WO2024243082A1

WO2024243082A1 - Nylon-degrading enzymes and methods of use

Info

Publication number: WO2024243082A1
Application number: PCT/US2024/030078
Authority: WO
Inventors: Nikita KHLYSTOV
Original assignee: Leland Stanford Junior University
Current assignee: Leland Stanford Junior University
Priority date: 2023-05-19
Filing date: 2024-05-17
Publication date: 2024-11-28
Anticipated expiration: 2025-11-19

Abstract

Provided are methods of degrading nylon. In certain embodiments, the methods comprise contacting a nylon substrate with a PET-degrading enzyme under conditions suitable for degradation of the nylon substrate by the PET-degrading enzyme. The PET-degrading enzyme may be a cutinase or cutinase-like enzyme, e.g., a wild-type or mutant cutinase from Humicola insolens (HiC), a wild-type or mutant leaf compost cutinase (LCC), a polyester hydrolase (PHL7) or the like. According to some embodiments, the PET-degrading enzyme is fused to a nylon-degrading enzyme, e.g., a wild-type or mutant nylB or nylC enzyme. In some instances, the nylon substrate is, or is comprised within, a nylon-containing textile, a net (e.g., a fishing net), or any other nylon-containing material which is desired to be degraded, e.g., for recycling, repurposing, and/or the like. Also provided are the fusion proteins, nucleic acids encoding same, cells comprising such nucleic acids, and methods of making the fusion proteins.

Description

NYLON-DEGRADING ENZYMES AND METHODS OF USE

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application No. 63/467,766, filed May 19, 2023, which application is incorporated herein by reference in its entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A SEQUENCE

LISTING XML FILE

A Sequence Listing is provided herewith as a Sequence Listing XML, “STAN- 2103WO_SEQ_LIST”, created on May 17, 2024, and having a size of 16,1 10 bytes. The contents of the Sequence Listing XML are incorporated herein by reference in their entirety.

INTRODUCTION

Over 360 million tons of plastic waste is generated around the world each year. Over 90% of plastic waste cannot be recycled, resulting in increasing ecological damage as well as human health hazards globally. Existing recycling methods are limited to well-sorted, pure streams of single plastic types, and the resulting recycled material has downgraded performance. Textiles are especially incompatible with current recycling methods due to their physically complex, highly multi-material nature. This means that less than 15% of all textile waste is recycled, with less than 1% going back into textiles.

The name "nylons" refers to the group of plastics known as “polyamides”. Nylons are typified by amide groups (CONH) and encompass a range of material types, e.g., Nylon 6; Nylon 6,6; Nylon 6,12; Nylon 4,6; Nylon 12; etc. Recycling of nylon-based textiles is particularly challenging, where nylon-containing blended textile waste cannot be recycled even using newer advanced chemical recycling methods, which are limited to relatively pure feedstocks containing only polyester and/or cotton.

Enzyme-based recycling technologies show promise in enabling recycling of mixed textile waste and have already been applied for polyester-containing mixed textile waste. However, few naturally occurring nylon-degrading enzymes have been characterized to date and engineering efforts towards enabling large-scale recycling of nylon have been limited, especially compared to polyester-focused efforts, meaning less than 2% of all polyamide textile fibers are presently recycled. SUMMARY

Provided are methods of degrading nylon. In certain embodiments, the methods comprise contacting a nylon substrate with a PET-degrading enzyme under conditions suitable for degradation of the nylon substrate by the PET-degrading enzyme. The PET-degrading enzyme may be a wild-type or mutant cutinase or cutinase-like enzyme, e.g., a wild-type or mutant cutinase from Humicola insolens (HiC), a wild-type or mutant leaf compost cutinase (LCC), a polyester hydrolase (PHL7) or the like. According to some embodiments, the PET-degrading enzyme is fused to a nylon-degrading enzyme. For example, the PET-degrading enzyme may be fused to a wild-type or mutant nylB enzyme. In some instances, the nylon substrate is, or is comprised within, a nylon-containing textile, a net (e.g., a fishing net), or any other nylon- containing material which is desired to be degraded, e.g., for recycling, repurposing, and/or the like. Also provided are the aforementioned fusion proteins, nucleic acids encoding same, cells comprising such nucleic acids, and methods of making the fusion proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1 B: Data demonstrating depolymerization of nylon 6 nanoparticles by cutinases and respective fusion proteins.

FIG. 2A-2B: Data demonstrating depolymerization of a copolymer monofilament consisting of nylon 6 and nylon 6,6 by cutinases and respective fusion proteins.

DETAILED DESCRIPTION

Before the methods and fusion proteins of the present disclosure are described in greater detail, it is to be understood that the methods and fusion proteins are not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the methods and fusion proteins will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the methods and fusion proteins. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the methods and fusion proteins, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the methods and fusion proteins. Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods and fusion proteins belong. Although any methods and fusion proteins similar or equivalent to those described herein can also be used in the practice or testing of the methods and fusion proteins, representative illustrative methods and fusion proteins are now described.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the materials and/or methods in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present methods and fusion proteins are not entitled to antedate such publication, as the date of publication provided may be different from the actual publication date which may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

It is appreciated that certain features of the methods and fusion proteins, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the methods and fusion proteins, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace operable processes and/or compositions. In addition, all sub-combinations listed in the embodiments describing such variables are also specifically embraced by the present methods and fusion proteins and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present methods. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

Herein, the terms “peptide”, “polypeptide”, “protein”, and “enzyme” refer to a chain of amino acids linked by peptide bonds, regardless of the number of amino acids forming said chain. The amino acids are herein represented by their one-letter or three-letters code according to the following nomenclature: A: alanine (Ala); C: cysteine (Cys); D: aspartic acid (Asp); E: glutamic acid (Glu); F: phenylalanine (Phe); G: glycine (Gly); H: histidine (His); I: isoleucine (He); K: lysine (Lys); L: leucine (Leu); M: methionine (Met); N: asparagine (Asn); P: proline (Pro); Q: glutamine (Gin); R: arginine (Arg); S: serine (Ser); T: threonine (Thr); V: valine (Vai); W: tryptophan (Trp) and Y: tyrosine (Tyr).

The term “wild-type” refers to the non-mutated version of a polypeptide as it appears naturally. The terms “mutant” and “variant” refer to polypeptides comprising at least one modification or alteration, i.e., a substitution, insertion, and/or deletion, at one or more (e.g., several) positions and having a nylon-degrading activity. The variants may be obtained by various techniques. In particular, examples of techniques for altering the DNA sequence encoding the wild-type protein, include, but are not limited to, site-directed mutagenesis, random mutagenesis and synthetic oligonucleotide construction. Thus, the terms “modification” and “alteration” as used herein in relation to a particular position means that the amino acid in this particular position has been modified compared to the amino acid in this particular position in the wild-type protein.

A “substitution” means that an amino acid residue is replaced by another amino acid residue. In some embodiments, the term “substitution” refers to the replacement of an amino acid residue by another selected from the naturally-occurring standard 20 amino acid residues (G, P, A, V, L, I, M, C, F, Y, W, H, K, R, Q, N, E, D, S and T).

As used herein, the term “sequence identity” or “identity” refers to the number (or fraction expressed as a percentage %) of matches (identical amino acid residues) between two polypeptide sequences. The sequence identity is determined by comparing the sequences when aligned so as to maximize overlap and identity while minimizing sequence gaps. In particular, sequence identity may be determined using any of a number of mathematical global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithm (e.g., Needleman and Wunsch algorithm; Needleman and Wunsch, 1970) which aligns the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g. Smith and Waterman algorithm (Smith and Waterman, 1981 ) or Altschul algorithm (Altschul et al., 1997; Altschul et al., 2005)). Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software available on internet web sites such as http://blast.ncbi.nlm.nih.gov/ or http://www.ebi.ac.uk/Tools/emboss/). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, % amino acid sequence identity values refers to values generated using the pair wise sequence alignment program EMBOSS Needle that creates an optimal global alignment of two sequences using the Needleman-Wunsch algorithm, wherein all search parameters are set to default values, i.e., Scoring matrix = BLOSUM62, Gap open = 10, Gap extend = 0.5, End gap penalty = false, End gap open = 10 and End gap extend = 0.5.

METHODS OF DEGRADING NYLON

Aspects of the present disclosure include methods of degrading nylon. In certain embodiments, the methods comprise contacting a nylon substrate with a polyethylene terephthalate (PET)-degrading enzyme under conditions suitable for degradation of the nylon substrate by the PET-degrading enzyme. As used herein, a “nylon substrate”, “nylon” or “nylons” refer(s) to the group of plastics known as “polyamides”. Nylons are typified by amide groups (CONH) and encompass a range of material types, e.g., Nylon 6; Nylon 6,6; Nylon 6,12; Nylon 4,6; Nylon 12; etc. According to the methods of the present disclosure, a “nylon substrate” may be comprised within a nylon-containing material, e.g., a nylon-containing textile, a nylon- containing net (e.g., a fishing net), a nylon-containing rope, nylon-containing carpet, a nylon- containing umbrella, a nylon-containing conveyor belt, a nylon-containing seat belt, a nylon- containing machine part, or the like. For example, the nylon substrate may be comprised within a nylon-containing textile.

In certain embodiments, the PET-degrading enzyme is a cutinase or cutinase-like enzyme. The term “esterase” refers to an enzyme which belongs to a class of hydrolases classified as EC 3.1 .1 according to Enzyme Nomenclature that catalyzes the hydrolysis of esters into an acid and an alcohol. The term “cutinase” refers to the esterases classified as EC 3.1 .1 .74 according to Enzyme Nomenclature that are able to catalyze the chemical reaction of production of cutin monomers from cutin and water. As used herein, a “cutinase-like enzyme” refers to any enzyme having properties similar to a lipase, an esterase, a serine protease, and/or a cutinase, including, but not limited to, sequence similarity, structural homology, a conserved GXSXG pentapeptide motif, a serine-histidine-aspartate catalytic active site, and/or ability to catalyze hydrolysis of ester- and amide-containing molecules.

According to some embodiments, the cutinase or cutinase-like enzyme is a Humicola insolens cutinase (HiC). The cutinase or cutinase-like enzyme may be a wild-type HiC. As used herein, the terms “wild-type” or “parent” refer to the non-mutated version of a polypeptide as it appears naturally. In other embodiments, the cutinase orcutinase-like enzyme is a mutant HiC. For example, a mutant HiC may comprise one or more amino acid substitutions relative to the amino acid sequence set forth in SEQ ID NO: 1. Non-limiting examples of such amino acid substitutions include those at position 1 , 3, 4, 1 1 , 15, 29, 37, 47, 164, 166, or any combination thereof, wherein numbering of positions is according to SEQ ID NO: 1 . According to some embodiments, the mutant HiC comprises the amino acid substitution Q1 A, Q1 C, Q1 D, Q1 E, Q1 F, Q1 G, Q1 H, Q1 1, Q1K, Q1 L, Q1 M, Q1 N, Q1 P, Q1 R, Q1 S, Q1T, Q1 V, Q1 Y, G3A, G3C, G3D, G3E, G3F, G3H, G3I, G3L, G3N, G3Q, G3R, G3S, G3T, G3V, G3Y, A4V, S11 N, N15D, T29K, T37A, T37I, T37L, T37M, T37N, T37P, T37S, T37V, E47A, E47C, E47F, E47G, E47H, E47I, E47L, E47M, E47N, E47Q, E47R, E47S, E47T, E47V, E47Y, T164S, T166A, or any combination thereof at different positions. In some instances, the mutant HiC comprises an amino acid substitution chosen from: Q1 R, G3S, T29K, T37L, E47Q, and any combination thereof. In certain embodiments, the mutant HiC comprises the amino acid substitutions Q1 R, G3S, T29K, and E47Q. In other embodiments, the mutant HiC comprises the amino acid substitutions Q1 R, G3T, T37L, and E47R.

In some instances, the PET-degrading enzyme is a cutinase or cutinase-like enzyme comprising an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% identity to the amino acid sequence set forth in SEQ ID NO:1 , SEQ ID NO:2, or SEQ ID NO:3, or a nylon-degrading enzyme fragment thereof.

According to some embodiments, the cutinase or cutinase-like enzyme is a leaf compost cutinase (LCC). The LCC may be a wild-type LCC or a mutant LCC. When the LCC is a mutant LCC, in certain embodiments, the mutant LCC comprises one or more amino acid substitutions relative to the amino acid sequence set forth in SEQ ID NO: 4, wherein numbering of positions is according to SEQ ID NO: 4. By way of example, the mutant LCC may comprise an amino acid substitution at position 73, 93, 166, 204, 209, 229, 249, or any combination thereof. In particular embodiments, the mutant LCC comprises an amino acid substitution chosen from: R73L, Y93G, V166A, D204C, F209I, W229R, S249C, and any combination thereof. When the cutinase or cutinase-like enzyme is an LCC, in some instances, the LCC comprises an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% identity to the amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:5, or a nylon-degrading enzyme fragment thereof.

In certain embodiments, the PET-degrading enzyme is a polyester hydrolase Leipzig 7 (PHL7) enzyme. Non-limiting examples of PHL7 enzymes that find use in the methods of the present disclosure include those comprising an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% identity to the amino acid sequence set forth in SEQ ID NO:6, or a nylon-degrading enzyme fragment thereof.

According to any of the embodiments of the present disclosure, the methods may employ a PET-degrading enzyme fused to a heterologous protein domain. The term “heterologous” refers to two components that are defined by structures that can be derived from different sources. For example, where “heterologous” is used in the context of a polypeptide, the polypeptide includes operably linked amino acid sequences that can be derived from polypeptides having different amino acid sequences (e.g., a first amino acid sequence from a first polypeptide and a second amino acid sequence from a second polypeptide). Similarly, “heterologous” in the context of a polynucleotide encoding a fusion polypeptide includes operably linked nucleic acid sequences that can be derived from different genes (e.g., a first component from a nucleic acid encoding a first portion of a polypeptide according to an embodiment disclosed herein and a second component from a nucleic acid encoding a second portion of a polypeptide disclosed herein).

In some instances, when the methods employ a PET-degrading enzyme fused to a heterologous protein domain, the heterologous protein domain is an enzyme. For example, the PET-degrading enzyme may be fused to a nylon-degrading enzyme. Non-limiting examples of nylon-degrading enzymes include a nylB enzyme (6-aminohexanoate-dimer hydrolase, EC 3.5.1.46) and a nylC enzyme (6-aminohexanoate-oligomer endo-hydrolase, EC3.5.1 .117). NylB hydrolyzes aminohexanoate (Ahx) oligomers by an exo-type mode, while NylC degrades Ahx- cyclic and -linear oligomers with a degree of polymerization greater than three by an endo-type mode.

Accordingly, in certain embodiments, the PET-degrading enzyme is fused to a nylB enzyme. Examples of suitable nylB enzymes include those comprising an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% identity to the amino acid sequence set forth in SEQ ID NO:7, or a nylon-degrading enzyme fragment thereof.

In other embodiments, the PET-degrading enzyme is fused to a nylC enzyme. In some instances, the nylC enzyme is a nylCk enzyme. Examples of nylCk enzymes include those comprising an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% identity to the amino acid sequence set forth in SEQ ID NO:8, or a nylondegrading enzyme fragment thereof.

According to some embodiments, when the PET-degrading enzyme is fused to a nylB enzyme or a nylC enzyme (e.g., a nylCk enzyme), the resulting fusion protein comprises an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% identity to the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 , or SEQ ID NO:12.

As summarized above, the methods of degrading nylon of the present disclosure comprise contacting a nylon substrate with the PET-degrading enzyme (optionally part of a fusion protein as described above) under conditions suitable for degradation of the nylon substrate by the PET-degrading enzyme. Nylons are synthetic polymers that contain recurring amide groups (R-CO-NH-R') as integral parts of their main polymer chains. The high strength, elasticity, abrasion resistance, chemical resistance, and shape-holding characteristics of nylons over wide temperature ranges make these polymers suitable for the production of fibers and plastics. Currently, the worldwide production of nylons is estimated to be three to four million tons per year. Nylons tend to be partially crystalline, and the degree of crystallinity affects the properties of nylons, such as the melting points, strength, and rigidity. Two forms of crystals (a and y) have been reported. In the a-form, each polymer chain is stabilized by hydrogen bonds with adjacent chains aligned in an antiparallel orientation, whereas the chains are parallel in the y-form. The a- form of nylon-6 is generally more stable. Nylon-6 is produced by the ring cleavage polymerization of e-caprolactam and consists of more than 100 units of 6-aminohexanoate (Ahx).

The methods comprise contacting the nylon substrate with the PET-degrading enzyme under conditions suitable for degradation of the nylon substrate by the PET-degrading enzyme. The contacting of the nylon substrate with the PET-degrading enzyme may include directly contacting the enzyme to the nylon, contacting the nylon with cells that express, secrete, display, or export the PET-degrading enzyme, or containing the nylon with a cell extract thereof.

The conditions suitable for degradation of the nylon can include a temperature between 0°C and 90°C, preferably between 20°C and 75°C, or more preferably between 30°C and 68°C. In a particular embodiment, the degrading process is implemented at 37°C. In another particular embodiment, the degrading process is implemented at 30°C. In another particular embodiment, the degrading process is implemented at 60°C.

More generally, the temperature is maintained below an inactivating temperature corresponding to the temperature at which the PET-degrading enzyme or fusion protein is inactivated, for example at which the PET-degrading enzyme or fusion protein has lost more than 80% of activity as compared to its activity at its optimum temperature and/or the recombinant organism is unable to express, produce, secrete, display, and/or export the PET-degrading enzyme or fusion protein. Preferably, the temperature is maintained below the crystallization temperature (Tc) of the targeted nylon, more preferably below the onset of crystallization of the targeted nylon, and more preferably below but near the glass transition temperature (Tg) of the targeted nylon.

The time required for degrading a nylon-containing material may vary depending on the nylon -containing material itself, for example the nature and origin of the nylon-containing material, its composition, and shape; the type and amount of PET-degrading enzyme or fusion protein used; and process parameters and conditions such as temperature, pH, applied forces, and additional agents. The process parameters may be adapted to the nylon-containing material and the envisioned degradation time.

In certain embodiments, the method is implemented in a continuous flow process, at a temperature at which the enzyme, fusion protein, or cells producing the enzyme or fusion protein can be used several times and/or recycled. In certain embodiments, the enzyme or fusion protein is immobilized on or bound to synthetic supports, for example, glass, plastic, polymers, filter, membranes, in the form of, for example, substrates, surfaces, particles, beads, columns, and/or plates. In certain embodiments, enzyme immobilization is achieved by chemical modification such as chemical crosslinking, non-covalent bonding or interaction(s), and/or physical adhesion and/or other interaction(s).

In certain embodiments, the method is implemented at a pH comprised between 5 and 11 , e.g., at a pH between 6 and 9, at a pH between 6.5 and 9, or at a pH between 6.5 and 8.

In certain embodiments, the nylon-containing material, for example, a nylon-containing textile, molded product, net, or the like, may be pretreated prior to contacting with the enzyme or fusion protein, in order to physically change its structure, so as to increase the surface of contact between the nylon and the enzyme or fusion protein, or to increase the activity of the enzyme or fusion protein acting on the nylon.

The methods of the present disclosure find use in degrading a wide variety of nylon substrates. In certain embodiments, the nylon substrate is a nylon fiber. In other embodiments, the nylon substrate is a molded nylon product. Non-limiting examples of nylon substrates which may be degraded according to the methods of the present disclosure include those in which the nylon substrate is, or is comprised within, a textile, a net (e.g., a fishing net), a rope, carpet, an umbrella, a conveyor belt, a seat belt, or a machine part. For example, the nylon substrate may be comprised within a nylon-containing textile. Nylon-containing textiles include, but are not limited to, tights, stockings, sportswear, yoga pants, and the like. Also by way of example, in some embodiments, the nylon substrate is, or is comprised within, a net.

In certain embodiments, the method is employed for surface hydrolysis or surface functionalization of a nylon-containing material, comprising exposing a nylon-containing material to an enzyme or fusion protein described herein, or corresponding recombinant cell or extract thereof, or composition.

Methods of Producing 6-Aminohexanoic Acid

In certain embodiments, monomers and/or oligomers are produced from a nylon- containing material, comprising of exposing a nylon-containing material to the enzyme or fusion protein, or corresponding cell or extract thereof, or composition, and optionally recovering monomers and/or oligomers. A single type of monomer and/or oligomer or several different types of monomers and/or oligomers may be recovered, depending on the starting nylon-containing material. The method is particularly useful for producing monomers such as 6-aminohexanoic acid. Accordingly, aspects of the present disclosure further include methods of producing 6- aminohexanoic acid. In some instances, the methods comprise performing any of the methods of degrading nylon of the present disclosure.

The present methods of producing 6-aminohexanoic acid may further comprise harvesting the produced 6-aminohexanoic acid. The harvested/recovered 6-aminohexanoic acid may be further purified (isolated), using any suitable purifying method and conditioned in a re- polymerizable form. Examples of purifying methods include stripping process, separation by aqueous solution, steam selective condensation, filtration and concentration of the medium after the bioprocess, separation, distillation, vacuum evaporation, extraction, electrodialysis, adsorption, ion exchange, precipitation, crystallization, concentration and acid addition dehydration and precipitation, nanofiltration, acid catalyst treatment, semi-continuous mode distillation or continuous-mode distillation, solvent extraction, evaporative concentration, evaporative crystallization, liquid/liquid extraction, hydrogenation, azeotropic distillation process, adsorption, column chromatography, simple vacuum distillation and microfiltration, combined or not.

Recovered 6-aminohexanoic acid may be reused for instance to synthesize nylon. In some embodiments, the recovered 6-aminohexanoic acid monomers and/or oligomers are mixed with other monomers and/or oligomers, for example to synthesize new copolymers. Alternatively, the recovered 6-aminohexanoic acid monomers may be used as chemical intermediates to produce other chemical compounds of interest.

In some instances, the methods of producing 6-aminohexanoic acid further comprise producing nylon from the produced 6-aminohexanoic acid. Such methods may further comprise producing a nylon-containing product with the produced nylon. Non-limiting examples of nylon- containing products that may be produced according to the present methods include a textile, a net (e.g., a fishing net), a rope, carpet, an umbrella, a conveyor belt, a seat belt, or a machine part.

FUSION PROTEINS

Aspects of the present disclosure further include fusion proteins comprising a PET- degrading enzyme fused to a nylon-degrading enzyme.

According to some embodiments, the PET-degrading enzyme of a fusion protein of the present disclosure is cutinase or cutinase-like enzyme. In some instances, the cutinase or cutinase-like enzyme is HiC. The cutinase or cutinase-like enzyme may be a wild-type HiC or a mutant HiC. When the PET-degrading enzyme of a fusion protein of the present disclosure is mutant HiC, the mutant HiC may comprise one or more amino acid substitutions relative to the amino acid sequence set forth in SEQ ID NO: 1 . Non-limiting examples of such amino acid substitutions include those at position 1 , 3, 4, 1 1 , 15, 29, 37, 47, 164, 166, or any combination thereof, wherein numbering of positions is according to SEQ ID NO: 1 . According to some embodiments, the mutant HiC comprises the amino acid substitution Q1 A, Q1 C, Q1 D, Q1 E, Q1 F, Q1 G, Q1 H, Q1 1, Q1K, Q1 L, Q1 M, Q1 N, Q1 P, Q1 R, Q1 S, Q1T, Q1 V, Q1 Y, G3A, G3C, G3D, G3E, G3F, G3H, G3I, G3L, G3N, G3Q, G3R, G3S, G3T, G3V, G3Y, A4V, S11 N, N15D, T29K, T37A, T37I, T37L, T37M, T37N, T37P, T37S, T37V, E47A, E47C, E47F, E47G, E47H, E47I, E47L, E47M, E47N, E47Q, E47R, E47S, E47T, E47V, E47Y, T164S, T166A, or any combination thereof at different positions. In some instances, the mutant HiC comprises an amino acid substitution chosen from: Q1 R, G3S, T29K, T37L, E47Q, and any combination thereof. In certain embodiments, the mutant HiC comprises the amino acid substitutions Q1 R, G3S, T29K, and E47Q. In other embodiments, the mutant HiC comprises the amino acid substitutions Q1 R, G3T, T37L, and E47R.

In some instances, when the PET-degrading enzyme of a fusion protein of the present disclosure is a cutinase or cutinase-like enzyme, the cutinase or cutinase-like enzyme comprises an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% identity to the amino acid sequence set forth in SEQ ID NO:1 , SEQ ID NO:2, or SEQ ID NO:3, or a nylon-degrading enzyme fragment thereof.

According to some embodiments, when the PET-degrading enzyme of a fusion protein of the present disclosure is a cutinase or cutinase-like enzyme, the cutinase or cutinase-like enzyme is a leaf compost cutinase (LCC). The LCC may be a wild-type LCC or a mutant LCC. When the LCC is a mutant LCC, in certain embodiments, the mutant LCC comprises one or more amino acid substitutions relative to the amino acid sequence set forth in SEQ ID NO: 4, wherein numbering of positions is according to SEQ ID NO: 4. By way of example, the mutant LCC may comprise an amino acid substitution at position 73, 93, 166, 204, 209, 229, 249, or any combination thereof. In particular embodiments, the mutant LCC comprises an amino acid substitution chosen from: R73L, Y93G, V166A, D204C, F209I, W229R, S249C, and any combination thereof. When the PET-degrading enzyme of a fusion protein of the present disclosure is an LCC, in some instances, the LCC comprises an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% identity to the amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:5, or a nylon-degrading enzyme fragment thereof.

In certain embodiments, the PET-degrading enzyme of a fusion protein of the present disclosure is a polyester hydrolase Leipzig 7 (PHL7) enzyme. Non-limiting examples of PHL7 enzymes that may be present in the fusion proteins of the present disclosure include those comprising an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% identity to the amino acid sequence set forth in SEQ ID NO:6, or a nylondegrading enzyme fragment thereof.

In some instances, the nylon-degrading enzyme to which the PET-degrading enzyme is fused is a nylB enzyme or a nylC enzyme, e.g., a nylCk enzyme.

Accordingly, in certain embodiments, a fusion protein of the present disclosure comprises the PET-degrading enzyme fused to a nylB enzyme. In some embodiments, the nylB enzyme is one comprising an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% identity to the amino acid sequence set forth in SEQ ID NOT, or a nylondegrading enzyme fragment thereof.

In some instances, the fusion protein comprises, from N-terminus to C-terminus, the nylB enzyme and the PET-degrading enzyme. In other instances, the fusion protein comprises, from N-terminus to C-terminus, the PET-degrading enzyme and the nylB enzyme. The nylB enzyme and the PET-degrading enzyme may be fused directly to each other. In other embodiments, the nylB enzyme and the PET-degrading enzyme are fused to each other indirectly via a linker. In some embodiments, the linker is a flexible linker. An example of a flexible linker that may be employed in a fusion protein of the present disclosure is a glycine-serine (GS) linker, a nonlimiting example of which is a linker comprising, consisting essentially of, or consisting of, the amino acid sequence GGGSGGSGGGSG (SEQ ID NO:13).

In other embodiments, a fusion protein of the present disclosure comprises the PET- degrading enzyme fused to a nylC enzyme. In some instances, the nylC enzyme is a nylCk enzyme. Examples of nylCk enzymes to which the PET-degrading enzyme may be fused include those comprising an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% identity to the amino acid sequence set forth in SEQ ID NO:8, or a nylon-degrading enzyme fragment thereof.

In some instances, the fusion protein comprises, from N-terminus to C-terminus, the nylC (e.g., nylCk) enzyme and the PET-degrading enzyme. In other instances, the fusion protein comprises, from N-terminus to C-terminus, the PET-degrading enzyme and the nylC (e.g., nylCk) enzyme. The nylC (e.g., nylCk) enzyme and the PET-degrading enzyme may be fused directly to each other. In other embodiments, the nylC (e.g., nylCk) enzyme and the PET-degrading enzyme are fused to each other indirectly via a linker. In some embodiments, the linker is a flexible linker. An example of a flexible linker that may be employed in a fusion protein of the present disclosure is a glycine-serine (GS) linker, a non-limiting example of which is a linker comprising, consisting essentially of, or consisting of, the amino acid sequence GGGSGGSGGGSG (SEQ ID NO:13).

According to some embodiments, when a fusion protein comprises a PET-degrading enzyme fused to a nylB enzyme or a nylC enzyme (e.g., a nylCk enzyme), the fusion protein comprises an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% identity to the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:1 1 , or SEQ ID NO:12.

The amino acid sequences of exemplary PET-degrading enzymes, nylon-degrading enzymes and fusion proteins of the present disclosure are provided below.

Amino Acid Sequences

SEQ ID NO: 1 : HiC, cutinase from Humicola insolens (HiC) - mature enzyme (no signal peptide)

QLGAIENGLESGSANACPDAILIFARGSTEPGNMGITVGPALANGLESHIRNIWIQGVGGPYDA ALATNFLPRGTSQANIDEGKRLFALANQKCPNTPVVAGGYSQGAALIAAAVSELSGAVKEQVK GVALFGYTQNLQNRGGIPNYPRERTKVFCNVGDAVCTGTLIITPAHLSYTIEARGEAARFLRDRI RALEHHHHHH

SEQ ID NO: 2: HiC mutant 1 , cutinase from Humicola insolens (HiC) containing mutations Q1R, G3S, T29K, E47Q (mutations shown in bold and underlined) RLSAIENGLESGSANACPDAILIFARGSKEPGNMGITVGPALANGLQSHIRNIWIQGVGGPYDA ALATNFLPRGTSQANIDEGKRLFALANQKCPNTPVVAGGYSQGAALIAAAVSELSGAVKEQVK GVALFGYTQNLQNRGGIPNYPRERTKVFCNVGDAVCTGTLIITPAHLSYTIEARGEAARFLRDRI RALEHHHHHH

SEQ ID NO: 3: HiC mutant 2, cutinase from Humicola insolens (HiC) containing mutations Q1R, G3T, T37L, E47R (mutations shown in bold and underlined)

RLTAIENGLESGSANACPDAILIFARGSTEPGNMGILVGPATANGLRSHIRNIWIQGVGGPYDAA LATNFLPRGTSQANIDEGKRLFALANQKCPNTPVVAGGYSQGAALIAAAVSELSGAVKEQVKG VALFGYTQNLQNRGGIPNYPRERTKVFCNVGDAVCTGTLIITPAHLSYTIEARGEAARFLRDRIR ALEHHHHHH

SEQ ID NO: 4: LCC, leaf compost cutinase - mature enzyme (no signal peptide)

MSNPYQRGPNPTRSALTADGPFSVATYTVSRLSVSGFGGGVIYYPTGTSLTFGGIAMSPGYTA DASSLAWLGRRLASHGFVVLVINTNSRFDYPDSRASQLSAALNYLRTSSPSAVRARLDANRLA VAGHSMGGGGTLRIAEQNPSLKAAVPLTPWHTDKTFNTSVPVLIVGAEADTVAPVSQHAIPFY QNLPSTTPKVYVELDNASHFAPNSNNAAISVYTISWMKLWVDNDTRYRQFLCNVNDPALSDFR TNNRHCQLEHHHHHH

SEQ ID NO: 5: LCC(ICCG), leaf compost cutinase containing mutations Y93G, D204C, F209I, S249C (mutations shown in bold and underlined) MSNPYQRGPNPTRSALTADGPFSVATYTVSRLSVSGFGGGVIYYPTGTSLTFGGIAMSPGYTA DASSLAWLGRRLASHGFVVLVINTNSRFDGPDSRASQLSAALNYLRTSSPSAVRARLDANRLA VAGHSMGGGGTLRIAEQNPSLKAAVPLTPWHTDKTFNTSVPVLIVGAEADTVAPVSQHAIPFY QNLPSTTPKVYVELCNASHIAPNSNNAAISVYTISWMKLWVDNDTRYRQFLCNVNDPALCDFR TNNRHCQLEHHHHHH

SEQ ID NO: 6: PHL7, polyester hydrolase Leipzig 7

MANPYERGPDPTESSIEAVRGPFAVAQTTVSRLQADGFGGGTIYYPTDTSQGTFGAVAISPGF TAGQESIAWLGPRIASQGFVVITIDTITRLDQPDSRGRQLQAALDHLRTNSVVRNRIDPNRMAV MGHSMGGGGALSAAANNTSLEAAIPLQGWHTRKNWSSVRTPTLVVGAQLDTIAPVSSHSEAF YNSLPSDLDKAYMELRGASHLVSNTPDTTTAKYSIAWLKRFVDDDLRYEQFLCPAPDDFAISEY RSTCPFLEHHHHHH

SEQ ID NO: 7: nylB, 6-aminohexanoate-dimer hydrolase

MNARSTGQHPARYPGAAAGEPTLDSWQEAPHNRWAFARLGELLPTAAVSRRDPATPAEPVV RLDALATRLPDLEQRLEETCTDAFLVLRGSEVLAEYYRAGFAPDDRHLLMSVSKSLCGTVVGA LIDEGRIDPAQPVTEYVPELAGSVYDGPSVLQVLDMQISIDYNEDYVDPASEVQTHDRSAGWR TRRDG DPADTYE FLTTLRG DGGTGE FQYCSANTD VLAWI VE RVTGLRYVE ALSTYLWAKLDA DRDATITVDQTGFGFANGGVSCTARDLARVGRMMLDGGVAPGGRVVSQGWVESVLAGGSR EAMTDEGFTSAFPEGSYTRQWWCTGNERGNVSGIGIHGQNLWLDPRTDSVIVKLSSWPDPD TRHWHGLQSGILLDVSRALDAVLEHHHHHH

SEQ ID NO: 8: nylCk, 6-aminohexanoate-oligomer hydrolase from Kocuria sp. strain KY2 MNTTPVHALTDIDGGIAVDPAPRLAGPPVFGGPGNAAFDLVPVRSTGRETLRFDFPGVSVGSA HYEEGPTGATVIHIPAGARTAVDARGGAVGLSGGYDFNHAICLAGGASYGLEAGAGVSGALLE RLEYRTGFAEAQLVSSAVIYDFSARSTAVYPDKALGRAALEFAVPGEFPQGRAGAGMSASAG KVDWDRTEITGQGAAFRRLGDVRILAVVVPNPVGVIMDRAGGIVRGNYDAQTGVRRHPVFDY QEAFAEQLPPVTQAGNTTISAIVTNVRMSPVELNQFAKQVHSSMHRGIQPFHTDMDGDTLFAV TTDEIDLPTTPGSSRGRLSVNATALGAIASEVMWDAVLEAAKLEHHHHHH

SEQ ID NO: 9: nylB-HiC. fusion of nylB and wildtype HiC via a 12-aa flexible linker (italicized) (HiC underlined)

MNARSTGQHPARYPGAAAGEPTLDSWQEAPHNRWAFARLGELLPTAAVSRRDPATPAEPVV RLDALATRLPDLEQRLEETCTDAFLVLRGSEVLAEYYRAGFAPDDRHLLMSVSKSLCGTVVGA LIDEGRIDPAQPVTEYVPELAGSVYDGPSVLQVLDMQISIDYNEDYVDPASEVQTHDRSAGWR TRRDG DPADTYE FLTTLRG DGGTGE FQYCSANTD VLAWI VE RVTGLRYVE ALSTYLWAKLDA DRDATITVDQTGFGFANGGVSCTARDLARVGRMMLDGGVAPGGRVVSQGWVESVLAGGSR EAMTDEGFTSAFPEGSYTRQWWCTGNERGNVSGIGIHGQNLWLDPRTDSVIVKLSSWPDPD TRHWHGLQSGILLDVSRALDAVGGGSGGSGGGSGQLGAIENGLESGSANACPDAILIFARGST EPGNMGITVGPALANGLESHIRNIWIQGVGGPYDAALATNFLPRGTSQANIDEGKRLFALANQK

CPNTPVVAGGYSQGAALIAAAVSELSGAVKEQVKGVALFGYTQNLQNRGGIPNYPRERTKVF CNVGDAVCTGTLIITPAHLSYTIEARGEAARFLRDRIRAL E H H H H H H

SEQ ID NO: 10: nylB-LCC(ICCG), fusion of nylB and leaf compost cutinase containing mutations Y93G, D204C, F209I, S249C via a 12-aa flexible linker (italicized) (LCC(ICCG) underlined and mutations shown in bold and italicized)

MNARSTGQHPARYPGAAAGEPTLDSWQEAPHNRWAFARLGELLPTAAVSRRDPATPAEPVV

RLDALATRLPDLEQRLEETCTDAFLVLRGSEVLAEYYRAGFAPDDRHLLMSVSKSLCGTVVGA LIDEGRIDPAQPVTEYVPELAGSVYDGPSVLQVLDMQISIDYNEDYVDPASEVQTHDRSAGWR TRRDG DPADTYE FLTTLRG DGGTGE FQYCSANTD VLAWI VE RVTGLRYVE ALSTYLWAKLDA DRDATITVDQTGFGFANGGVSCTARDLARVGRMMLDGGVAPGGRVVSQGWVESVLAGGSR

EAMTDEGFTSAFPEGSYTRQWWCTGNERGNVSGIGIHGQNLWLDPRTDSVIVKLSSWPDPD TRHWHGLQSGILLDVSRALDAVGGGSGGSGGGSGSNPYQRGPNPTRSALTADGPFSVATYT

VSRLSVSGFGGGVIYYPTGTSLTFGGIAMSPGYTADASSLAWLGRRLASHGFVVLVINTNSRF

DGPDSRASQLSAALNYLRTSSPSAVRARLDANRLAVAGHSMGGGGTLRIAEQNPSLKAAVPL

TPWHTDKTFNTSVPVLIVGAEADTVAPVSQHAIPFYQNLPSTTPKVYVELCNASH/APNSNNAAI

SVYTISWMKLWVDNDTRYRQFLCNVNDPALCDFRTNNRHCQ LEHHHHHH

SEQ ID NO: 11 : nylB-PHL7, fusion of nylB and polyester hydrolase Leipzig 7 via a 12-aa flexible linker (italicized) (PHL7 underlined) MNARSTGQHPARYPGAAAGEPTLDSWQEAPHNRWAFARLGELLPTAAVSRRDPATPAEPVV RLDALATRLPDLEQRLEETCTDAFLVLRGSEVLAEYYRAGFAPDDRHLLMSVSKSLCGTVVGA

LIDEGRIDPAQPVTEYVPELAGSVYDGPSVLQVLDMQISIDYNEDYVDPASEVQTHDRSAGWR TRRDGDPADTYEFLTTLRGDGGTGEFQYCSANTDVLAWIVERVTGLRYVEALSTYLWAKLDA DRDATITVDQTGFGFANGGVSCTARDLARVGRMMLDGGVAPGGRVVSQGWVESVLAGGSR EAMTDEGFTSAFPEGSYTRQWWCTGNERGNVSGIGIHGQNLWLDPRTDSVIVKLSSWPDPD TRHWHGLQSGILLDVSRALDAVGGGSGGSGGGSGNPYERGPDPTESSIEAVRGPFAVAQTT VSRLQADGFGGGTIYYPTDTSQGTFGAVAISPGFTAGQESIAWLGPRIASQGFVVITIDTITRLD QPDSRGRQLQAALDHLRTNSVVRNRIDPNRMAVMGHSMGGGGALSAAANNTSLEAAIPLQG WHTRKNWSSVRTPTLVVGAQLDTIAPVSSHSEAFYNSLPSDLDKAYMELRGASHLVSNTPDTT TAKYSIAWLKRFVDDDLRYEQFLCPAPDDFAISEYRSTCPF LEHHHHHH

SEQ ID NO: 12: nylB-LCC(ICCG) mut. 1, fusion of nylB and leaf compost cutinase containing mutations R73L, Y93G, V166A, D204C, F209I, W229R, S249C via a 12-aa flexible linker (italicized) (LCC(ICCG) underlined and mutations shown in bold and italicized)

MNARSTGQHPARYPGAAAGEPTLDSWQEAPHNRWAFARLGELLPTAAVSRRDPATPAEPVVR LDALATRLPDLEQRLEETCTDAFLVLRGSEVLAEYYRAGFAPDDRHLLMSVSKSLCGTVVGALI DEGRIDPAQPVTEYVPELAGSVYDGPSVLQVLDMQISIDYNEDYVDPASEVQTHDRSAGWRTR

RDGDPADTYEFLTTLRGDGGTGEFQYCSANTDVLAWIVERVTGLRYVEALSTYLWAKLDADRD ATITVDQTGFGFANGGVSCTARDLARVGRMMLDGGVAPGGRVVSQGWVESVLAGGSREAMT DEGFTSAFPEGSYTRQWWCTGNERGNVSGIGIHGQNLWLDPRTDSVIVKLSSWPDPDTRHW HGLQSGILLDVSRALDAVGGGSGGSGGGSGSNPYQRGPNPTRSALTADGPFSVATYTVSRLS VSGFGGGVIYYPTGTSLTFGGIAMSPGYTADASSLAWLGLRLASHGFVVLVINTNSRFDGPDSR ASQLSAALNYLRTSSPSAVRARLDANRLAVAGHSMGGGGTLRIAEQNPSLKAAVPLTPWHTDK TFNTSAPVLIVGAEADTVAPVSQHAIPFYQNLPSTTPKVYVELCNASH/APNSNNAAISVYTISW MKLBVDNDTRYRQFLCNVNDPALCDFRTNNRHCQ LEHHHHHH

The present disclosure provides each of the polypeptides provided above (SED ID NOs. 1 -12), and each of the individual domains therein, as well as nucleic acids that encode such polypeptides and individual domains. Cells comprising such polypeptides and nucleic acids are also provided. As will be appreciated, the present disclosure also provides variants of any of the polypeptides and individual domains therein, where in some instances a variant polypeptide or domain thereof comprises an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 91 % or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater amino acid sequence identity to the parental/reference sequence, or a functional fragment thereof, where the variant retains the functionality (e.g., nylon-degrading functionality) of the parental/reference sequence.

In certain embodiments, variants having one or more amino acid substitutions are provided. Conservative substitutions are shown in the table below (right-most column) More substantial changes are provided in the table below under the heading of "exemplary substitutions," and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into an enzyme of interest and the products screened for a desired activity, e.g., retained/improved nylon-degrading activity, or the like.

Amino Acid Substitutions

Amino acids may be grouped according to common side-chain properties:

(1 ) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

Assessing a variant enzyme or fusion protein for the retention (e.g., enhancement) of nylon-degrading activity may be performed in a variety of ways. For example, high-throughput screening of nylon-degrading activity may be performed using any device that involves an array having a plurality of physically separate wells or containers for liquid, for example microtiter spectroscopic plates or microcapillary arrays, where the wells or containers may have closed ends or open ends. Screening may also be performed using devices employing both liquids and solids, such as deposition of a single liquid mixture of nylon-degrading enzymes or fusion proteins and/or a single liquid suspension of cells encoding, expressing, secreting, displaying, and/or exporting nylon-degrading enzymes or fusion proteins on a homogeneous nylon substrate, and/or deposition of individual liquid volumes containing and/or encapsulating individual nylondegrading enzymes or fusion proteins or cells encoding, expressing, secreting, displaying, and/or exporting nylon-degrading enzymes or fusion proteins on said homogeneous nylon substrate, where the nylon substrate may be comprised of a solid film of a specific type of nylon; a solid sheet of a specific type of nylon; and/or a microscopically heterogeneous but macroscopically homogeneous nylon-containing substrate such as fibers, fabrics, and/or textiles consisting of one or more material types in addition to nylon (e.g., polyester, cotton, nylon, elastane). The screening methods may be used in tandem and/or in combination with other embodiments of screening methods including those described herein to produce datasets enabling the identification and characterization of enzymes with improved nylon-degrading capabilities.

Libraries of nylon-degrading enzymes or fusion proteins may be generated via random, site-directed, or site-saturation mutagenesis. For example, mutagenesis PCR may be performed using DNA base pair orthologs, using an error-prone DNA polymerase (e.g., GeneMorph II, Agilent Technologies), or using degenerate primers for site-saturation mutagenesis. The products of this PCR reaction may have homology of 10 to 50 base-pairs to a vector that allows for the expression, secretion, display, and/or export of the gene product out of the cell. Homologous recombination of the expression vector and the insert library may be achieved by transformation into yeast and subsequent selection for transformants via an appropriate selection marker. Variant libraries may be produced in other host organisms using appropriate transformation and assembly methods.

Halo-based screening may be used for identifying transformants encoding, secreting, or displaying enzymes with nylon-degrading capabilities. This may be achieved using nylon- containing agar growth plates that simultaneously enable organism growth, production of enzyme, and indication of nylon degradation. Recombinant organisms encoding, expressing, secreting, displaying, and/or exporting one or more nylon-degrading enzymes or fusion proteins may be comprised of organisms of bacterial or fungal origin, for example Escherichia ccii, Saccharomyces cerevisiae, Pichia pastoris, etc. Growth plates may be prepared by incorporating the nylon substrate with tryptone, peptone, yeast extract, salts, and other growth medium suitable for the culture of recombinant organisms capable of producing nylon-degrading enzymes or fusion proteins. Nylon-degrading activity may be assayed by visual inspection, photography, image processing, or other means of identifying and characterizing halo production.

Screening for nylon-degrading activity may also be achieved using a suspension, mixture, emulsion, gel, sol, or any combination of the preceding of nylon-containing material in a liquid medium such as water or buffer without use of growth media components. Screening of nylondegrading activity may be achieved in combination with halo-based activity screening. The nylon- containing suspension, mixture, emulsion, gel, sol, or any combination of the preceding may consist of a buffering agent with appropriate buffering capacity at the desired pH corresponding to optimal activity of the nylon-degrading enzyme, for example pH 7, 8, or 9 for polyesterdegrading enzymes. Surfactants, dispersants, emulsifiers, and other compounds may be used to achieve homogeneous dispersion of the nylon-containing material. The nylon-containing material may be in the form of solids, film, fibers, micronized powder, nanoparticles, or any combination of the preceding. Recombinant organisms encoding nylon-degrading enzyme(s), extracts thereof containing nylon-degrading enzymes or fusion proteins, or other samples containing nylondegrading enzyme(s) may be prepared in high-throughput devices, for example microtiter plates, 96-deep-well plates, or microcapillary arrays, as to contain sufficient quantities of enzyme for activity detection. In one non-limiting set of examples, the cell culture, cell extract, cell lysate, or culture medium depleted of cells is brought in contact with an appropriate nylon-containing material in the context of high-throughput devices.

Enzymes with nylon-degrading activity may be identified by extracting the DNA vector containing the gene encoding the nylon-degrading enzyme and sequencing the relevant part or whole of the DNA vector by, for example, Sanger DNA sequencing or Illumina next-generation sequencing, to identify advantageous mutations. Nylon-degrading enzymes or fusion proteins may be expressed in recombinant organisms to produce sufficient quantities of nylon-degrading enzyme for activity analysis. Nylon-degrading enzymes or fusion proteins may be purified using methods including but not limited to affinity chromatography, size exclusion chromatography, and/or ion exchange chromatography, to enable and/or facilitate activity analysis.

In some embodiments, the purified enzyme or fusion protein variants are tested on nylon- containing materials and assessed by an appropriate method to calculate, quantify, or otherwise characterize nylon-degrading activity. Nylon-degrading activity may be assessed using a method such as fluorescence, gravimetry, liquid chromatography mass spectroscopy (LCMS), or Fourier transform infrared spectroscopy (FTIR).

In certain embodiments, nylon-degrading activity is assessed using a spectroscopic method, for example optical density, colorimetry, optical absorbance, or fluorescence. In a particular embodiment, nylon-degrading activity is assessed using fluorescence and calculated as the fluorescence increase before and after contact with a nylon-degrading enzyme or fusion protein.

In certain embodiments, nylon-degrading activity is assessed using gravimetry and calculated as the mass change of a nylon-containing material before and after contact with a nylon-degrading enzyme or fusion protein. In certain embodiments, nylon-degrading activity is assessed using gravimetry and calculated as the mass change of a nylon-containing material after being contacted with a nylon-degrading enzyme or fusion protein relative to a nylon- containing material without being contacted with a nylon-degrading enzyme or fusion protein. In a particular embodiment, nylon-degrading activity is assessed using gravimetry and calculated as the mass change of a nylon-containing fabric before and after contact with a nylon-degrading enzyme or fusion protein and relative to a nylon-containing fabric without being contacted by the nylon-degrading enzyme or fusion protein.

In certain embodiments, nylon-degrading activity is assessed using Fourier transform infrared spectroscopy (FTIR) and calculated as the change in the absorbance measurement at one or more wavelengths representative of the nylon-containing material to be degraded. In a particular embodiment, nylon-degrading activity is assessed using Fourier transform infrared spectroscopy (FTIR) and calculated as the change in the absorbance at specific wavelengths representing specific bonds (e.g., 1641 cm ¹ representing the C=O bond) present in a nylon- containing material after contacting the nylon-containing material with the nylon-degrading enzyme or fusion protein.

In certain embodiments, nylon-degrading activity is assessed using liquid chromatography mass spectroscopy (LCMS) as the quantity of one or more products caused by nylon-degrading activity, such as a monomer, oligomer, or polymer, where the quantity may be the arithmetic sum of multiple products. In a particular embodiment, nylon-degrading activity is assessed using LCMS as the quantity of 6-aminohexanoic acid, or any sum of monomers and oligomers, produced after contacting the nylon-containing material with the nylon-degrading enzyme or fusion protein.

NUCLEIC ACIDS, EXPRESSION CONSTRUCTS, CELLS AND COMPOSITIONS

Aspects of the present disclosure further include nucleic acids and expression constructs. For example, provided are nucleic acids encoding any of the nylon-degrading enzymes or fusion proteins of the present disclosure. Because of the knowledge of the codons corresponding to the various amino acids, availability of an amino acid sequence of a polypeptide of interest provides a description of all the polynucleotides capable of encoding the polypeptide of interest. The degeneracy of the genetic code, where the same amino acids are encoded by alternative or synonymous codons allows an extremely large number of nucleic acids to be made, all of which encode the enzymes disclosed herein. Thus, having identified a particular amino acid sequence, those of ordinary skill in the art could make any number of different nucleic acids by simply modifying the sequence of one or more codons in a way which does not change the amino acid sequence of the polypeptide of interest. In this regard, the present disclosure specifically contemplates each and every possible variation of polynucleotides that could be made by selecting combinations based upon the possible codon choices, and all such variations are to be considered specifically disclosed for any polypeptide disclosed herein, including the amino acid sequences of SEQ ID NOs. 1 -12.

The nucleotide sequences of the nucleic acids of the present may be codon optimized. “Codon-optimized” refers to changes in the codons of the polynucleotide encoding a polypeptide to those preferentially used in a particular organism such that the encoded protein is efficiently expressed in the organism of interest. Although the genetic code is degenerate in that most amino acids are represented by several codons, called “synonyms” or “synonymous” codons, codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome. In some embodiments, a nucleic acid of the present disclosure encoding a polypeptide may be codon-optimized for optimal production from the host organism selected for expression, e.g., bacterial cells, such as E. coli cells or yeast cells, such as, Saccharomyces cerevisiae.

Also provided are expression constructs comprising any of the nucleic acids of the present disclosure. As used herein, an “expression construct” is a circular or linear polynucleotide (a polymer composed of naturally occurring and/or non-naturally occurring nucleotides) comprising a region that encodes a fusion protein of the present disclosure, operably linked to a suitable promoter, e.g., a constitutive or inducible promoter. In some embodiments, expression of the polypeptide is under the control of one or more heterologous regulatory elements, e.g., promoter, enhancer, etc., present in the expression construct. In some embodiments, expression of the polypeptide may be controlled by one or more endogenous regulatory elements, e.g., promoter, enhancer, etc., at or near a genomic locus into which the expression construct is inserted.

The expression constructs (e.g., vectors) can be suitable for replication and integration in prokaryotes, eukaryotes, or both. The expression constructs may contain functionally appropriately oriented transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the nucleic acid encoding the enzymes. The expression constructs optionally contain generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in both eukaryotes and prokaryotes, e.g., as found in shuttle vectors, and selection markers for both prokaryotic and eukaryotic systems.

To obtain high levels of expression of a cloned nucleic acid one may construct expression constructs which typically contain a strong promoter to direct transcription, a ribosome binding site for translational initiation, and a transcription/translation terminator, each in functional orientation to each other and to the protein-encoding sequence. Examples of regulatory regions suitable for this purpose in E. coli are the promoter and operator region of the E. coli tryptophan biosynthetic pathway, the leftward promoter of phage lambda (PL), and the L-arabinose (araBAD) operon. The inclusion of selection markers in DNA vectors transformed in E. coli 's also useful. Examples of such markers include genes specifying resistance to ampicillin, tetracycline, or chloramphenicol. Expression systems for expressing the enzyme are available for use in, for example, E. coli, Bacillus sp. and Salmonella. E. coli systems may also be used. Transducing cells with nucleic acids (e.g., expression constructs) can involve, for example, incubating lipidic microparticles containing nucleic acids with cells or incubating viral vectors containing nucleic acids with cells.

In certain embodiments, upon delivery of an expression construct to cells, one or more of the expression constructs are episomal (e.g., extra-chromosomal), where by “episome” or “episomal” is meant a polynucleotide that replicates independently of the cell’s chromosomal DNA. A non-limiting example of an episome that may be employed is a plasmid.

Aspects of the present disclosure further include cells comprising a nucleic acid of the present disclosure, as well as cells comprising an expression construct of the present disclosure. In certain embodiments, the cells are prokaryotic cells (e.g., bacteria, E. coli), yeast cells (e.g., Saccharomyces species, e.g., Saccharomyces cerevisiae), insect (e.g., drosophila) cells, amphibian (e.g., frog, e.g., Xenopus) cells, plant cells, etc. According to some embodiments, the cells are mammalian cells. Mammalian cells of interest include human cells, rodent cells, and the like and include cell lines, such as, CHO cells, HEK293 cells, etc. In a particular embodiment, the cells of interest are Saccharomyces cerevisiae. In another particular embodiment, the cells of interest are Escherichia coli.

Also provided by the present disclosure are compositions. According to some embodiments, provided are compositions comprising any of the cells, polypeptides, nucleic acids, and/or expression constructs of the present disclosure.

Such compositions may comprise the polypeptides, nucleic acids, expression constructs, and/or cells present in a liquid medium. The liquid medium may be an aqueous liquid medium, such as water, a buffered solution, or the like. In certain embodiments, the liquid medium is a cell culture medium. The cell culture medium may be bacterial or yeast cell culture mediums. Non- limiting examples of cell culture media include peptone, yeast extract, and dextrose or glucose; Minimal Essential Media, DMEM, a-MEM, RPMI Media, Clicks, F-12, X-Vivo 15, X-Vivo 20, Optimizer, and the like.

In certain embodiments, the compositions may be frozen or lyophilized. In certain embodiments, the cell(s), cell extract(s), polypeptide(s), and/or enzyme(s) of the present disclosure may be formulated into suspensions, sprayable solutions, hydrogels, or otherwise easily dispersible formulations.

In certain embodiments, the composition may be liquid or dry, for instance in the form of a powder. In certain embodiments, the composition is a lyophilizate. The composition may further comprise excipients and/or reagents. Appropriate excipients encompass buffers commonly used in biochemistry, and/or agents for adjusting pH. The composition may be obtained by mixing the cell(s), cell extract(s), polypeptide(s), and/or enzyme(s) of the present disclosure with one or several excipients.

In certain embodiments, the composition may further comprise additional cell(s), cell extract(s), enzyme(s), and/or polypeptide(s) exhibiting an enzymatic activity. In certain embodiments, the enzymatic activity is a nylon-degrading activity. The amounts of cell(s), cell extract(s), enzyme(s), and/or polypeptide(s) may be adapted depending e.g., on the nature of the nylon to degrade and/or the additional cell(s), cell extract(s), enzyme(s), and/or polypeptide(s) contained in the composition.

In certain embodiments, the composition is solubilized in an aqueous medium together with one or several excipients, especially excipients which are able to stabilize or protect the nylon-degrading cell(s), cell extract(s), enzyme(s), and/or polypeptide(s) from degradation. For instance, the cell(s), cell extract(s), enzyme(s), and/or polypeptide(s) of the present disclosure may be solubilized in water, eventually with additional components. The resulting mixture may then be dried so as to obtain a powder. Methods for drying such mixture are well known to the one skilled in the art and include, without limitation, lyophilization, freeze-drying, spray-drying, supercritical drying, down-draught evaporation, thin-layer evaporation, centrifugal evaporation, conveyer drying, fluidized bed drying, drum drying or any combination thereof.

PRODUCTION OF NYLON-DEGRADING ENZYMES OR FUSION PROTEINS

Methods of producing the nylon-degrading enzymes or fusion proteins of the present disclosure are also provided. The methods may include, expressing a nucleic acid encoding the enzyme or fusion protein and optionally recovering the enzyme or fusion protein.

In an exemplary embodiment, an in vitro method of producing an enzyme or fusion protein of the present disclosure may include (a) contacting a nucleic acid, cassette or vector encoding the enzyme with an in vitro expression system; and (b) recovering the enzyme or fusion protein produced. In vitro expression systems are well-known by the person skilled in the art and are commercially available. In another embodiment, the method of production comprises (a) culturing a cell that comprises a nucleic acid encoding an enzyme or fusion protein of the present disclosure under conditions suitable to express the nucleic acid; and optionally (b) recovering the enzyme or fusion protein from the cell culture.

Exemplary cells include recombinant Bacillus, recombinant E. coli, recombinant Aspergillus, recombinant Trichoderma, recombinant Streptomyces, recombinant Saccharomyces, recombinant Pichia, recombinant Vibrio or recombinant Yarrowia.

The cells are cultivated in a nutrient medium suitable for production of polypeptides. For example, the cell may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid-state fermentations) in laboratory or industrial fermenters performed in a suitable medium and under conditions allowing the enzyme to be expressed and/or isolated. The cultivation can take place in a suitable nutrient medium, from commercial suppliers or prepared according to published compositions (e.g., in catalogs of the American Type Culture Collection).

In certain embodiments, the enzyme or fusion protein is excreted into the nutrient medium and is recovered directly from the culture supernatant. Alternatively, the enzyme or fusion protein can be recovered from cell lysates or after permeabilization. The enzyme or fusion protein may be recovered from the nutrient medium by procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. Optionally, the enzyme or fusion protein may be partially or totally purified by a variety of procedures including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction to obtain substantially pure polypeptides.

The nylon-degrading enzyme or fusion protein may be used as such, in purified form, either alone or in combinations with additional enzymes, to catalyze enzymatic reactions involved in the degradation and/or recycling of nylon(s) and/or nylon-containing material. The nylondegrading enzyme or fusion protein may be in soluble form, or on solid phase. In certain embodiments, it may be bound to cell membranes or lipid vesicles, or to synthetic supports such as glass, plastic, polymers, filter, membranes, for example in the form of beads, columns, and/or plates. In certain embodiments, the nylon-degrading enzyme or fusion protein may be obtained as cell culture or cell extract derived from recombinant cells expressing, secreting, displaying, or exporting the nylon-degrading enzyme or fusion protein.

For purposes of completeness, non-limiting aspects and embodiments of the present disclosure are further disclosed in the following numbered clauses. 1 . A method of degrading nylon, the method comprising contacting a nylon substrate with a PET-degrading enzyme under conditions suitable for degradation of the nylon substrate by the PET-degrading enzyme.

2. The method according to clause 1 , wherein the PET-degrading enzyme is a cutinase or cutinase-like enzyme.

3. The method according to clause 2, wherein the cutinase or cutinase-like enzyme is a cutinase from Humicola insolens (HiC).

4. The method according to clause 3, wherein the cutinase or cutinase-like enzyme is a wild-type cutinase from HiC.

5. The method according to clause 3, wherein the cutinase or cutinase-like enzyme is a mutant cutinase from HiC.

6. The method according to clause 5, wherein the mutant cutinase from HiC comprises one or more amino acid substitutions relative to the amino acid sequence set forth in SEQ ID NO: 1.

7. The method according to clause 6, wherein the mutant cutinase from HiC comprises an amino acid substitution at position 1 , 3, 4, 11 , 15, 29, 37, 47, 164, 166, or any combination thereof, wherein numbering of positions is according to SEQ ID NO: 1.

8. The method according to clause 7, wherein the mutant cutinase from HiC comprises the amino acid substitution Q1A, Q1 C, Q1 D, Q1 E, Q1 F, Q1 G, Q1 H, Q1 I, Q1 K, Q1 L, Q1 M, Q1 N, Q1 P, Q1 R, Q1S, Q1T, Q1V, Q1Y, G3A, G3C, G3D, G3E, G3F, G3H, G3I, G3L, G3N, G3Q, G3R, G3S, G3T, G3V, G3Y, A4V, S11 N, N15D, T29K, T37A, T37I, T37L, T37M, T37N, T37P, T37S, T37V, E47A, E47C, E47F, E47G, E47H, E47I, E47L, E47M, E47N, E47Q, E47R, E47S, E47T, E47V, E47Y, T164S, T166A, or any combination thereof at different positions.

9. The method according to clause 7, wherein the mutant cutinase from HiC comprises an amino acid substitution chosen from: Q1 R, G3S, T29K, T37L, E47Q, and any combination thereof.

10. The method according to clause 7, wherein the mutant cutinase from HiC comprises the amino acid substitutions Q1 R, G3S, T29K, and E47Q.

11 . The method according to clause 7, wherein the mutant cutinase from HiC comprises the amino acid substitutions Q1 R, G3T, T37L, and E47R.

12. The method according to any one of clauses 3 to 11 , wherein the cutinase or cutinase- like enzyme comprises an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% identity to the amino acid sequence set forth in SEQ ID NO:1 , SEQ ID NO:2, or SEQ ID NO:3, or a nylon-degrading enzyme fragment thereof. 13. The method according to clause 2, wherein the cutinase or cutinase-like enzyme is a leaf compost cutinase (LCC).

14. The method according to clause 13, wherein the LCC is a wild-type LCC.

15. The method according to clause 13, wherein the LCC is a mutant LCC.

16. The method according to clause 15, wherein the mutant LCC comprises one or more amino acid substitutions relative to the amino acid sequence set forth in SEQ ID NO: 4, wherein numbering of positions is according to SEQ ID NO: 4.

17. The method according to clause 15, wherein the mutant LCC comprises an amino acid substitution at position 93, 204, 209, 249, or any combination thereof.

18. The method according to clause 17, wherein the mutant LCC comprises an amino acid substitution chosen from: Y93G, D204C, F209I, S249C, and any combination thereof.

19. The method according to any one of clauses 13 to 18, wherein the LCC comprises an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% identity to the amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:5, or a nylon-degrading enzyme fragment thereof.

20. The method according to clause 1 , wherein the PET-degrading enzyme is a polyester hydrolase Leipzig 7 (PHL7) enzyme.

21 . The method according to clause 20, wherein the PHL7 enzyme comprises an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% identity to the amino acid sequence set forth in SEQ ID NO:6, or a nylon-degrading enzyme fragment thereof.

22. The method according to any one of clauses 1 to 21 , wherein the PET-degrading enzyme is fused to a heterologous protein domain.

23. The method according to clause 22, wherein the heterologous protein domain is an enzyme.

24. The method according to clause 23, wherein the enzyme is a nylon-degrading enzyme.

25. The method according to clause 24, wherein the nylon-degrading enzyme is a nylB enzyme.

26. The method according to clause 25, wherein the nylB enzyme comprises an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% identity to the amino acid sequence set forth in SEQ ID NO:7, or a nylon-degrading enzyme fragment thereof. 27. The method according to clause 24, wherein the nylon-degrading enzyme is a nylC enzyme.

28. The method according to clause 27, wherein the nylC enzyme is a nylCk enzyme.

29. The method according to clause 28, wherein the nylCk enzyme comprises an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% identity to the amino acid sequence set forth in SEQ ID NO:8, or a nylon-degrading enzyme fragment thereof.

30. The method according to any one of clauses 25 to 29, wherein the fusion protein comprises an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% identity to the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , or SEQ ID NO:12.

31 . The method according to any one of clauses 1 to 30, wherein the nylon substrate is a nylon fiber.

32. The method according to any one of clauses 1 to 30, wherein the nylon substrate is a molded nylon product.

33. The method according to any one of clauses 1 to 30, wherein the nylon substrate is, or is comprised within, a textile, a net (e.g., a fishing net), a rope, carpet, an umbrella, a conveyor belt, a seat belt, or a machine part.

34. The method according to any one of clauses 1 to 30, wherein the nylon substrate is comprised within a nylon-containing textile.

35. The method according to any one of clauses 1 to 30, wherein the nylon substrate is, or is comprised within, a net.

36. A method of producing 6-aminohexanoic acid, the method comprising performing the method according to any one of clauses 1 to 35.

37. The method according to clause 36, further comprising harvesting the produced 6- aminohexanoic acid.

38. The method according to clause 37, further comprising isolating the harvested 6- aminohexanoic acid.

39. The method according to any one of clauses 36 to 38, further comprising producing nylon from the produced 6-aminohexanoic acid.

40. The method according to clause 39, further comprising producing a nylon-containing product with the produced nylon. 41 . The method according to clause 40, wherein the nylon-containing product is a textile, a net (e.g., a fishing net), a rope, carpet, an umbrella, a conveyor belt, a seat belt, or a machine part.

42. The method according to clause 40, wherein the nylon-containing product is a nylon- containing textile.

43. A fusion protein comprising a PET-degrading enzyme fused to a nylon-degrading enzyme.

44. The fusion protein of clause 43, wherein the PET-degrading enzyme is a cutinase or cutinase-like enzyme.

45. The fusion protein of clause 44, wherein the cutinase or cutinase-like enzyme is a cutinase from Humicola insolens (HiC).

46. The fusion protein of clause 45, wherein the cutinase or cutinase-like enzyme is a wildtype cutinase from HiC.

47. The fusion protein of clause 45, wherein the cutinase or cutinase-like enzyme is a mutant cutinase from HiC.

48. The fusion protein of clause 47, wherein the mutant cutinase from HiC comprises one or more amino acid substitutions relative to the amino acid sequence set forth in SEQ ID NO: 1 .

49. The fusion protein of clause 48, wherein the mutant cutinase from HiC comprises an amino acid substitution at position 1 , 3, 4, 11 , 15, 29, 37, 47, 164, 166, or any combination thereof, wherein numbering of positions is according to SEQ ID NO: 1.

50. The fusion protein of clause 49, wherein the mutant cutinase from HiC comprises the amino acid substitution Q1A, Q1 C, Q1 D, Q1 E, Q1 F, Q1 G, Q1 H, Q1 I, Q1 K, Q1 L, Q1 M, Q1 N, Q1 P, Q1 R, Q1S, Q1T, Q1V, Q1Y, G3A, G3C, G3D, G3E, G3F, G3H, G3I, G3L, G3N, G3Q, G3R, G3S, G3T, G3V, G3Y, A4V, S11 N, N15D, T29K, T37A, T37I, T37L, T37M, T37N, T37P, T37S, T37V, E47A, E47C, E47F, E47G, E47H, E47I, E47L, E47M, E47N, E47Q, E47R, E47S, E47T, E47V, E47Y, T164S, T166A, or any combination thereof at different positions.

51 . The fusion protein of clause 49, wherein the mutant cutinase from HiC comprises an amino acid substitution chosen from: Q1 R, G3S, T29K, T37L, E47Q, and any combination thereof.

52. The fusion protein of clause 49, wherein the mutant cutinase from HiC comprises the amino acid substitutions Q1 R, G3S, T29K, and E47Q.

53. The fusion protein of clause 49, wherein the mutant cutinase from HiC comprises the amino acid substitutions Q1 R, G3T, T37L, and E47R.

54. The fusion protein of any one of clauses 45 to 53, wherein the cutinase or cutinase-like enzyme comprises an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% identity to the amino acid sequence set forth in SEQ ID NO:1 , SEQ ID NO:2, or SEQ ID NO:3, or a nylon-degrading enzyme fragment thereof.

55. The fusion protein of clause 44, wherein the cutinase or cutinase-like enzyme is a leaf compost cutinase (LCC).

56. The fusion protein of clause 55, wherein the LCC is a wild-type LCC.

57. The fusion protein of clause 55, wherein the LCC is a mutant LCC.

58. The fusion protein of clause 57, wherein the mutant LCC comprises one or more amino acid substitutions relative to the amino acid sequence set forth in SEQ ID NO: 4, wherein numbering of positions is according to SEQ ID NO: 4.

59. The fusion protein of clause 58, wherein the mutant LCC comprises an amino acid substitution at position 93, 204, 209, 249, or any combination thereof.

60. The fusion protein of clause 59, wherein the mutant LCC comprises an amino acid substitution chosen from: Y93G, D204C, F209I, S249C, and any combination thereof.

61 . The fusion protein of any one of clauses 58 to 60, wherein the mutant LCC further comprises an amino acid substitution at position 73, 166, 229, or any combination thereof.

62. The fusion protein of clause 61 , wherein the mutant LCC comprises an amino acid substitution chosen from: R73L, V166A, W229R, and any combination thereof.

63. The fusion protein of any one of clauses 55 to 62, wherein the LCC comprises an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% identity to the amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:5, or a nylon-degrading enzyme fragment thereof.

64. The fusion protein of clause 43, wherein the PET-degrading enzyme is a polyester hydrolase Leipzig 7 (PHL7) enzyme.

65. The fusion protein of clause 64, wherein the PHL7 enzyme comprises an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% identity to the amino acid sequence set forth in SEQ ID NO:6, or a nylon-degrading enzyme fragment thereof.

66. The fusion protein of any one of clauses 43 to 65, wherein the nylon-degrading enzyme is a nylB enzyme.

67. The fusion protein of clause 66, wherein the nylB enzyme comprises an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% identity to the amino acid sequence set forth in SEQ ID NO:7, or a nylon-degrading enzyme fragment thereof. 68. The fusion protein of any one of clauses 43 to 65, wherein the nylon-degrading enzyme is a nylC enzyme.

69. The fusion protein of clause 68, wherein the nylC enzyme is a nylCk enzyme.

70. The fusion protein of clause 69, wherein the nylCk enzyme comprises an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% identity to the amino acid sequence set forth in SEQ ID NO:8, or a nylon-degrading enzyme fragment thereof.

71 . The fusion protein of any one of clauses 43 to 70, wherein the fusion protein comprises an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% identity to the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , or SEQ ID NO:12.

72. A nucleic acid encoding the fusion protein of any one of clauses 43 to 71 .

73. A cell comprising the nucleic acid of clause 72.

74. The cell of clause 73, wherein the cell is a prokaryotic cell.

75. The cell of clause 74, wherein the prokaryotic cell is an E. coli cell or a B. subtilis cell.

76. The cell of clause 73, wherein the cell is a eukaryotic cell.

77. The cell of clause 76, wherein the eukaryotic cell is a S. cerevisiae cell, a P. pastoris cell, an A. niger cell, an A. nidulans cell, or a T. reesei cell.

78. The cell of any one of clauses 73 to 77, wherein the nucleic acid is present in an expression vector.

79. A method of producing the fusion protein of any one of clauses 43 to 71 , comprising culturing the cell of clause 78 under conditions suitable for expression of the fusion protein, wherein the fusion protein is produced.

The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL

Example 1 - Depolymerization of nylon 6 nanoparticles, and copolymer monofilament consisting of nylon 6 and nylon 6,6, by cutinases and respective fusion proteins

Polyester-degrading enzymes were found to depolymerize solid, unprocessed nylon 6 substrates, yielding the valuable nylon monomer 6-aminohexanoic acid. Fusions comprising a PET-degrading enzyme fused to a nylon-degrading enzyme (in this example, nylB) were observed to synergistically improve overall depolymerization of nylon. The observed conversion of nylB-containing fusions was found to exceed even a previously described nylon-6-oligomer- degrading enzyme, nylC. Improved conversion was demonstrated for two different types of nylon- 6-containing samples, namely nylon-6 nanoparticles as well as a commercially available nylon- 6-nylon-6,6 copolymer monofilament fiber.

The enzymes and fusion proteins studied here were expressed in E. coli BL21 (DE3) and purified via affinity chromatography. Strains harboring expression plasmids encoding SEQ ID NOS. 1 -1 1 were grown for 18 hours at 20°C in ZYM auto inducible medium (Studier et al., 2005, Prot. Exp. Pur. 41 , 207-234). Cells were harvested by centrifugation (3700xg, 30 minutes, 4°C) and resuspended in lysis buffer (20 mM sodium phosphate, 10 mM imidazole, 10% glycerol, pH 7.4). The cell suspension was then sonicated (5 minutes, 30% amplitude, 66% duty cycle). Following centrifugation (30 minutes at 15000xg, 4°C), the soluble fraction was used for purification of the desired enzyme using Talon Metal Affinity Resin (Clontech, CA, USA) following manufacturer’s guidelines. Protein elution was carried out with lysis buffer supplemented with 300 mM imidazole. Purified protein was buffer exchanged into lysis buffer without imidazole and protein concentration quantified using absorbance at 280 nm.

Enzymes and fusion proteins were diluted to a final concentration of 0.2 mg/ml in 250 mM potassium phosphate, pH 8, and incubated with either 1 mg/ml nylon-6 nanoparticles or 80 mg/ml nylon-6-nylon-6,6 copolymer monofilament fiber at 37°C. The breakdown products at multiple timepoints as well as the final amounts were quantified using Liquid Chromatography Mass Spectrometry (LC-MS), as shown in FIG. 1 A-1 B (nylon-6 nanoparticles) and FIG. 2A-2B (nylon- 6-nylon-6,6 copolymer monofilament fiber). The y-axes indicate product amount in pM. Error bars represent standard deviation of triplicate reactions.

Abbreviations:

“Monomer” refers to the nylon 6 monomer 6-aminohexanoic acid;

“Dimer” refers to 6-(6-aminohexanamido)hexanoic acid;

“Trimer” refers to 6-(6-(6-aminohexanamido)aminohexanamido)hexanoic acid;

“Total” refers to the sum of monomer, dimer, and trimer product amounts.

HiC, cutinase from Humicola insolens, SEQ ID NO: 1 ;

HiC mut. 1 , HiC containing mutations Q1 R, G3S, T29K, E47Q, SEQ ID NO: 2;

HiC mut. 2, HiC containing mutations Q1 R, G3T, T37L, E47R, SEQ ID NO: 3;

LCC(ICCG), leaf compost cutinase containing mutations Y93G, D204C, F209I, S249C, SEQ ID NO: 5;

PHL7, polyester hydrolase Leipzig 7, SEQ ID NO: 6; nylB, SEQ ID NO: 7; nylCk, SEQ ID NO: 8; nylB-HiC, fusion of nylB and wildtype HiC via a 12-aa flexible linker, SEQ ID NO: 9; nylB-LCC(ICCG), fusion of nylB and leaf compost cutinase containing mutations Y93G, D204C, F209I, S249C via a 12-aa flexible linker, SEQ ID NO: 10; nylB-PHL7, fusion of nylB and polyester hydrolase Leipzig 7, SEQ ID NO: 1 1 .

Example 2 - Depolymerization of Nylon 6 Nanoparticles by a NvIB-Cutinase Fusion Mutant

Error-prone PCR was used to generate random mutations in the sequence encoding the nylB-LCC(ICCG) fusion (SEQ ID NO: 10). Enzyme variants were expressed and purified as described in Example 1 . Enzymes and fusion proteins were diluted to a final concentration of 0.2 mg/ml in 250 mM potassium phosphate, pH 8, and incubated with 1 mg/ml nylon-6 nanoparticles at 37°C. The breakdown products after 6 days were quantified using Liquid Chromatography Mass Spectrometry (LC-MS). A triple mutant containing mutations R73L, V166A, and W229R (SEQ ID NO: 12) was found to have 117% breakdown product release relative to the original LCC(ICCG) fusion (SEQ ID NO: 10).

Accordingly, the preceding merely illustrates the principles of the present disclosure. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein.

Claims

WHAT IS CLAIMED IS:

1 . A method of degrading nylon, the method comprising contacting a nylon substrate with a PET-degrading enzyme under conditions suitable for degradation of the nylon substrate by the PET-degrading enzyme.

2. The method according to claim 1 , wherein the PET-degrading enzyme is a cutinase or cutinase-like enzyme.

3. The method according to claim 2, wherein the cutinase or cutinase-like enzyme is a cutinase from Humicola insolens (HiC).

4. The method according to claim 3, wherein the cutinase or cutinase-like enzyme is a wild-type cutinase from HiC.

5. The method according to claim 3, wherein the cutinase or cutinase-like enzyme is a mutant cutinase from HiC.

6. The method according to claim 5, wherein the mutant cutinase from HiC comprises one or more amino acid substitutions relative to the amino acid sequence set forth in SEQ ID NO: 1.

7. The method according to claim 6, wherein the mutant cutinase from HiC comprises an amino acid substitution at position 1 , 3, 4, 11 , 15, 29, 37, 47, 164, 166, or any combination thereof, wherein numbering of positions is according to SEQ ID NO: 1.

8. The method according to claim 7, wherein the mutant cutinase from HiC comprises the amino acid substitution Q1A, Q1 C, Q1 D, Q1 E, Q1 F, Q1 G, Q1 H, Q1 I, Q1 K, Q1 L, Q1 M, Q1 N, Q1 P, Q1 R, Q1S, Q1T, Q1V, Q1Y, G3A, G3C, G3D, G3E, G3F, G3H, G3I, G3L, G3N, G3Q, G3R, G3S, G3T, G3V, G3Y, A4V, S11 N, N15D, T29K, T37A, T37I, T37L, T37M, T37N, T37P, T37S, T37V, E47A, E47C, E47F, E47G, E47H, E47I, E47L, E47M, E47N, E47Q, E47R, E47S, E47T, E47V, E47Y, T164S, T166A, or any combination thereof at different positions.

9. The method according to claim 7, wherein the mutant cutinase from HiC comprises an amino acid substitution chosen from: Q1 R, G3S, T29K, T37L, E47Q, and any combination thereof.

10. The method according to claim 7, wherein the mutant cutinase from HiC comprises the amino acid substitutions Q1 R, G3S, T29K, and E47Q.

11 . The method according to claim 7, wherein the mutant cutinase from HiC comprises the amino acid substitutions Q1 R, G3T, T37L, and E47R.

12. The method according to any one of claims 3 to 11 , wherein the cutinase or cutinase- like enzyme comprises an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% identity to the amino acid sequence set forth in SEQ ID NO:1 , SEQ ID NO:2, or SEQ ID NO:3, or a nylon-degrading enzyme fragment thereof.

13. The method according to claim 2, wherein the cutinase or cutinase-like enzyme is a leaf compost cutinase (LCC).

14. The method according to claim 13, wherein the LCC is a wild-type LCC.

15. The method according to claim 13, wherein the LCC is a mutant LCC.

16. The method according to claim 15, wherein the mutant LCC comprises one or more amino acid substitutions relative to the amino acid sequence set forth in SEQ ID NO: 4, wherein numbering of positions is according to SEQ ID NO: 4.

17. The method according to claim 15, wherein the mutant LCC comprises an amino acid substitution at position 93, 204, 209, 249, or any combination thereof.

18. The method according to claim 17, wherein the mutant LCC comprises an amino acid substitution chosen from: Y93G, D204C, F209I, S249C, and any combination thereof.

19. The method according to any one of claims 13 to 18, wherein the LCC comprises an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% identity to the amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:5, or a nylon-degrading enzyme fragment thereof.

20. The method according to claim 1 , wherein the PET-degrading enzyme is a polyester hydrolase Leipzig 7 (PHL7) enzyme.

21 . The method according to claim 20, wherein the PHL7 enzyme comprises an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% identity to the amino acid sequence set forth in SEQ ID NO:6, or a nylon-degrading enzyme fragment thereof.

22. The method according to any one of claims 1 to 21 , wherein the PET-degrading enzyme is fused to a heterologous protein domain.

23. The method according to claim 22, wherein the heterologous protein domain is an enzyme.

24. The method according to claim 23, wherein the enzyme is a nylon-degrading enzyme.

25. The method according to claim 24, wherein the nylon-degrading enzyme is a nylB enzyme.

26. The method according to claim 25, wherein the nylB enzyme comprises an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% identity to the amino acid sequence set forth in SEQ ID NO:7, or a nylon-degrading enzyme fragment thereof.

27. The method according to claim 24, wherein the nylon-degrading enzyme is a nylC enzyme.

28. The method according to claim 27, wherein the nylC enzyme is a nylCk enzyme.

29. The method according to claim 28, wherein the nylCk enzyme comprises an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% identity to the amino acid sequence set forth in SEQ ID NO:8, or a nylon-degrading enzyme fragment thereof.

30. The method according to any one of claims 25 to 29, wherein the fusion protein comprises an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% identity to the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 , or SEQ ID NO:12.

31 . The method according to any one of claims 1 to 30, wherein the nylon substrate is a nylon fiber.

32. The method according to any one of claims 1 to 30, wherein the nylon substrate is a molded nylon product.

33. The method according to any one of claims 1 to 30, wherein the nylon substrate is, or is comprised within, a textile, a net (e.g., a fishing net), a rope, carpet, an umbrella, a conveyor belt, a seat belt, or a machine part.

34. The method according to any one of claims 1 to 30, wherein the nylon substrate is comprised within a nylon-containing textile.

35. The method according to any one of claims 1 to 30, wherein the nylon substrate is, or is comprised within, a net.

36. A method of producing 6-aminohexanoic acid, the method comprising performing the method according to any one of claims 1 to 35.

37. The method according to claim 36, further comprising harvesting the produced 6- aminohexanoic acid.

38. The method according to claim 37, further comprising isolating the harvested 6- aminohexanoic acid.

39. The method according to any one of claims 36 to 38, further comprising producing nylon from the produced 6-aminohexanoic acid.

40. The method according to claim 39, further comprising producing a nylon-containing product with the produced nylon.

41 . The method according to claim 40, wherein the nylon-containing product is a textile, a net (e.g., a fishing net), a rope, carpet, an umbrella, a conveyor belt, a seat belt, or a machine part.

42. The method according to claim 40, wherein the nylon-containing product is a nylon- containing textile.

44. The fusion protein of claim 43, wherein the PET-degrading enzyme is a cutinase or cutinase-like enzyme.

45. The fusion protein of claim 44, wherein the cutinase or cutinase-like enzyme is a cutinase from Humicola insolens (HiC).

46. The fusion protein of claim 45, wherein the cutinase or cutinase-like enzyme is a wildtype cutinase from HiC.

47. The fusion protein of claim 45, wherein the cutinase or cutinase-like enzyme is a mutant cutinase from HiC.

48. The fusion protein of claim 47, wherein the mutant cutinase from HiC comprises one or more amino acid substitutions relative to the amino acid sequence set forth in SEQ ID NO: 1 .

49. The fusion protein of claim 48, wherein the mutant cutinase from HiC comprises an amino acid substitution at position 1 , 3, 4, 11 , 15, 29, 37, 47, 164, 166, or any combination thereof, wherein numbering of positions is according to SEQ ID NO: 1.

50. The fusion protein of claim 49, wherein the mutant cutinase from HiC comprises the amino acid substitution Q1 A, Q1 C, Q1 D, Q1 E, Q1 F, Q1 G, Q1 H, Q11, Q1 K, Q1 L, Q1 M, Q1 N, Q1 P, Q1 R, Q1S, Q1T, Q1V, Q1Y, G3A, G3C, G3D, G3E, G3F, G3H, G3I, G3L, G3N, G3Q, G3R, G3S, G3T, G3V, G3Y, A4V, S11 N, N15D, T29K, T37A, T37I, T37L, T37M, T37N, T37P, T37S, T37V, E47A, E47C, E47F, E47G, E47H, E47I, E47L, E47M, E47N, E47Q, E47R, E47S, E47T, E47V, E47Y, T164S, T166A, or any combination thereof at different positions.

51 . The fusion protein of claim 49, wherein the mutant cutinase from HiC comprises an amino acid substitution chosen from: Q1 R, G3S, T29K, T37L, E47Q, and any combination thereof.

52. The fusion protein of claim 49, wherein the mutant cutinase from HiC comprises the amino acid substitutions Q1 R, G3S, T29K, and E47Q.

53. The fusion protein of claim 49, wherein the mutant cutinase from HiC comprises the amino acid substitutions Q1 R, G3T, T37L, and E47R.

54. The fusion protein of any one of claims 45 to 53, wherein the cutinase or cutinase-like enzyme comprises an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% identity to the amino acid sequence set forth in SEQ ID NO:1 , SEQ ID NO:2, or SEQ ID NO:3, or a nylon-degrading enzyme fragment thereof.

55. The fusion protein of claim 44, wherein the cutinase or cutinase-like enzyme is a leaf compost cutinase (LCC).

56. The fusion protein of claim 55, wherein the LCC is a wild-type LCC.

57. The fusion protein of claim 55, wherein the LCC is a mutant LCC.

58. The fusion protein of claim 57, wherein the mutant LCC comprises one or more amino acid substitutions relative to the amino acid sequence set forth in SEQ ID NO: 4, wherein numbering of positions is according to SEQ ID NO: 4.

59. The fusion protein of claim 58, wherein the mutant LCC comprises an amino acid substitution at position 93, 204, 209, 249, or any combination thereof.

60. The fusion protein of claim 59, wherein the mutant LCC comprises an amino acid substitution chosen from: Y93G, D204C, F209I, S249C, and any combination thereof.

61 . The fusion protein of any one of claims 58 to 60, wherein the mutant LCC further comprises an amino acid substitution at position 73, 166, 229, or any combination thereof.

62. The fusion protein of claim 61 , wherein the mutant LCC comprises an amino acid substitution chosen from: R73L, V166A, W229R, and any combination thereof.

63. The fusion protein of any one of claims 55 to 62, wherein the LCC comprises an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% identity to the amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:5, or a nylon-degrading enzyme fragment thereof.

64. The fusion protein of claim 43, wherein the PET-degrading enzyme is a polyester hydrolase Leipzig 7 (PHL7) enzyme.

65. The fusion protein of claim 64, wherein the PHL7 enzyme comprises an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% identity to the amino acid sequence set forth in SEQ ID NO:6, or a nylon-degrading enzyme fragment thereof.

66. The fusion protein of any one of claims 43 to 65, wherein the nylon-degrading enzyme is a nylB enzyme.

67. The fusion protein of claim 66, wherein the nylB enzyme comprises an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% identity to the amino acid sequence set forth in SEQ ID NOT, or a nylon-degrading enzyme fragment thereof.

68. The fusion protein of any one of claims 43 to 65, wherein the nylon-degrading enzyme is a nylC enzyme.

69. The fusion protein of claim 68, wherein the nylC enzyme is a nylCk enzyme.

70. The fusion protein of claim 69, wherein the nylCk enzyme comprises an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% identity to the amino acid sequence set forth in SEQ ID NO:8, or a nylon-degrading enzyme fragment thereof.

71 . The fusion protein of any one of claims 43 to 70, wherein the fusion protein comprises an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% identity to the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , or SEQ ID NO:12.

72. A nucleic acid encoding the fusion protein of any one of claims 43 to 71 .

73. A cell comprising the nucleic acid of claim 72.

74. The cell of claim 73, wherein the cell is a prokaryotic cell.

75. The cell of claim 74, wherein the prokaryotic cell is an E. coli cell or a B. subtilis cell.

76. The cell of claim 73, wherein the cell is a eukaryotic cell.

77. The cell of claim 76, wherein the eukaryotic cell is a S. cerevisiae cell, a P. pastoris cell, an A. niger cell, an A. nidulans cell, or a T. reesei cell.

78. The cell of any one of claims 73 to 77, wherein the nucleic acid is present in an expression vector.

79. A method of producing the fusion protein of any one of claims 43 to 71 , comprising culturing the cell of claim 78 under conditions suitable for expression of the fusion protein, wherein the fusion protein is produced.