Enzyme variants and uses thereof

Abstract

The present disclosure relates generally to polypeptides capable of hydrolysing an amide bond in a polyamide, including polyamides of nylon polymers; methods of producing said polypeptides and uses thereof, including for hydrolysing said polyamide.

Description

ENZYME VARIANTS AND USES THEREOF

[0001] This application claims priority from Australian Provisional Patent Application No. 2024904187 filed 18 December 2024, the entire contents of which are incorporated herein by cross-reference.

[0002] The present invention relates to enzymes, in particular synthetic enzymes that catalyse the hydrolysis of nylon polymers, and uses thereof.BACKGROUND

[0003] All references, including any patent or patent application cited in this specification are hereby incorporated by reference to enable full understanding of the invention. Nevertheless, such references are not to be read as constituting an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

[0004] Global industrialization has had significant environmental impact, not least of which is an increase in the manufacture and reliance on plastic and plastic products. These plastics include a range of synthetic or semi-synthetic polymers, such as polyethylene terephthalate, PVC, polypropylene, polystyrene and polyamides.

[0005] Polyamide (also known generically as nylon) is a silk-like thermoplastic that can be melt-processed into fibres, films or shapes, including in combination with a variety of additives. Polyamides have many significant commercial applications, such as in clothing, the production of resins and molded shapes, in food packaging and industrial fibre and rope.

[0006] Whilst there is a growing effort to find suitable and environmentally sustainable alternatives to plastics, including nylon, including their manufacture and disposal, such products remain a significant problem and contribute to a vast majority of environmental pollutants. The environmental significance of this problem is attributed, at least in part, to the chemical nature of plastics, as they do not readily decompose in nature. Approaches to deal with the problem of plastic waste products have typically included incineration, disposal in landfill and mechanical disintegration. However, these approaches also have significant environmental impact. For instance, incineration of plastics produces potentially harmful byproducts that are released into the atmosphere; the decomposition rate of plastics in landfill is typically very slow and there is a risk that toxic materials will leach intogroundwater; and mechanical disintegration is relatively expensive and inefficient and there is often limited use for its byproducts.

[0007] Nylon can take many decades to decompose, and while there are ways to recycle nylon, these methods require energy-intensive grinding and re-melting of the polymer. Depending on the nylon and the intended applications, the recycled nylon polymer may need to be mixed with virgin nylon, limiting its flexibility and sustainability.

[0008] More recently, biological (enzymatic) degradation of plastics has been considered as an alternative approach to reducing plastic waste accumulation. In the case of nylon, a strain of bacterium found in the waste water of a nylon factory was discovered to have unique enzymes (NylA, NylB and NylC) that could digest by-products of nylon 6 (a nylon homopolymer of caprolactam) manufacture.

[0009] Whilst enzymatic degradation of plastics such as nylon is an attractive alternative to alleviating the environmental impact of plastic waste products and their disposal, it has not yet seen widespread adoption, including because of its relative inefficiency, slow rate of enzymatic degradation and low levels of enzyme expression in common industrial host organisms.SUMMARY OF THE INVENTION

[0010] In one aspect disclosed herein, there is provided a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the polypeptide comprises amino acid residues 2-394 of SEQ ID NO: 1, or an amino acid sequence that has at least 70% sequence identity thereto, and wherein the amino acid sequence of the polypeptide differs from SEQ ID NO: 1 at one or more amino acid positions selected from the group consisting of amino acid positions 220, 222, 331, 334, 336, 348, 352 and 380 of SEQ ID NO: 1.

[0011] In an embodiment, the amino acid residue at position 220 is selected from the group consisting of Y, C, S, I, D, A, E and conservative amino acid substitutions of any of the foregoing; the amino acid residue at position 222 is selected from the group consisting of S, E and conservative amino acid substitutions of any of the foregoing; the amino acid residue at position 331 is selected from the group consisting of Y, W, D and conservative amino acid substitutions of any of the foregoing; the amino acid residue at position 334 is selected from the group consisting of K, Y, E, H, D and conservative amino acid substitutions of anyof the foregoing; the amino acid residue at position 336 is selected from the group consisting of Y, D, W, E and conservative amino acid substitutions of any of the foregoing; the amino acid residue at position 348 is selected from the group consisting of I, H, E, W and conservative amino acid substitutions of any of the foregoing; the amino acid residue at position 352 is selected from the group consisting of G, C and conservative amino acid substitutions of any of the foregoing; and / or the amino acid residue at position 380 is selected from the group consisting of S, Y and conservative amino acid substitutions of any of the foregoing.

[0012] In an embodiment, the amino acid residue at position 220 is selected from the group consisting of Y, C, S, I, D, A, and E; the amino acid residue at position 222 is selected from the group consisting of S or E; the amino acid residue at position 331 is selected from the group consisting of Y, W and D; the amino acid residue at position 334 is selected from the group consisting of K, Y, E, H and D; the amino acid residue at position 336 is selected from the group consisting of Y, D, W and E; the amino acid residue at position 348 is selected from the group consisting of I, H, E and W; the amino acid residue at position 352 is selected from the group consisting of G and C; and / or the amino acid residue at position 380 is selected from the group consisting of S and Y.

[0013] In an embodiment, the polyamide is a nylon polymer or a nylon oligomer. In an embodiment, the polypeptide is capable of hydrolysing an amide bond in a nylon oligomer. In an embodiment, the nylon polymer or nylon oligomer is a nylon 6 polymer or a nylon 6 oligomer. In another embodiment, the nylon polymer or nylon oligomer is a nylon 6, 6 polymer or a nylon 6,6 oligomer.

[0014] In an embodiment, the nylon oligomer is a nylon dimer, a nylon trimer, a nylon tetramer, a nylon pentamer, a nylon hexamer or a nylon heptamer. In another embodiment, the nylon oligomer is an aqueous soluble nylon oligomer. In another embodiment, the nylon oligomer is a carboxylic acid terminated oligomer of nylon.

[0015] The present disclosure also extends to a composition comprising the polypeptide as described herein.

[0016] The present disclosure also extends to a nucleic acid sequence encoding the polypeptide described herein.

[0017] The present disclosure also extends to an expression vector comprising the nucleic acid sequence described herein.

[0018] The present disclosure also extends to a host cell comprising the nucleic acid sequence or the expression vector described herein.

[0019] In another aspect, the present disclosure provides a method of producing a polypeptide capable of hydrolysing an amide bond in a polyamide, the method comprising: i) providing the polynucleotide described herein; ii) expressing the polynucleotide in a host cell under conditions sufficient to allow the host cell to produce the polypeptide; and iii) collecting the polypeptide produced by the host cell in ii).

[0020] In another aspect, the present disclosure provides a method of hydrolysing nylon polymer or nylon oligomer, the method comprising exposing the nylon polymer or nylon oligomer to the polypeptide, the composition or the host cell disclosed herein under conditions sufficient to convert the nylon-6, 6 polymer to adipic acid and / or hexamethyl enedi amine .

[0021] In another aspect, the present disclosure provides a method of degrading a nylon- containing product, the method comprising exposing the nylon-containing product to the polypeptide, the composition or the host cell as disclosed herein.

[0022] In an embodiment, the methods disclosed herein comprises i) chemical processing of nylon polymer, nylon oligomer or nylon-containing product to generate nylon oligomers, ii) exposing the nylon oligomers generated in step (i) to the polypeptide, the composition or the host cell as disclosed herein, under conditions sufficient to produce adipic acid and / or hexamethylenediamine. In one embodiment, the nylon oligomers are nylon dimers, nylon trimers, nylon tetramers, nylon pentamers and nylon hexamers. In another embodiment, the nylon oligomers are aqueous soluble nylon oligomers.

[0023] In an embodiment, the nylon in the nylon polymer, nylon oligomer or the nylon- containing product is a nylon 6 or a nylon 6,6. In another embodiment, the nylon is a nylon 6,6.

[0024] In another embodiment, the methods described herein further comprise recovering the adipic acid and / or the hexamethylenediamine produced in step (ii).

[0025] In another aspect, the present disclosure provides a composition comprising the adipic acid and / or the hexamethylenediamine recovered by the methods disclosed herein.

[0026] In another aspect, the present disclosure provides a method of producing a nylon polymer using the composition of adipic acid and / or the hexamethylenediamine recovered by the methods disclosed herein.

[0027] In another aspect, the present disclosure extends to a nylon polymer when produced using the composition of adipic acid and / or the hexamethylenediamine recovered by the methods disclosed herein.BRIEF DESCRIPTION OF THE FIGURES

[0028] Figure 1 shows activity of the polypeptides having the amino acid sequence of SEQ ID NOs: 3-22 on PA-6,6 trimer. Activity is reported as fold-change difference from SEQ ID NO: 1.

[0029] Figure 2 shows an alignment of polypeptides disclosed herein.

[0030] Figure 3 shows activity of polypeptide having the amino acid sequence of SEQ ID NO: 23 when compared to an extant nylonase of SEQ ID NO:24.

[0031] Figure 4 shows the melting temperature of polypeptides having the amino acid sequence of SEQ ID NOs: 1, 23 and 24.DETAILED DESCRIPTION OF THE INVENTION

[0032] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.

[0033] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article, unless explicitly stated otherwise. By way of example, “an element” means one element or more than one element.

[0034] As used herein, the term "about" refers to a quantity, level, value, dimension, size, or amount that varies by as much as 10% (e.g, by 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%) to a reference quantity, level, value, dimension, size, or amount.

[0035] Throughout this specification, unless the context requires otherwise, the words "has", "have", "having" "comprise", "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0036] The present disclosure relates generally to the design and production of engineered polypeptides, including those with improved hydrolase activity, such as high activity, or broader activity on nylon polymers and nylon oligomers, particularly nylon dimers, trimers, tetramers, and pentamers of nylon. The present disclosure is predicated, at least in part, on the inventors' surprising finding that said engineered polypeptides, from the ancestral sequence reconstruction of extant and ancestral variants of the NylB family, have one or more increased or enhanced properties relative to one or more of the extant enzymes. For example, in certain embodiments, the engineered polypeptides disclosed herein have activity in hydrolysing an amide bond in a polyamide. In some embodiments, the engineered polypeptides disclosed herein have an improved ability to hydrolyse amide bonds in nylon polyamide. Without being bound by theory or mode of application, the engineered polypeptides disclosed herein show enhanced hydrolysis of amide bonds of acid-terminated nylon oligomers, including nylon 6,6 oligomers. In certain embodiments, the engineered polypeptides disclosed herein have increased thermal stability. This is a highly surprising discovery, including because the temperature conditions to which a hypothetical ancestral enzyme may have been exposed would not be too dissimilar to the temperature or conditions to which one or more corresponding extant enzymes are exposed. In certain embodiments, the engineered polypeptides disclosed herein are associated with increased recombinant expression in a host cell system, where such host cells are modified by insertion of a polynucleotide sequence encoding said enzymes. This is particularly advantageous, as the presently known nylonase enzymes only demonstrate low levels of enzyme expression in common industrial host organisms.

[0037] In one aspect disclosed herein, there is provided a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the polypeptide comprises amino acid residues 2-394 of SEQ ID NO: 1, or an amino acid sequence that has at least 70% sequenceidentity thereto, and wherein the amino acid sequence of the polypeptide differs from SEQ ID NO: 1 at one or more amino acid positions selected from the group consisting of amino acid positions 220, 222, 331, 334, 336, 348, 352 and 380 of SEQ ID NO: 1.

[0038] In an embodiment, the amino acid sequence of the polypeptide differs from SEQ ID NO: 1 at position 220. In an embodiment, the amino acid sequence of the polypeptide differs from SEQ ID NO: 1 at position 222. In an embodiment, the amino acid sequence of the polypeptide differs from SEQ ID NO: 1 at position 331. In an embodiment, the amino acid sequence of the polypeptide differs from SEQ ID NO: 1 at position 334. In an embodiment, the amino acid sequence of the polypeptide differs from SEQ ID NO: 1 at position 336. In an embodiment, the amino acid sequence of the polypeptide differs from SEQ ID NO: 1 at position 348. In an embodiment, the amino acid sequence of the polypeptide differs from SEQ ID NO: 1 at position 352. In an embodiment, the amino acid sequence of the polypeptide differs from SEQ ID NO: 1 at position 380.

[0039] In an embodiment, i. the amino acid residue at position 220 is selected from the group consisting of Y, C, S, I, D, A, E and conservative amino acid substitutions of any of the foregoing; ii. the amino acid residue at position 222 is selected from the group consisting of S, E and conservative amino acid substitutions of any of the foregoing; iii. the amino acid residue at position 331 is selected from the group consisting of Y, W, D and conservative amino acid substitutions of any of the foregoing; iv. the amino acid residue at position 334 is selected from the group consisting of K, Y, E, H, D and conservative amino acid substitutions of any of the foregoing; v. the amino acid residue at position 336 is selected from the group consisting of Y, D, W, E and conservative amino acid substitutions of any of the foregoing;vi. the amino acid residue at position 348 is selected from the group consisting of I, H, E, W and conservative amino acid substitutions of any of the foregoing; vii. the amino acid residue at position 352 is selected from the group consisting of G, C and conservative amino acid substitutions of any of the foregoing; and / or viii. the amino acid residue at position 380 is selected from the group consisting of S, Y and conservative amino acid substitutions of any of the foregoing.

[0040] In an embodiment, i. the amino acid residue at position 220 is selected from the group consisting of Y, C, S, I, D, A, and E; ii. the amino acid residue at position 222 is selected from the group consisting of S and E; iii. the amino acid residue at position 331 is selected from the group consisting of Y, W, and D; iv. the amino acid residue at position 334 is selected from the group consisting of K, Y, E, H, and D; v. the amino acid residue at position 336 is selected from the group consisting of Y, D, W, and E; vi. the amino acid residue at position 348 is selected from the group consisting of I, H, E, and W; vii. the amino acid residue at position 352 is selected from the group consisting of G and C; and / or viii. the amino acid residue at position 380 is selected from the group consisting of S and Y.

[0041] In an embodiment, i. the amino acid residue at position 220 is selected from the group consisting of C, S, I, D, A, and E; ii. the amino acid residue at position 222 is S; iii. the amino acid residue at position 331 is Y; iv. the amino acid residue at position 334 is K; v. the amino acid residue at position 336 is Y; vi. the amino acid residue at position 348 is I; vii. the amino acid residue at position 352 is G; and viii. the amino acid residue at position 380 is S.

[0042] In an embodiment, i. the amino acid residue at position 220 is Y; ii. the amino acid residue at position 222 is E; iii. the amino acid residue at position 331 is Y; iv. the amino acid residue at position 334 is K; v. the amino acid residue at position 336 is Y; vi. the amino acid residue at position 348 is I; vii. the amino acid residue at position 352 is G; and viii. the amino acid residue at position 380 is S.

[0043] In an embodiment, i. the amino acid residue at position 220 is Y; ii. the amino acid residue at position 222 is S; iii. the amino acid residue at position 331 is selected from the group consisting of W and D; iv. the amino acid residue at position 334 is K;v. the amino acid residue at position 336 is Y; vi. the amino acid residue at position 348 is I; vii. the amino acid residue at position 352 is G; and viii. the amino acid residue at position 380 is S.

[0044] In an embodiment, i. the amino acid residue at position 220 is Y; ii. the amino acid residue at position 222 is E; iii. the amino acid residue at position 331 is Y; iv. the amino acid residue at position 334 is selected from the group consisting of Y, E, H and D; v. the amino acid residue at position 336 is Y; vi. the amino acid residue at position 348 is I; vii. the amino acid residue at position 352 is G; and viii. the amino acid residue at position 380 is S.

[0045] In an embodiment, i. the amino acid residue at position 220 is Y; ii. the amino acid residue at position 222 is E; iii. the amino acid residue at position 331 is Y; iv. the amino acid residue at position 334 is K; v. the amino acid residue at position 336 is selected from the group consisting of D, W and E; vi. the amino acid residue at position 348 is I; vii. the amino acid residue at position 352 is G; and viii. the amino acid residue at position 380 is S.

[0046] In an embodiment, i. the amino acid residue at position 220 is Y; ii. the amino acid residue at position 222 is E; iii. the amino acid residue at position 331 is Y; iv. the amino acid residue at position 334 is K; v. the amino acid residue at position 336 is Y; vi. the amino acid residue at position 348 is selected from the group consisting ofH, E and W;vii. the amino acid residue at position 352 is G; and viii. the amino acid residue at position 380 is S.

[0047] In an embodiment, i. the amino acid residue at position 220 is Y; ii. the amino acid residue at position 222 is E; iii. the amino acid residue at position 331 is Y; iv. the amino acid residue at position 334 is K; v. the amino acid residue at position 336 is Y; vi. the amino acid residue at position 348 is I; vii. the amino acid residue at position 352 is C; and viii. the amino acid residue at position 380 is selected from the group consisting of S or and Y.

[0048] In an embodiment, i. The polypeptide of any one of claims 1-3, wherein ii. the amino acid residue at position 220 is Y; iii. the amino acid residue at position 222 is E; iv. the amino acid residue at position 331 is Y; v. the amino acid residue at position 334 is K; vi. the amino acid residue at position 336 is Y; vii. the amino acid residue at position 348 is I; viii. the amino acid residue at position 352 is selected from the group consisting of G and C; and ix. the amino acid residue at position 380 is Y.

[0049] In an embodiment, the polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1.

[0050] In an embodiment, the polypeptide comprises an amino acid sequence of amino acid residues 2-394 of SEQ ID NO:2. In an embodiment, the polypeptide consists of an amino acid sequence of amino acid residues 2-394 of SEQ ID NO:2.

[0051] In an embodiment, the polypeptide comprises residues 2-394 of an amino acid sequence selected from the group consisting of SEQ ID Nos: 3-22. In an embodiment, the polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID Nos: 3-22.

[0052] In one aspect, there is provided a polypeptide that comprises an amino acid sequence of amino acid residues 2-394 of SEQ ID NO:23. In an embodiment, the polypeptide consists of an amino acid sequence of SEQ ID NO:23.

[0053] Herein, the terms "peptide", "polypeptide", "protein", "enzyme" are to be understood as referring to a chain of amino acids linked by peptide bonds, irrespective of the number of amino acids forming said chain. The amino acids are typically 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).

[0054] The terms "mutant" and "variant" may be used interchangeably herein to refer to a polypeptide comprising an amino acid sequence that is derived from a wildtype or extant enzyme or from the ancestral sequence reconstruction process and may further comprise a modification or alteration (e.g., a substitution, insertion, and / or deletion), at one or more (e.g., several) positions and having enhanced activity in catalysing the hydrolysis of nylon, nylon polymer or nylon oligomers, when compared to extant nylonases. Such variants may be obtained by various techniques well known in the art, illustrative examples of which include site-directed mutagenesis, random mutagenesis and synthetic oligonucleotide construction. The terms "modification", "alteration", "substitution" and the like, as used herein in relation to an amino acid residue or position, typically mean that the amino acid in the particular position has been modified compared to the amino acid of the wild-type or parent polypeptide.

[0055] Suitable substitutions may include the replacement of an amino acid residue by another selected from the naturally-occurring standard 20 amino acid residues, rare naturally occurring amino acid residues (e.g., hydroxyproline, hydroxylysine, allohydroxylysine, 6- N-methylysine, N-ethylglycine, N-methylglycine, N-ethylasparagine, allo-isoleucine, N- methylisoleucine, N-methylvaline, pyroglutamine, aminobutyric acid, ornithine, norleucine, norvaline), and non-naturally occurring amino acid residue, often made synthetically, (e.g., cyclohexyl-alanine). Preferably, the substitution comprises 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). The modification or alteration may be identified herein using the following terminology: Y197V denotes that amino acid residue Tyrosine (Y) at position 197 of the parent polypeptide sequence is substituted for a Valine (V). Y197V / I / M denotes that amino acid residue Tyrosine (Y) at position 197 of the parent sequence may be substituted for one of the following amino acids: Valine (V), Isoleucine (I), or Methionine (M). The substitution can be a conservative or nonconservative substitution. Examples of conservative substitutions will be familiar to persons skilled in the art, illustrative examples of which include substitutions within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine, asparagine and threonine), hydrophobic amino acids (methionine, leucine, isoleucine, cysteine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine and serine).

[0056] Unless otherwise specified, the positions disclosed in the present application are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, both of which are 394 amino acids in length. In this context, the term "corresponding to", when used in reference to an amino acid position, is intended to mean an amino acid position in a polypeptide sequence when that position is aligned with the equivalent or corresponding position in the sequence set forth in SEQ ID NO: 1 or SEQ ID NO:2.

[0057] 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. In a preferred embodiment, the sequence identity is determined by comparing the sequences when aligned so as to maximize overlap and identity while minimizing sequence gaps. Sequence identity may be determined using any of a number ofmathematical global or local alignment algorithms known to persons skilled in the art, depending on the length of the two sequences. Sequences of similar lengths may be aligned using a global alignment algorithms (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 the purposes of determining percent amino acid sequence identity can be achieved by any means available to persons skilled in the art, illustrative examples of which include publicly available computer software, such as is available at http: / / blast.ncbi.nlm.nih.gov / or http: / / www.ebi.ac.uk / Tools / emboss / ). Persons skilled in the art can readily determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. As used herein, % sequence identity typically refers to values generated using pair wise sequence alignment that creates an optimal global alignment of two sequences (e.g., using the Needleman-Wunsch algorithm), where all search parameters are set to default values, e.g., Scoring matrix = BLOSUM62, Gap open = 10, Gap extend = 0.5, End gap penalty = false, End gap open = 10 and End gap extend = 0.5.

[0058] The present disclosure also extends to a composition comprising the polypeptide as described herein. The present disclosure also extends to a nucleic acid sequence encoding the polypeptide described herein. The present disclosure also extends to an expression vector comprising the nucleic acid sequence described herein. The present disclosure also extends to a host cell comprising the nucleic acid sequence or the expression vector described herein.

[0059] The term "recombinant", as used herein, typically refers to a nucleic acid construct, a vector, a polypeptide or a cell produced by genetic engineering.

[0060] The term "expression", as used herein, typically refers to any step involved in the production of a polypeptide, such as by transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0061] The term "expression cassette" denotes a nucleic acid construct comprising a coding region, and suitably a regulatory region to which the coding region is operably linked.

[0062] The term "expression vector" typically means a DNA or RNA molecule that comprises an expression cassette. The expression vector may be a linear or circular double stranded DNA molecule.

[0063] As used herein, the term "nucleic acid", "nucleic sequence" "polynucleotide", "oligonucleotide" and "nucleotide sequence" are used interchangeably and refer to a sequence of deoxyribonucleotides and / or ribonucleotides. The nucleic acids can be DNA (cDNA or gDNA), RNA, or a mixture of the two. It can be in single stranded form or in duplex form or a mixture of the two. It can be of recombinant, artificial and / or synthetic origin and it can comprise modified nucleotides, comprising for example a modified bond, a modified purine or pyrimidine base, or a modified sugar. The nucleic acids of the invention can be in isolated or purified form, and made, isolated and / or manipulated by techniques known per se in the art, e.g., cloning and expression of cDNA libraries, amplification, enzymatic synthesis or recombinant technology. The nucleic acids can also be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Belousov (1997) Nucleic Acids Res. 25:3440-3444.

[0064] The nucleic acid sequences disclosed herein may suitably be codon optimized. Suitable methods for codon optimization will be familiar to persons skilled in the art, illustrative examples of which are described in the reference manual Sambrook et al. (Sambrook et al., 2001).

[0065] The nucleic acid sequences described herein may be suitably deduced from the amino acid sequence of the polypeptides described herein and codon usage may be adapted according to the host cell in which the nucleic acid shall be transcribed.

[0066] In some embodiments, the nucleic acid sequences described herein may suitably comprise additional nucleotide sequences, such as regulatory regions, i.e., promoters, enhancers, silencers, terminators, signal peptides and the like that can be used to cause or regulate expression of the polypeptide in a selected host cell or system. Alternatively, or in addition, the nucleic acid sequences described herein may further comprise additional nucleotide sequences encoding fusion proteins, such as maltose binding protein (MBP) or glutathion S transferase (GST) that can be used to favor polypeptide expression and / or solubility.

[0067] As noted elsewhere herein, the present disclosure also extends to expression vectors and expression cassettes comprising the nucleic acid sequence described herein, optionally operably linked to one or more control sequences that direct the expression of the nucleic acid sequence in a suitable host cell. Typically, the expression vector or cassette comprises the nucleic acid sequence described herein operably linked to a control sequence such as transcriptional promoter and / or transcription terminator. The control sequence may include a promoter that is recognized by a host cell or an in vitro expression system for expression of the nucleic acid encoding the polypeptide described herein. The promoter will typically comprise a transcriptional control sequence that mediates the expression of the polypeptide. The promoter may be any polynucleotide that shows transcriptional activity in a host cell, including mutant, truncated, and hybrid promoters, and may suitably be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell. The control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription. The terminator is typically operably linked to the 3 '-terminus of the nucleic acid encoding the polypeptide. Any terminator that is functional in the host cell may be used in this context. Typically, the expression vector or cassette comprises the nucleic acid sequence described herein operably linked to a transcriptional promoter and a transcription terminator.

[0068] The term "vector" typically refers to a DNA molecule used as a vehicle to transfer recombinant genetic material into a host cell. Suitable vectors include plasmids, bacteriophages, viruses, fosmids, cosmids, and artificial chromosomes. The vector is typically a DNA sequence that comprises an insert (a heterologous nucleic acid sequence, transgene) and a larger sequence that serves as the "backbone" of the vector. The purpose of a vector which transfers genetic information to the host is typically to isolate, multiply, or express the insert in the target cell. Expression vectors (also referred to as expression constructs) are specifically adapted for the expression of the heterologous sequences in the target cell, and generally have a promoter sequence that drives expression of the heterologous sequences encoding a polypeptide.

[0069] Generally, the regulatory elements that are used in an expression vector include a transcriptional promoter, a ribosome binding site, a terminator, and optionally present operator. An expression vector may further comprise an origin of replication for autonomous replication in a host cell, a selectable marker, a limited number of useful restriction enzymesites, and a potential for high copy number. Suitable expression vectors will be familiar to persons skilled in the art, illustrative examples of which include cloning vectors, modified cloning vectors, plasmids and viruses. Expression vectors that are capable of providing suitable levels of polypeptide expression in different hosts are also well known in the art. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.

[0070] The present disclosure also extends to a host cell comprising the nucleic acid sequence described herein. The host cell may be transformed, transfected or transduced in a transient or stable manner. The nucleic acid, expression cassette or vector is introduced into a host cell so that the nucleic acid, cassette or vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector. The term "host cell" encompasses any progeny of a parent host cell that is not identical to the parent host cell due to mutations that occur during replication. The host cell may be any cell useful in the production of a variant of the present invention, e.g., a prokaryote or a eukaryote. The prokaryotic host cell may be any Gram-positive or Gram-negative bacterium. The host cell may also be a eukaryotic cell, such as a yeast, fungal, mammalian, insect or plant cell. In a particular embodiment, the host cell is selected from the group of Escherichia coli, Pseudomonas, Bacillus, Streptomyces, Trichoderma, Aspergillus, Saccharomyces, Pichia, Thermus or Yarrowia.

[0071] The nucleic acid, expression cassette or expression vector according to the invention may be introduced into the host cell by any suitable method known to persons skilled in the art, illustrative examples of which include electroporation, conjugation, transduction, competent cell transformation, protoplast transformation, protoplast fusion, biolistic "gene gun" transformation, PEG-mediated transformation, lipid-assisted transformation or transfection, chemically mediated transfection, lithium acetate-mediated transformation and liposome-mediated transformation.

[0072] In an embodiment, the host cell is a genetically modified host cell or microorganism. In this context, a host cell or microorganism may be genetically modified to enhance the expression of the polypeptide in which it is expressed and / or activity of the host cell. For example, the polypeptide described herein may be used to complement a wild type strain of a fungus or bacteria known to be capable of nylonase activity, in order to improve and / or increase the nylonase activity of that strain.

[0073] The polypeptides as disclosed herein are capable of hydrolysing an amide bond in a polyamide. In an embodiment, the polypeptides are capable of hydrolysing a polyamide. In an embodiment, the polyamide is a nylon. In an embodiment, the polyamide is a nylon polymer or a nylon oligomer. In an embodiment, the polyamide is nylon 6. In an embodiment, the polyamide is nylon 6,6. In another embodiment, the polypeptides are capable of hydrolysing nylon, including nylon 6,6, as shown in the illustrative examples. In an embodiment, the polypeptide is capable of hydrolysing an amide bond in a nylon oligomer. In an embodiment, the nylon polymer or nylon oligomer is a nylon 6 polymer or a nylon 6 oligomer. In another embodiment, the nylon polymer or nylon oligomer is a nylon 6, 6 polymer or a nylon 6,6 oligomer. Enzymes capable of digesting, hydrolysing or breaking down nylon, are broadly called nylonases. Nylonases were first discovered in bacteria capable of digesting byproducts of nylon 6 manufacture. The three nylonases were 6- aminohexanoate-cyclic-dimer hydrolase (El, NylA, UniProt: Pl 3398) 6-aminohexanoate- dimer hydrolase (Eli, NylB, UniProt: P07061) and 6-aminohexanoate-oligomer endohydrolase (EIII, NylC, UniProt: Q57326).

[0074] In some embodiments, the polypeptides disclosed herein have adipic acid mono- and di- N-alkyl amide hydrolase activity. In some embodiments, the polypeptides disclosed herein are capable to hydrolysing nylon 6,6 polymer and nylon 6,6 oligomers. Methods for determining and measuring such activities, including adipic acid mono- and di- N-alkyl amide hydrolase activity, will be known to persons skilled in the art, illustrative examples of which are disclosed elsewhere herein.

[0075] The term "nylon" or "nylon polymer" or "nylon oligomers" refers to synthetic polymers composed of polyamides (repeating units linked by amide links). In one embodiment, the nylon or nylon polymer or nylon oligomer is nylon 6 (polycaprolactam). In another embodiment, the nylon or nylon polymer or nylon oligomer is nylon 6,6 (containing diamines and dicarboxylic acids). In one embodiment, the nylon or nylon polymer or nylon oligomer is a copolymer of different nylons, comprising of nylon 6 and / or nylon 6, 6. In another embodiment, the nylon or nylon polymer or nylon oligomer is a nylon blend of different nylons comprising of nylon 6 and / or nylon 6,6.

[0076] The term "polymer", as used herein, typically refers to a chemical compound or a mixture of compounds whose structure is made up of multiple monomers (repeat units) linked by covalent chemical bonds. Within the context of the invention, the term polymerincludes natural or synthetic polymers, constituted of a single type of repeat unit (z.e., homopolymers) or of a mixture of different repeat units (z.e., copolymers or heteropolymers).

[0077] Oligomers are low molecular weight polymers containing few repeated units whose physical properties are typically dependent upon the length of the chain. The term "nylon oligomers" as used herein refers to a molecule of nylon containing a discrete number of repeating nylon monomer unit. In an embodiment, the nylon oligomer contains up to 50- mers of nylon. In another embodiment, the nylon oligomer contains up to 40-mers of nylon. In another embodiment, the nylon oligomer contains up to 30-mers of nylon. In another embodiment, the nylon oligomer contains up to 20-mers of nylon. In another embodiment, the nylon oligomer contains 20-mers, 19-mers, 18-mers, 17-mers, 16-mers, 15-mers, 14- mers,13-mers, 12-mers, 11-mers, 10-mers of nylon. In another embodiment, the nylon oligomer contains 9-mers, 8-mers, 7-mers, or 6-mers, of nylon. In another embodiment, the nylon oligomer are pentamers, tetramers, trimers or dimers of nylon. In an embodiment, the nylon oligomer is a solid nylon oligomer. In an embodiment, the nylon oligomer is a solubilised nylon oligomer. In an embodiment, the nylon oligomer is an aqueous soluble nylon oligomer. A solubilised nylon oligomer is an oligomer that is not in solid form, but is in solution (z.e., fluid or liquid form). That is the oligomer has dissolved in a liquid solution, usually through the addition of a solvent. In some embodiments, the oligomers are solubilised in an aqueous solution. In some embodiments, the oligomers are solubilised in a non-aqueous solution. In an embodiment, the solubilised nylon oligomer comprises 10-mers, 9-mers, 8-mers, 7-mers, or 6-mers, pentamers, tetramers, trimers and / or dimers of nylon. In an embodiment, the solubilised nylon oligomer comprises 10-mers, 9-mers, 8-mers, 7-mers, or 6-mers, pentamers, tetramers, trimers and / or dimers of nylon 6,6. In an embodiment, the solubilised nylon oligomer comprises 10-mers, 9-mers, 8-mers, 7-mers, or 6-mers, pentamers, tetramers, trimers and / or dimers of nylon 6. In an embodiment, the solubilised nylon oligomer contains pentamers, tetramers, trimers, or dimers. In an embodiment, the solubilised nylon oligomer contains pentamers, tetramers, trimers, or dimers of nylon 6,6. In an embodiment, the solubilised nylon oligomer contains pentamers, tetramers, trimers, or dimers of nylon 6. In some embodiments, the solubilised nylon oligomers is a mix of 10- mers, 9-mers, 8-mers, 7-mers, or 6-mers, pentamers, tetramers, trimers and / or dimers of nylon. In some embodiments, the solubilised nylon oligomers is a mix of 10-mers, 9-mers, 8-mers, 7-mers, or 6-mers, pentamers, tetramers, trimers and / or dimers of nylon 6,6. In someembodiments, the solubilised nylon oligomers is a mix of 10-mers, 9-mers, 8-mers, 7-mers, or 6-mers, pentamers, tetramers, trimers and / or dimers of nylon 6. In an embodiment, the solubilised nylon oligomer is a mix of pentamers, tetramers, trimers, or dimers of nylon. In an embodiment, the solubilised nylon oligomer is a mix of pentamers, tetramers, trimers, or dimers of nylon 6. In an embodiment, the solubilised nylon oligomer is a mix of pentamers, tetramers, trimers, or dimers of nylon 6,6. In an embodiment, the nylon oligomer is a tetramer. In an embodiment, the nylon oligomer is a trimer. In an embodiment, the nylon oligomer is a dimer. In another embodiment, the solubilised nylon oligomers contains tetramers, trimers or dimers. In an embodiment, the nylon oligomer is a tetramer of nylon 6. In an embodiment, the nylon oligomer is a trimer of nylon 6. In an embodiment, the nylon oligomer is a dimer of nylon 6. In another embodiment, the solubilised nylon oligomers contains tetramers, trimers or dimers of nylon 6. In an embodiment, the nylon oligomer is a tetramer of nylon 6,6. In an embodiment, the nylon oligomer is a trimer of nylon 6,6. In an embodiment, the nylon oligomer is a dimer of nylon 6,6. In another embodiment, the solubilised nylon oligomers contains tetramers, trimers or dimers of nylon 6,6. Without being bound by theory or mode of application, the polypeptides disclosed herein hydrolyse an amide bond in a carboxylic acid terminated nylon oligomer. In an embodiment, the nylon oligomer is an acid terminated oligomer of nylon. In another embodiment, the nylon oligomer is a carboxylic acid terminated oligomer of nylon. In an embodiment, the nylon oligomer is nylon dimer, nylon trimer, tetramer, pentamer, 6-mers, 7-mers, 8-mers, 9-mers or 10-mers, wherein at least one terminus of the nylon oligomer is carboxylic acid terminated. In another embodiment, the nylon oligomer is a carboxylic acid terminated oligomer of nylon 6,6. It will be appreciated the oligomer can be terminated at one or both of its terminal ends with a carboxylic acid group. For example, a nylon 6,6 dimer will be terminated at one end with a carboxylic acid group and terminated at the other end with an amine group. In another example, a nylon 6,6 trimer may be terminated at both of its terminal ends with a carboxylic acid group or it may be terminated at both of its terminal ends with an amine group. In another example of a carboxylic acid end capped oligomer as described herein, such as the nylon 6, 6 trimer described herein, the oligomer is terminated at both of its terminal ends with a carboxylic acid group. As used herein, the terms "nylon-containing material", "nylon-containing product" and the like are to be understood as refers to a product, such as nylon product, comprising at least one nylon in crystalline, semi-crystalline or totally amorphous form. The nylon- containing material may refer to any item made from at least one nylon, such as nylon sheet, tube, rod, profile, shape, film, massive block, fibre, textiles,etc., which contains at least one nylon, and possibly other substances or additives, such as plasticizers, mineral or organic fillers. In an embodiment, the nylon-containing material is a textile or fabric comprising at least one nylon containing fibre. In another embodiment, the nylon-containing material is a nylon compound, or nylon formulation, in a molten or solid state, suitable for making a nylon product.

[0078] The nylon-containing material or nylon-containing product may comprise of nylon and one or more synthetic and / or natural materials. The nylon-containing material or nylon- containing product may comprise of nylon and another plastics such as polyesters, acrylics, polyurethanes, polyethylene (PE), polypropylene (PP), polystyrene (PS), and polyvinyl chloride (PVC). The nylon-containing material or nylon-containing product may comprise of nylon and polyethylene terephthalate (PET). The nylon-containing material or nylon- containing product may comprise of nylon and natural materials such as cotton, silk, cellulose, linen and wool.

[0079] In another aspect, the present disclosure provides a method of producing a polypeptide capable of hydrolysing an amide bond in a polyamide, the method comprising: i) providing the polynucleotide described herein; ii) expressing the polynucleotide in a host cell under conditions sufficient to allow the host cell to produce the polypeptide; and iii) collecting the polypeptide produced by the host cell in ii).

[0080] In another aspect, the present disclosure provides a method of hydrolysing nylon polymer or nylon oligomer, the method comprising exposing the nylon polymer or nylon oligomer to the polypeptide, the composition or the host cell disclosed herein under conditions sufficient to convert the nylon-6, 6 polymer to adipic acid and / or hexamethyl enedi amine .

[0081] In another aspect, the present disclosure provides a method of degrading a nylon- containing product, the method comprising exposing the nylon-containing product to the polypeptide, the composition or the host cell as disclosed herein.

[0082] In an embodiment, the methods disclosed herein comprises i) chemical processing of nylon polymer, nylon oligomer or nylon-containing product to generate nylon dimers, trimers, tetramers, pentamers and hexamers and ii) exposing the nylon dimers, trimers, tetramers, pentamers and hexamers generated in step (i) to the polypeptide, the composition or the host cell as disclosed herein, under conditions sufficient to produce adipic acid and / or hexamethylenediamine. In an embodiment, the methods disclosed herein comprises i) chemical processing of nylon polymer, nylon oligomer or nylon-containing product to generate nylon dimers, trimers, tetramers, pentamers and hexamers, wherein the nylon dimers, trimers, tetramers, pentamers and hexamers are acid terminated nylon dimers, trimers, tetramers, pentamers and hexamers, and ii) exposing the nylon dimer, trimer, tetramer, pentamers and hexamers generated in step (i) to the polypeptide, the composition or the host cell as disclosed herein, under conditions sufficient to produce adipic acid and / or hexamethylenediamine. In an embodiment, the methods disclosed herein comprises i) chemical processing of nylon polymer, nylon oligomer or nylon-containing product to generate nylon dimers, trimers, tetramers, pentamers and hexamers, wherein the nylon dimers, trimers, tetramers, pentamers and hexamers are carboxylic acid terminated oligomer nylon dimers, trimers, tetramers, pentamers and hexamers, and ii) exposing the nylon dimer, trimer, tetramer, pentamers and hexamers generated in step (i) to the polypeptide, the composition or the host cell as disclosed herein, under conditions sufficient to produce adipic acid and / or hexamethylenediamine. Also contemplated herein are embodiments in which the step (i) of the methods disclosed herein comprises chemical processing of nylon polymer, nylon oligomer or nylon-containing product to generate larger nylon oligomers, acid terminated nylon oligomers or carboxylic acid terminated nylon oligomers, including, for example, heptamers, octamers, nanomers, decamers, hendecamers, dodecamers, and so on.

[0083] In an embodiment, the nylon in the nylon polymer, nylon oligomer or the nylon- containing product is a nylon 6 or a nylon 6,6. In another embodiment, the nylon is a nylon 6,6.

[0084] In another embodiment, the methods described herein further comprise recovering the adipic acid and / or the hexamethylenediamine produced in step (ii).

[0085] In another aspect, the present disclosure provides a composition comprising the adipic acid and / or the hexamethylenediamine recovered by the methods disclosed herein.

[0086] In another aspect, the present disclosure provides a method of producing a nylon polymer using the composition of adipic acid and / or the hexamethylenediamine recovered by the methods disclosed herein.

[0087] In the context of the present disclosure, reference to increased or enhanced activity hydrolysis of an amide bond in a polyamide, indicates an increased ability of thepolypeptides / novel engineered polypeptides and its variants to hydrolyse nylon, nylon polymers or nylon oligomers, when compared to extant or wild-type NylB or nylonase enzymes, such as the polypeptides having the amino acid sequence of SEQ ID NOs: 24-38. In an embodiment, the activity of the polypeptide described herein is increased by at least about 1%. In an embodiment, the activity of the polypeptide described herein is increased by at least about 5%, preferably by at least about 10%, preferably by at least about 20%, preferably by at least about 30%, preferably by at least about 40%, preferably by at least about 50%, preferably by at least about 100%, preferably by at least about 200%, preferably by at least about 300%, preferably by at least about 400%, preferably by at least about 500%, preferably by at least about 600%, preferably by at least about 700%, preferably by at least about 800%, preferably by at least about 900%, or more preferably by at least about 1,000% or more in comparison to extant or wild-type NylB or nylonase enzymes, such as the polypeptides having the amino acid sequence of SEQ ID NOs: 24-38.

[0088] As noted elsewhere herein, the present inventors have engineered polypeptides with improved, increased or enhanced hydrolase activity, such as high activity, or broader activity on nylon polymers and nylon oligomers. These nylon oligomers may be solubilised nylon oligomers. The solubilised nylon oligomers may be dimers, trimers, tetramers, pentamers, hexamers, 7-mers, 8-mers, 9-mers or 1 Omers of nylon. The present disclosure is predicated, at least in part, on the inventors' surprising finding that said engineered polypeptides, from the ancestral sequence reconstruction of extant and ancestral variants of the NylB family, have one or more increased or enhanced properties relative to one or more of the extant enzymes. This increased or enhanced activity is also seen with substrates of nylon 6,6 oligomers. For example, in certain embodiments, the engineered polypeptides disclosed herein have increased activity in hydrolysing an amide bond in a polyamide; as shown in an example provided herein, an improved ability to hydrolyse amide bonds in nylon polyamide. In certain embodiments, the engineered polypeptides disclosed herein have increased activity in hydrolysing an amide bond in a carboxylic acid terminated nylon oligomer. In certain embodiments, the engineered polypeptides disclosed herein have increased activity in hydrolysing an amide bond in a carboxylic acid terminated nylon oligomer, wherein the carboxylic acid terminated nylon oligomer is a carboxylic acid terminated nylon6,6 trimer. In certain embodiments, the engineered polypeptides disclosed herein have increased thermal stability. This is a highly surprising discovery, including because the temperature conditions to which a hypothetical ancestral enzyme may have been exposed would not betoo dissimilar to the temperature or conditions to which one or more corresponding extant enzymes are exposed. In certain embodiments, the engineered polypeptides disclosed herein are associated with increased recombinant expression in a host cell system, where such host cells are modified by insertion of a polynucleotide sequence encoding said enzymes. Presently known nylonase enzymes only demonstrate low levels of enzyme expression in common industrial host organisms.

[0089] The polypeptides disclosed here in have superior properties for use in industrial processes. With the aim of improving the activity of hydrolases, in particular those that can hydrolyse nylon, in conditions (i.e. heat, pH and pressure conditions) at which industrial degradation of nylon polymer and / or nylon-containing products are typically performed. The polypeptides disclosed herein capable of hydrolysing an amide bond in a polyamide, and are particularly suited for the degradation of nylon 6,6 polymers, nylon 6,6, oligomers and / or nylon 6,6-containing materials or products. The engineered polypeptides as described herein have adipic acid mono- and di- N-alkyl amide hydrolase activity.

[0090] In the context of the present disclosure, reference to increased or enhanced activity may include one or more of the following: increased ability of the polypeptide to hydrolyse an amide bond in a polyamide, including nylons such as nylon 6 and nylon 6,6 when compared to extant or wild-type NylB or nylonase enzymes; increased recombinant expression in a host cell system when compared to extant or wild-type NylB or nylonase enzymes; increased whole cell activity when compared to extant or wild-type NylB or nylonase enzymes; and increased thermostability when compared to extant or wild-type NylB or nylonase enzymes. Examples of extant, wildtype NylB or nylonase enzymes include the polypeptides having the amino acid sequences of SEQ ID NOs: 24-38.

[0091] In an embodiment, the polypeptide capable of hydrolysing an amide bond in a polyamide as disclosed herein demonstrates increased recombinant expression in a host cell system, when compared to extant or wild-type NylB or nylonase enzymes.

[0092] In another embodiment, the polypeptide capable of hydrolysing an amide bond in a polyamide as disclosed herein demonstrates increased thermostability when compared to extant or wild-type NylB or nylonase enzymes.

[0093] In another embodiment, the polypeptide capable of hydrolysing an amide bond in a polyamide as disclosed herein comprises increased whole cell activity when compared toextant or wild-type NylB or nylonase enzymes. As used herein, the term whole cell activity typically refers to the ability of the polyamide, just as nylon polymer or nylon oligomers when expressed in a host cell system.

[0094] In an embodiment, the activity of the polypeptide in hydrolysing an amide bond in a polyamide or in hydrolysing nylon 6 oligomers or nylon 6,6 oligomers as disclosed herein is similar to an extant or wild-type NylB or nylonase enzymes. In an embodiment, the activity of the polypeptide in hydrolysing an amide bond in a polyamide or in hydrolysing nylon 6 oligomers or nylon 6,6 oligomers as disclosed herein described herein of the polypeptide described herein is increased by at least about 5%, preferably by at least about 10%, preferably by at least about 20%, preferably by at least about 30%, preferably by at least about 40%, preferably by at least about 50%, preferably by at least about 100%, preferably by at least about 200%, preferably by at least about 300%, preferably by at least about 400%, preferably by at least about 500%, preferably by at least about 600%, preferably by at least about 700%, preferably by at least about 800%, preferably by at least about 900%, or more preferably by at least about 1,000% or more to a polypeptide having the amino acid sequence of a extant or wildtype NylB enzyme. In an embodiment, the activity of the polypeptide in hydrolysing nylon 6,6 oligomers as disclosed herein is similar to an extant or wild-type NylB or nylonase enzymes. In an embodiment, the activity of the polypeptide in hydrolysing nylon 6,6 oligomers as disclosed herein described herein of the polypeptide described herein is increased by at least about 5%, preferably by at least about 10%, preferably by at least about 20%, preferably by at least about 30%, preferably by at least about 40%, preferably by at least about 50%, preferably by at least about 100%, preferably by at least about 200%, preferably by at least about 300%, preferably by at least about 400%, preferably by at least about 500%, preferably by at least about 600%, preferably by at least about 700%, preferably by at least about 800%, preferably by at least about 900%, or more preferably by at least about 1,000% or more when compared to extant or wild-type NylB or nylonase enzymes. Suitable methods of determining or measuring enzyme activity of a polypeptide will be familiar to persons skilled in the art, an illustrative example of which is described elsewhere herein. Other methods of measuring nylon hydrolysis are described in Kiumarsi and Parvinzadeh, 2010 J Appl Polymer Sci, 116:3140 and Gashti et al., 2013 Preparative Biochemistry & Biotechnology, 43: 798, the contents of which are incorporated herein by reference in their entirety. In an embodiment, the activity of the polypeptide in hydrolysing nylon 6,6 oligomers as disclosed herein is increased by at least about 5%,preferably by at least about 10%, preferably by at least about 20%, preferably by at least about 30%, preferably by at least about 40%, preferably by at least about 50%, preferably by at least about 100%, preferably by at least about 200%, preferably by at least about 300%, preferably by at least about 400%, preferably by at least about 500%, preferably by at least about 600%, preferably by at least about 700%, preferably by at least about 800%, preferably by at least about 900%, or more preferably by at least about 1,000% or more when compared to extant or wild-type NylB or nylonase enzymes, when determined by a colorimetric assay or LC-MC methods using solubilised nylon 6,6 oligomers, including dimers, trimers and tetramers as a substrate.

[0095] The activity of the polypeptide in hydrolysing an amide bond in a polyamide or in hydrolysing nylon 6 oligomers or nylon 6,6 oligomers as disclosed herein may be assigned an absolute value or a value relative to the activity of a comparator (e.g., extant or wild-type NylB or nylonase enzymes). In an embodiment, the activity of the polypeptide is measured as the rate of monomers and / or oligomers is released over time, under suitable conditions of temperature, pH and buffer. In another embodiment, the activity of the polypeptide is measured as the rate of digestion of the substrate (i.e. measuring rate of change in the concentration or amounts of substrate) over time under suitable conditions of temperature, pH and buffer.

[0096] The activity of the polypeptide in hydrolysing an amide bond in a polyamide or in hydrolysing nylon 6 oligomers or nylon 6,6 oligomers can be measured or assayed using a purified enzyme. Alternatively, enzyme activity can be measured as a function of the activity of the enzyme when recombinantly expressed in a host cell system (also referred to herein as cellular catalytic activity or whole cell activity).

[0097] Advantageously, the polypeptide described herein exhibits increased or enhanced recombinant expression in a host cell by at least about 5%, preferably by at least about 10%, preferably by at least about 20%, preferably by at least about 30%, preferably by at least about 40%, preferably by at least about 50%, preferably by at least about 100%, preferably by at least about 200%, preferably by at least about 300%, preferably by at least about 400%, preferably by at least about 500%, preferably by at least about 600%, preferably by at least about 700%, preferably by at least about 800%, preferably by at least about 900%, or more preferably by at least about 1,000% when compared to extant or wild-type NylB or nylonase enzymes.

[0098] The polypeptide here displays increased thermostability, that is the ability of to resist irreversible change in enzyme function or activity (i.e. denature) when exposed to extreme temperatures. The polypeptide described herein retains its ability to hydrolyse an amide bond in a polyamide or in hydrolysing nylon 6 oligomers or nylon 6,6 oligomers after exposure to a range of temperatures from about 4°C to about 70°C, preferably from about 10°C to about 70°C, preferably from about 20°C to about 60°C, more preferably from about 35°C to about 55°C, even more preferably from about 40°C to about 50°C, even more preferably at about 45°C. In an embodiment, the polypeptide described herein exhibits activity at a temperature from about 10°C to about 60°C, preferably from about 20°C to about 60°C, preferably from about 30°C to about 60°C, more preferably from about 40°C to about 60°C, even more preferably from about 40°C to about 50°C, or even more preferably at about 45 °C. Advantageously, the polypeptide described herein exhibits the ability to hydrolyse an amide bond in a polyamide or in hydrolysing nylon 6 oligomers or nylon 6,6 oligomers in a range of temperatures from about 10°C to about 80°C, preferably from about 20°C to about 70°C, preferably from about 30°C to about 60°C, more preferably from about 35°C to about 55°C, even more preferably from about 40°C to about 50°C, even more preferably at about 45°C. In an embodiment, the polypeptide described herein exhibits activity at a temperature from about 10°C to about 70°C, preferably from about 20°C to about 60°C, preferably from about 30°C to about 60°C, more preferably from about 40°C to about 60°C, even more preferably from about 40°C to about 50°C, or even more preferably at about 45°C. In an embodiment, the ability to hydrolyse an amide bond in a polyamide or in hydrolysing nylon 6 oligomers or nylon 6,6 oligomers is measurable at a temperature between about 40°C and about 70°C, preferably between about 40°C and about 50°C, or even more preferably at about 45°C. In another particular embodiment, the ability to hydrolyse an amide bond in a polyamide or in hydrolysing nylon 6 oligomers or nylon 6,6 oligomers is still measurable at a temperature between about 10°C and about 30°C, preferably between about 15°C and about 28°C, corresponding to the mean temperature in the natural environment (ambient temperature).

[0099] In an embodiment, the polypeptide comprises the ability to hydrolyse an amide bond in a polyamide or in hydrolysing nylon 6 oligomers or nylon 6,6 oligomers is measurable at a temperature from about 10°C to about 60°C, preferably from about 20°C to about 60°C, preferably from about 30°C to about 60°C, more preferably from about 40°C to about 60°C, even more preferably from about 40°C to about 50°C, or even more preferably at about 45°Cof at least about 5%, preferably by at least about 10%, preferably by at least about 20%, preferably by at least about 30%, preferably by at least about 40%, preferably by at least about 50%, preferably by at least about 100%, preferably by at least about 200%, preferably by at least about 300%, preferably by at least about 400%, preferably by at least about 500%, preferably by at least about 600%, preferably by at least about 700%, preferably by at least about 800%, preferably by at least about 900%, or more preferably by at least about 1,000% when compared to extant or wild-type NylB or nylonase enzymes .

[0100] In another particular embodiment, the polypeptide described herein has increased activity in hydrolysing an amide bond in a polyamide or in hydrolysing nylon 6 oligomers or nylon 6,6 oligomers, when compared to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 24-38 or a wildtype NylB enzyme, at a temperature of between about 10°C and about 70°C, preferably between about 20°C and about 60°C, preferably from about 30°C to about 60°C, preferably between about 40°C and about 60°C, preferably between about 40°C and about 50°C, or more preferably at about 45°C. In an embodiment, the polypeptide described herein is capable of hydrolysing an amide bond in a polyamide or in hydrolysing nylon 6 oligomers or nylon 6,6 oligomers at between about 20°C to about 70°C of at least about 5%, preferably by at least about 10%, preferably by at least about 20%, preferably by at least about 30%, preferably by at least about 40%, preferably by at least about 50%, preferably by at least about 100%, preferably by at least about 200%, preferably by at least about 300%, preferably by at least about 400%, preferably by at least about 500%, preferably by at least about 600%, preferably by at least about 700%, preferably by at least about 800%, preferably by at least about 900%, or more preferably by at least about 1,000% or more when compared to extant or wild-type NylB or nylonase enzymes.

[0101] In another embodiment, the polypeptide described herein has increased activity in hydrolysing an amide bond in a polyamide or in hydrolysing nylon 6 oligomers or nylon 6,6 oligomers, when compared to extant or wild-type NylB or nylonase enzymes, at a temperature between about 10°C and about 60°C, preferably between about 20°C and about 50°C, even more preferably between about 20°C and about 40°C, or even more preferably at about 40°C. In an embodiment, the polypeptide described herein has activity in hydrolysing an amide bond in a polyamide or in hydrolysing nylon 6 oligomers or nylon 6,6 oligomers at a temperature between about 10°C and about 40°C of at least about 5%, preferably by at least about 10%, preferably by at least about 20%, preferably by at leastabout 30%, preferably by at least about 40%, preferably by at least about 50%, preferably by at least about 100%, preferably by at least about 200%, preferably by at least about 300%, preferably by at least about 400%, preferably by at least about 500%, preferably by at least about 600%, preferably by at least about 700%, preferably by at least about 800%, preferably by at least about 900%, or more preferably by at least about 1,000% or more when compared to extant or wild-type NylB or nylonase enzymes.

[0102] The stability, activity and / or half-life of the polypeptides described herein under a range of different conditions (pH, temperature and ionic conditions) can be advantageous when selecting an enzyme for use under different processing or industrial conditions. In an embodiment, the polypeptide described herein exhibits a measurable activity in hydrolysing an amide bond in a polyamide or in hydrolysing nylon 6 oligomers or nylon 6,6 oligomers at least in a range of pH from 5 to 11, preferably in a range of pH from 6 to 10, more preferably in a range of pH from 6.5 to 9, even more preferably in a range of pH from 7 to 8. In some embodiments, the stability of the polypeptide described herein is not significantly improved when compared to the stability of the polypeptide of SEQ ID NO: 1. In some embodiments, the stability of the polypeptide described herein is improved when compared to the stability of the polypeptide of SEQ ID NO: 1. In some embodiments, the isoelectric point of the polypeptide described herein is not significantly different when compared to the stability of the polypeptide of SEQ ID NO: 1. In some embodiments, the isoelectric point of the polypeptide described herein is different when compared to that of the polypeptide of SEQ ID NO: 1. The stability and activity of the polypeptides here under a range of different conditions

[0103] Advantageously, the thermostability of the polypeptide described herein is not significantly impaired when compared to extant or wild-type NylB or nylonase enzymes. In some embodiments, the thermostability of the polypeptide described herein is improved when compared to the thermostability of an extant or wild-type NylB or nylonase enzymes. In some embodiments, the thermostability of the polypeptide described herein is not significantly improved when compared to the thermostability of the polypeptide of SEQ ID NO: 1. In some embodiments, the thermostability of the polypeptide described herein is improved when compared to the thermostability of the polypeptide of SEQ ID NO: 1. The term "improved thermostability" or "increased thermostability", as used herein, indicates an increased ability of the enzyme to resist changes in its chemical and / or physical structure athigher temperatures, more specifically at temperature between 40°C and 70°C, when compared to extant or wild-type NylB or nylonase enzymes. In an embodiment, the polypeptides described herein have an increased half-life at a temperature between 40°C and 70°C, when compared to extant or wild-type NylB or nylonase enzymes. The polypeptides described herein may exhibit a higher or equivalent melting temperature (Tm) when compared to extant or wild-type NylB or nylonase enzymes (see for example, Figure 4). In some embodiments, the polypeptide described herein shows improved thermostability at a temperature of between 40°C and 70°C when compared to extant or wild-type NylB or nylonase enzymes.

[0104] The thermostability of a polypeptide may be evaluated by any suitable means known to persons skilled in the art. For example, thermostability can be assessed by measuring the residual enzyme activity of the polypeptide after incubation at different temperatures. The ability to perform multiple rounds of hydrolysis at different temperatures can also be evaluated. Differential Scanning Fluorimetry (DSF) may also be used to assess the thermostability of the polypeptide. Circular dichroism may also be used to measure thermostability of the polypeptides described herein, including their melting temperatures (Tm). The term "melting temperature (Tm)" is understood to mean a given protein corresponds to the temperature at which 50% of said protein is denatured.

[0105] In an embodiment, the polypeptide described herein exhibits a melting temperature (Tm) of from about 45°C to about 80°C, preferably from about 50°C to about 75°C, preferably from about 52°C to about 75°C. In an embodiment, the polypeptide described herein exhibits a melting temperature (Tm) that is lower than the melting temperature (Tm) exhibited of an extant or wild-type NylB or nylonase enzymes. In one embodiment, the polypeptide described herein exhibits a melting temperature (Tm) that is higher than the melting temperature (Tm) exhibited by an extant or wild-type NylB or nylonase enzymes (an illustrative example is shown in Figure 4).

[0106] The present disclosure also extends to a method of producing a polypeptide capable of hydrolysing an amide bond in a polyamide or in hydrolysing nylon 6 oligomers or nylon 6,6 oligomers, the method comprising: a) providing a nucleic acid sequence as described herein; b) expressing the nucleic acid sequence in a host cell culture, thereby producing the polypeptide; andc) recovering the polypeptide produced in (b) from the host cell culture.

[0107] The present invention disclosure also extends to in vitro methods of producing the polypeptide described herein, the method comprising (a) contacting a nucleic acid, cassette or vector of the invention with an in vitro expression system; and (b) recovering the polypeptide produced. In vitro expression systems are well-known by the person skilled in the art and are commercially available.

[0108] Suitable host cells will be familiar to persons skilled in the art, illustrative examples of which include a recombinant Bacillus, recombinant E. coli, recombinant Pseudomonas, recombinant Aspergillus, recombinant Trichoderma, recombinant Streptomyces, recombinant Saccharomyces, recombinant Pichia, recombinant Thermus or recombinant Yarrowia. In an embodiment, the host cell is an E. coli.

[0109] The host cells may be cultivated in a nutrient medium suitable for production of polypeptides, using methods that will be known to persons skilled in the art. Suitable examples include cultivating the host cells by shake flask cultivation, or small-scale or large- scale fermentation (including continuous, batch, fed- batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the enzyme to be expressed and / or isolated. The cultivation will typically take place in a suitable nutrient medium, from commercial suppliers or prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection) or any other culture medium suitable for cell growth. Where the polypeptide is expressed and / or secreted into the nutrient medium, the polypeptide can be used in the form of a cellular / supernatant mixture, or in the form of a crude cell lysate. Alternatively, the polypeptide can be recovered directly from the culture supernatant. Conversely, the polypeptide can be recovered from cell lysates or after permeabilisation of the host cell membrane. The polypeptide may be recovered using any suitable method known to persons skilled in the art, illustrative examples of which include collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. Optionally, the polypeptide may be partially or totally purified by a variety of procedures known in the art including, but not limited to, thermal shock, 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.

[0110] The polypeptide may be used, in purified form, either alone or in combination with additional enzymes (e.g., PETases. MHETases or nylonases), to catalyze enzymatic reactions involved in the degradation and / or recycling of a nylon containing material. The polypeptides described herein may be in soluble form, or on solid phase. In particular, they may be bound to cell membranes or lipid vesicles, or to synthetic supports such as glass, plastic, polymers, filter membranes, e.g., in the form of beads, columns, plates and the like.[OHl] The present disclosure also extends to compositions comprising the polypeptide, the nucleic acid or the host cell described herein.

[0112] The composition may be liquid or dry, for instance in the form of a powder. In some embodiments, the composition is a lyophilisate. For instance, the composition may comprise the polypeptide, nucleic acid and / or host cells and optionally excipients and / or reagents etc. Suitable excipients may include buffers commonly used in biochemistry, agents for adjusting pH, preservatives such as sodium benzoate, sodium sorbate or sodium ascorbate, conservatives, protective or stabilizing agents such as starch, dextrin, arabic gum, salts, sugars e.g., sorbitol, trehalose or lactose, glycerol, polyethylene glycol, polyethene glycol, polypropylene glycol, propylene glycol, divalent ions such as calcium, sequestering agent such as EDTA, reducing agents (e.g., beta-mercaptoethanol, dithiothreitol, ascorbic acid, tris(2-carboxyethyl)phosphine), amino acids, a carrier such as a solvent or an aqueous solution, and the like.

[0113] In an embodiment, the composition comprises the polypeptide described herein (the polypeptide may be present in the composition in an isolated or at least partially purified form). In an embodiment, the composition comprises the polypeptide described herein in an amount of from about 0.1% to about 99.9%, preferably from about 0.1% to about 50%, preferably from about 0.1% to about 30%, preferably from about 0.1% to about 5% by weight of the total weight of the composition. In a preferred embodiment, the composition comprises the polypeptide described herein in an amount of from about 0.1 to about 5% by weight of the total weight of the composition. In another embodiment, the composition comprises the polypeptide described herein in an amount of from about 0.1 to about 0.2% by weight of the total weight of the composition. The amount of polypeptide in the composition may be suitably adapted by persons skilled in the art, depending e.g., on the nature and / or amount of the polyester containing material to be degraded (hydrolysed) and / or the presence or absence of any additional enzymes / polypeptides in the composition.

[0114] The compositions described herein may further comprise additional polypeptide(s) exhibiting enzymatic activity, not limited to nylonases.

[0115] In an embodiment, the polypeptide described herein is solubilized in an aqueous medium together with one or more excipients, such as excipients that may suitably stabilize or protect the polypeptide from degradation. For example, the polypeptides described herein may be solubilized in water and then admixed with excipients such as glycerol, sorbitol, dextrin, starch, glycol such as propanediol, salt, etc. The resulting admixture 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, lyophilisation, freeze-drying, spray-drying, supercritical drying, down-draught evaporation, thin-layer evaporation, centrifugal evaporation, conveyor drying, fluidized bed drying, drum drying or any combination thereof.

[0116] In an embodiment, the composition comprises at least one host cell expressing the polypeptide described herein, or an extract thereof. By "extract of a cell" is meant any fraction obtained from a cell, such as cell supernatant, cell debris, cell walls, DNA extract, enzymes or enzyme preparation or any preparation derived from cells by chemical, physical and / or enzymatic treatment, which is essentially free of living cells. Preferred extracts are enzymatically-active extracts. The composition may comprise one or several host cells or extract thereof containing the polypeptide described herein, and optionally one or several additional cells.

[0117] As noted elsewhere herein, the present inventors have surprisingly found that the polypeptides described herein have greater activity in hydrolysing an amide bond in a polyamide or in hydrolysing nylon 6 oligomers or nylon 6,6 oligomers when compared to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 24-38or a wildtype NylB enzyme. Thus, disclosed herein is a method of hydrolysing nylon polymer or nylon oligomer or a nylon-containing product, the method comprising exposing nylon polymer or nylon oligomer or a nylon-containing product to the polypeptide, the composition or the host cell described herein, under conditions sufficient to convert the nylon polymer or nylon oligomer or a nylon-containing product, adipic acid and / or hexamethylenediamine.

[0118] The present disclosure extends to the use of the polypeptide, the composition or the host cell described herein for degrading a nylon polymer or nylon oligomer or a nylon-containing product in aerobic or anaerobic conditions and / or recycling nylon containing material, as plastic products made of or containing nylon and / or producing biodegradable plastic products containing nylon. Such methods are particularly useful for degrading a nylon polymer or nylon oligomer or a nylon-containing product comprising nylon 6,6 polymer or nylon 6,6 oligomer or a nylon 6,6-containing product.

[0119] Advantageously, the nylon(s) of the nylon-containing material or nylon-containing product is (are) depolymerized up to monomers and / or oligomers. In an embodiment, at least one nylon polymer or nylon oligomer is degraded to yield re-polymerizable monomers and / or oligomers, which are advantageously retrieved or recovered for further use.

[0120] In an embodiment, nylon(s) of the nylon-containing material or nylon-containing product is (are) fully degraded.

[0121] As noted elsewhere herein, the nylon product may comprise at least one polyester selected from the group consisting of polyethylene terephthalate (PET), polylactic acid (PLA), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polyethylene terephthalate (PET), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), polycaprolactone (PCL), poly(ethylene adipate) (PEA) and combinations of any of the foregoing.

[0122] The time required for degrading a nylon-containing material or nylon-containing product may vary depending on the nylon-containing material or nylon-containing product itself (i.e., nature and origin of the material / product, its composition, shape etc.), the type and amount of polypeptide used, as well as various process parameters (i.e., temperature, pH, additional agents, etc.). One skilled in the art may easily adapt the process parameters to the polyester containing material.

[0123] Advantageously, the degrading process is implemented at a temperature from about 10°C to about 70°C, preferably from about 20°C to about 60°C, preferably from about 30°C to about 60°C, more preferably from about 40°C to about 60°C, even more preferably from about 40°C to about 50°C, or even more preferably at about 45°C. The temperature is typically be maintained below an inactivating temperature, which corresponds to thetemperature at which the polypeptide is inactivated and / or the recombinant microorganism does not synthesize, produce or release the polypeptide described herein.

[0124] In an embodiment, the nylon polymer, a nylon oligomer or a nylon-comprising product or nylon-comprising material may be pretreated or pre-processed prior to being contacted with the polypeptide in order to physically change its structure or solubility, so as to increase the surface of contact between the nylon and the enzyme. In an embodiment, the nylon polymer, a nylon oligomer or a nylon-comprising product or nylon-comprising material may be pre-treated or pre-processed so that the nylon is solubilised prior to being contacted with the polypeptide.

[0125] Monomers resulting from the depolymerization or degradation process or method may be suitably recovered, sequentially or continuously. A single type of monomers or several different types of monomers may be recovered, depending on the starting nylon polymer, nylon oligomer or nylon-containing material / product.

[0126] The recovered monomers may be further purified, using any suitable purifying method and conditioned in a repolymerizable form. Illustrative examples of suitable 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, acid or heat catalysed lactamisation, adsorption, column chromatography, simple vacuum distillation and microfiltration, combined or not.

[0127] The repolymerizable monomers may be used to synthesize new nylon polymers. Advantageously, nylons of the same nature are repolymerized. However, it is possible to mix the recovered monomers with other monomers, for example, in order to synthesize new copolymers of nylon. Alternatively, the recovered monomers may be used as chemical intermediates in order to produce new chemical compounds of interest.

[0128] The present disclosure also extends to a compound comprising the polypeptide, composition and / or host cell expressing said polypeptide or an extract thereof containing said polypeptide.

[0129] The present disclosure also extends to a masterbatch composition comprising the polypeptide, composition and / or host cell expressing said polypeptide or an extract thereof containing said polypeptide.

[0130] Advantageously, such compound or masterbatch composition described herein can be used for the production of a nylon containing material and / or plastic article that will include the polypeptide described herein.

[0131] In an embodiment, the resulting compound, masterbatch composition or nylon article is a biodegradable plastic compound, masterbatch composition or plastic article complying with at least one of the relevant standards and / or labels known by the person skilled in the art, such as standard EN 13432, standard ASTM D6400, OK Biodegradation Soil (Label Vincotte), OK Biodegradation Water (Label Vincotte), OK Compost (Label Vincotte), OK Home Compost (Label Vincotte).

[0132] The polypeptides disclosed herein are suitable for a range of applications, including industrial applications, illustrative examples of which include as additives in detergents, compositions, textiles production, electronics and biomedical applications. For example, the polypeptides disclosed herein can be employed in textile production, where it can be used as an exonuclease to suitably modify the properties of textile fibres.

[0133] The invention will now be described with reference to the following Examples which illustrate some preferred aspects of the present invention. However, it is to be understood that the particularity of the following description of the invention is not to supersede the generality of the preceding description of the inventionEXAMPLESExample 1: Ancestral sequence reconstruction

[0134] Based on a polypeptide of SEQ ID NO: 1, amino acid substitutions at specific amino acid positions were designed to test how they affected enzyme activity on nylon 6,6 trimer.

[0135] Assays were performed at 40°C, pH 8. Screening of candidates were performed using 200 pL reaction mixtures containing 50 pL distilled water, 50 pL of eluate from 96-well purification of E. Coli cells expressing the polypeptide variants, and 100 pL of substrate solution containing a mixture of soluble PA-6,6 oligomers (comprising adipic acid, dimers and trimers). The substrate solution containing a mixture of PA-6,6 oligomers was generated by incubating solid PA-6,6 pellets with 50% v / v sulfuric acid at room temperature before heating at reflux for 6 hours. This mixture was cooled and neutralised using NaOH. The substrate solution containing a mixture of PA-6,6 oligomers comprised 39.3 g L-l AA, 41.2 g L-l dimer, 4.7 g L-l BAB trimer (base-terminated trimer), and 3.0 g L-l ABA trimer (acid-terminated trimer). The enzymatic reaction ran for 2 hours. The samples were passed through a 10 kDa MWCO centrifugal filters (13,000 x g, 5 min) to remove the enzyme and thereby quench the reaction. To assay for enzyme activity, the decrease in oligomer concentration and increase in adipic acid was quantified by high performance liquid chromatography (HPLC).

[0136] Many of the variant sequences demonstrated ability to hydrolyse amide bonds in a polyamide (SEQ ID NOs: 3-21, see Table 1) with similar levels of activity as SEQ ID NO: L (See Figure 1).

[0137] An alignment of these variants indicated that the variants shared a consensus sequence of SEQ ID NO: 2:SEQ ID NO:2MTGTTAFETRYGFRRNQVTLDNWRTAPFNRWSFQNVGELVPSARIAAASGAPEA PAADMSGLLAETVSLAGGSETVAAFLERSHTDALTVMKGGRFVGDWFAPHMDF GARHIIFSISKSLTAILAGILEGEGLLDPDAPVTQYIPEAAGSAYGDATVRHVLDMT VSLDFEEAYLDPESAFARYRRATLWNPSTGGGAESLREFLLTLQRLPEPHGETFRX 1RX2PNSDLLGILVERASGQRYADLMREKLWKPLGAKSEASITVDREGTARAAGGI SVTPRDLARVGEMMRQGGVANGRRIVPEAWVRDTTTGGSAEAWQRGDMAHLFP QGRX3RNX4WX5QTGAASGAYCGX6GIHX7QWLYVDPAAEVVIVKMSSQPEPVDD PLXxHENVAFFEALARMV (wherein Xi-Xs is any amino acid)

[0138] Table 2 shows the amino acid substitutions at the specified positions of the variant polypeptides, where the amino acid numbering is relative to the amino acid positions of SEQ ID NO: 1 or 2.

[0139] Figure 3 depicts the activity of the polypeptide having the amino acid sequence SEQ ID NO:23 in hydrolysing nylon 6,6 dimer when compared to the polypeptide of SEQ ID NO:24. Figure 4 depicts the melting temperature of polypeptides having the amino acid sequence of SEQ ID NOs: 1, 23 and 24.

[0140] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.

[0141] The citation of any reference herein should not be construed as an admission that such reference is available as “Prior Art” to the instant application.

[0142] Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Those of skill in the art will therefore appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All such modifications and changes are intended to be included within the scope of the appended claims.Table 1: Amino acid sequences of the disclosed polypeptides.Table 2: Amino acid substitutions across variant polypeptides

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the polypeptide comprises amino acid residues 2-394 of SEQ ID NO: 1, or an amino acid sequence that has at least 70% sequence identity thereto, and wherein the amino acid sequence of the polypeptide differs from SEQ ID NO: 1 at one or more amino acid positions selected from the group consisting of amino acid positions 220, 222, 331, 334, 336, 348, 352 and 380 of SEQ ID NO: 1.

2. The polypeptide of claim 1, wherein i. the amino acid residue at position 220 is selected from the group consisting of Y, C, S, I, D, A, E and conservative amino acid substitutions of any of the foregoing; ii. the amino acid residue at position 222 is selected from the group consisting of S, E and conservative amino acid substitutions of any of the foregoing; iii. the amino acid residue at position 331 is selected from the group consisting of Y, W, D and conservative amino acid substitutions of any of the foregoing; iv. the amino acid residue at position 334 is selected from the group consisting of K, Y, E, H, D and conservative amino acid substitutions of any of the foregoing; v. the amino acid residue at position 336 is selected from the group consisting of Y, D, W, E and conservative amino acid substitutions of any of the foregoing; vi. the amino acid residue at position 348 is selected from the group consisting of I, H, E, W and conservative amino acid substitutions of any of the foregoing; vii. the amino acid residue at position 352 is selected from the group consisting of G, C and conservative amino acid substitutions of any of the foregoing; and / or viii. the amino acid residue at position 380 is selected from the group consisting of S, Y and conservative amino acid substitutions of any of the foregoing.

3. The polypeptide of claim 1 or claim 2, wherein i. the amino acid residue at position 220 is selected from the group consisting of Y, C, S, I, D, A, and E; ii. the amino acid residue at position 222 is selected from the group consisting of S or E; iii. the amino acid residue at position 331 is selected from the group consisting of Y, W and D; iv. the amino acid residue at position 334 is selected from the group consisting of K, Y, E, H and D; v. the amino acid residue at position 336 is selected from the group consisting of Y, D, W and E; vi. the amino acid residue at position 348 is selected from the group consisting of I, H, E and W; vii. the amino acid residue at position 352 is selected from the group consisting of G and C; and / or viii. the amino acid residue at position 380 is selected from the group consisting of S and Y.

4. The polypeptide of any one of claims 1-3, wherein i. the amino acid residue at position 220 is selected from the group consisting of C, S, I, D, A, and E; ii. the amino acid residue at position 222 is S; iii. the amino acid residue at position 331 is Y; iv. the amino acid residue at position 334 is K; v. the amino acid residue at position 336 is Y; vi. the amino acid residue at position 348 is I; vii. the amino acid residue at position 352 is G; andviii. the amino acid residue at position 380 is S.

5. The polypeptide of any one of claims 1-3, wherein i. the amino acid residue at position 220 is Y; ii. the amino acid residue at position 222 is E; iii. the amino acid residue at position 331 is Y; iv. the amino acid residue at position 334 is K; v. the amino acid residue at position 336 is Y; vi. the amino acid residue at position 348 is I; vii. the amino acid residue at position 352 is G; and viii. the amino acid residue at position 380 is S.

6. The polypeptide of any one of claims 1-3, wherein i. the amino acid residue at position 220 is Y; ii. the amino acid residue at position 222 is S; iii. the amino acid residue at position 331 is selected from the group consisting ofW and D; iv. the amino acid residue at position 334 is K; v. the amino acid residue at position 336 is Y; vi. the amino acid residue at position 348 is I; vii. the amino acid residue at position 352 is G; and viii. the amino acid residue at position 380 is S.

7. The polypeptide of any one of claims 1-3, wherein i. the amino acid residue at position 220 is Y;ii. the amino acid residue at position 222 is E; iii. the amino acid residue at position 331 is Y; iv. the amino acid residue at position 334 is selected from the group consisting ofY, E, H and D; v. the amino acid residue at position 336 is Y; vi. the amino acid residue at position 348 is I; vii. the amino acid residue at position 352 is G; and viii. the amino acid residue at position 380 is S.

8. The polypeptide of any one of claims 1-3, wherein i. the amino acid residue at position 220 is Y; ii. the amino acid residue at position 222 is E; iii. the amino acid residue at position 331 is Y; iv. the amino acid residue at position 334 is K; v. the amino acid residue at position 336 is selected from the group consisting ofD, W and E; vi. the amino acid residue at position 348 is I; vii. the amino acid residue at position 352 is G; and viii. the amino acid residue at position 380 is S.

9. The polypeptide of any one of claims 1-3, wherein i. the amino acid residue at position 220 is Y; ii. the amino acid residue at position 222 is E; iii. the amino acid residue at position 331 is Y; iv. the amino acid residue at position 334 is K;v. the amino acid residue at position 336 is Y; vi. the amino acid residue at position 348 is selected from the group consisting ofH, E and W; vii. the amino acid residue at position 352 is G; and viii. the amino acid residue at position 380 is S.

10. The polypeptide of any one of claims 1-3, wherein i. the amino acid residue at position 220 is Y; ii. the amino acid residue at position 222 is E; iii. the amino acid residue at position 331 is Y; iv. the amino acid residue at position 334 is K; v. the amino acid residue at position 336 is Y; vi. the amino acid residue at position 348 is I; vii. the amino acid residue at position 352 is C; and viii. the amino acid residue at position 380 is selected from the group consisting ofS or and Y.

11. The polypeptide of any one of claims 1-3, wherein i. the amino acid residue at position 220 is Y; ii. the amino acid residue at position 222 is E; iii. the amino acid residue at position 331 is Y; iv. the amino acid residue at position 334 is K; v. the amino acid residue at position 336 is Y; vi. the amino acid residue at position 348 is I; vii. the amino acid residue at position 352 is selected from the group consisting ofG and C; andviii. the amino acid residue at position 380 is Y.

12. The polypeptide of any one of claims 1-11, wherein the polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1.

13. The polypeptide of any one of claims 1-12, wherein the polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1.

14. The polypeptide of any one of claims 1-13, wherein the polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1.

15. The polypeptide of any one of claims 1-14, wherein the polypeptide comprises an amino acid sequence of amino acid residues 2-394 of SEQ ID NO:2.

16. The polypeptide of any one of claims 1-15, wherein the polypeptide comprises an amino acid sequence of SEQ ID NO:2.

17. The polypeptide of claim 16, wherein the polypeptide consists of an amino acid sequence of SEQ ID NO:2.

18. The polypeptide of any one of claims 1-17, comprising the amino acid sequence of amino acid residues 2-394 of any one of SEQ ID NOs: 3-22.

19. The polypeptide of any one of claims 1-18, consists of the amino acid sequence of any one of SEQ ID NOs: 3-22.

20. A polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the polypeptide comprises an amino acid sequence of amino acid residues 2-392 of SEQ ID NO:23.

21. The polypeptide of claim 20 comprising the amino acid sequence of SEQ ID NO:23.

22. The polypeptide of any one of claims 1-21, wherein the polypeptide has adipic acid mono- and di-N-alkyl amide hydrolase activity.

23. The polypeptide of any one of claims 1-22, wherein the polypeptide is capable of hydrolysing a nylon polymer.

24. The polypeptide of claim 23, wherein the nylon polymer is a nylon oligomer.

25. The polypeptide of claim 23 or claim 24, wherein the nylon polymer or nylon oligomer is a nylon 6 polymer, a nylon 6 oligomer, a nylon 6,6 polymer or a nylon 6,6 oligomer.

26. The polypeptide of claim 25, wherein the nylon polymer or the nylon oligomer is a nylon 6,6 polymer or a nylon 6,6 oligomer.

27. The polypeptide of claim 26, wherein the nylon 6,6 oligomer is an aqueous soluble nylon 6,6 oligomer.

28. The polypeptide of claim 26 or claim 27, wherein the nylon 6,6 oligomer is selected from the group consisting of a dimer, a trimer, a tetramer, a pentamer and hexamer of nylon 6,6.

29. The polypeptide of claim 28, wherein the nylon 6,6 oligomer is a nylon 6,6 trimer.

30. The polypeptide of claim 28, wherein the nylon 6,6 oligomer is a nylon 6,6 dimer.

31. The polypeptide of claim 28, wherein the nylon 6,6 oligomer is a nylon 6,6 tetramer.

32. The polypeptide of any one of claims 27-31, wherein the polypeptide is capable of hydrolysing the nylon 6,6 oligomer to produce adipic acid and hexamethylenediamine.

33. The polypeptide of any one of claims 1-32, wherein the polypeptide is characterised by: i. increased recombinant expression in a host cell system; ii. increased enzyme activity in hydrolysing an amide bond in a polyamide; and / or iii. increased thermostability, when compared to a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 24-38.

34. The polypeptide of claim 33, wherein the polypeptide is characterised by increased enzyme activity when compared to an extant or wildtype nylonase.

35. A composition comprising the polypeptide of any one of claims 1-34.

36. A polynucleotide comprising a nucleic acid sequence encoding the polypeptide of any one of claims 1-34.

37. An expression vector comprising the polynucleotide of claim 36.

38. A host cell comprising the polynucleotide of claim 36, or the expression vector of claim 37.

39. A method of producing a polypeptide capable of hydrolysing an amide bond in a polyamide, the method comprising: i. providing the polynucleotide of claim 36; ii. expressing the nucleic acid sequence in a host cell culture, thereby producing the polypeptide; and iii. collecting the polypeptide produced in (ii) from the host cell culture.

40. A method of hydrolysing nylon polymer or nylon oligomer, the method comprising exposing the nylon polymer or nylon oligomer to the polypeptide of any one of claims 1 to 34, the composition of claim 35 or the host cell of claim 38 under conditions sufficient to convert the nylon-6, 6 polymer to adipic acid and / or hexamethyl enedi amine .

41. A method of degrading a nylon-containing product, the method comprising exposing the nylon-containing product to the polypeptide of any one of claims 1 to 34, the composition of claim 35 or the host cell of claim 38.

42. The method of claim 40 or claim 41, comprising: i. chemical processing of nylon polymer, nylon oligomer or nylon-containing product to generate nylon oligomers; ii. exposing the nylon oligomers generated in step (i) to the polypeptide of any one of claims 1 to 34, the composition of claim 35 or the host cell of claim 38, under conditions sufficient to produce adipic acid and / or hexamethyl enedi amine .

43. The method of any one of claims 40-42, wherein the nylon in the nylon polymer, nylon oligomer or the nylon-containing product is nylon 6 or nylon 6,6.

44. The method of claim 43, wherein the nylon is nylon 6,6.

45. The method of claim 44, wherein the nylon 6,6 oligomer is an aqueous soluble nylon 6,6 oligomer.

46. The method of any one of claims 42-45, wherein the nylon 6,6 oligomer is selected from the group consisting of a dimer, a trimer, a tetramer, a pentamer and hexamer of nylon 6,6.

47. The method of claim 46, wherein the nylon 6,6 oligomer is a nylon 6,6 trimer.

48. The method of claim 46, wherein the nylon 6,6 oligomer is a nylon 6,6 dimer.

49. The method of claim 46, wherein the nylon 6,6 oligomer is a nylon 6,6 tetramer.

50. The method of any one of claims 42-49, further comprising recovering the adipic acid and / or the hexamethylenediamine produced in step (ii).

51. A composition comprising the adipic acid and / or the hexamethylenediamine recovered by the method of claim 50.

52. A method of producing a nylon polymer using the composition of claim 51.

53. A nylon polymer when produced using the composition of claim 51.

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