate, polyester-polyurethane, polytrimethylene terephthalate, polybutylene terephthalate, polyethylene isosorbide terephthalate, polylactic acid, polyhydroxyalkanoate, polybutylene succinate, polybutylene succinate adipate, polybutylene adipate terephthalate, polyethylene furanoate, polycaprolactone, poly(ethylene adipate), polyethylene naphthalate, or a combination thereof.

46. The method of any one of claims 43-45, wherein degradation of the organic polymer by the hydrolase produces one or more degradation products comprising monomers and / or oligomers.

47. The method of claim 46, further comprising: recovering the one or more degradation products.

48. The method of claim 46 or 47, wherein the one or more degradation products are selected from the group consisting of hydrolyzed polyols, hydrolyzed polycarboxylic acids, and hydrolyzed polyamines.

49. The method of any one of claims 46-48, wherein the one or more degradation products comprise one or more hydrolyzed 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.

50. The method of any one of claims 46-49, wherein the one or more degradation products comprise one or more hydrolyzed 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.

51. The method of any one of claims 46-50, wherein the one or more degradation products comprise one or more hydrolyzed polyamines selected from the group consisting of methylene-4,4′-diamine, methylene-2,4′-diamine, methylene-2,2′-diamine, naphthylene-1,4-Docket No.: B1704.70002WO00 diamine, naphthylene-1,5-diamine, naphthylene-1,6-diamine, tolylene-2,4-diamine, and tolylene-2,6-diamine.

52. The method of any one of claims 43-51, wherein the hydrolase is purified.

53. The method of any one of claims 43-51, wherein the organic polymer is contacted with a host cell expressing and secreting the hydrolase.

54. The method of any one of claims 43-53, wherein the conditions comprise an incubation temperature of between about 30 °C and about 80 °C.

55. The method of any one of claims 43-54, wherein the conditions comprise an incubation temperature of about 70 °C.

56. The method of any one of claims 43-55, wherein the conditions comprise a pH of between about 3.5 and about 9.

0.

57. The method of any one of claims 43-56, wherein the conditions comprise a pH of between about 4.5 and about 5.

5.

58. The method of any one of claims 43-56, wherein the conditions comprise a pH of between about 7.5 and about 8.5.

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