Processes and enzymes for use therein

Abstract

The present invention relates to novel amidase enzymes, their use in the hydrolysis of polyamide oligomers and their use in processes for recycling polyamide polymers. The invention finds particular use in the hydrolysis of oligomers of polyamide 6,6 and therefore in processes for the recycling of this polymer.

Description

[0001] PROCESSES AND ENZYMES FOR USE THEREIN

[0002] TECHNICAL FIELD

[0003] The present invention relates, inter alia, to novel amidase enzymes, their use in the hydrolysis of polyamide oligomers and their use in processes for recycling polyamide polymers. The invention finds particular use in the hydrolysis of oligomers of polyamide 6,6 and therefore in processes for the recycling of this polymer.

[0004] BACKGROUND TO THE INVENTION

[0005] The production of synthetic polymers (or plastics) has increased rapidly since the late 1940s and is now of the order of 400 million metric tonnes annually. At the end of product life, much of this production is either disposed of in land fill or is burnt. Increasingly however, attention is turning to methods of recycling these polymers. In one approach, thermoplastic polymers can be separated, cleaned, repelletized and reformed into new products, however, such reformed polymers retain the additives of the original polymer article, are typically impure, often containing greater or lesser quantities of contaminating polymers and are not suitable for higher value products requiring controlled polymer properties. Progressive breakdown of polymer chains during repeated processing also leads to poorer quality feedstock. The preferred approach would be to de -polymerise the polymer into constituent monomers, purify them and reintroduce these monomers into the manufacturing process.

[0006] Polyamides are ubiquitous synthetic thermoplastic polymers used in the preparation of a wide variety of products including fibres, films and moulded objects. The most common polyamides are Nylon 6 (polyamide 6 or PA6), a homopolymer of repeating 6-aminohexanoic acid units (formula I); and Nylon 6,6 (polyamide 6,6 or PA66), a co-polymer of hexamethylenediamine and adipic acid (formula II).

[0007] Formula I Formula II

[0008] Chemical processes for depolymerising these polyamides include acid or alkaline hydrolysis at high temperatures, although hydrolysis by exposure to water at high temperatures and pressures is also known (e.g. WO2023 / 149514). These processes typically require high temperatures and extended reaction times and so require a higher energy input.

[0009] The use of enzymes to hydrolyse synthetic polymers is attractive because enzymatic hydrolysis doesn’t require the use of high temperatures and so energy input is reduced. However, industrial processes often operate under conditions that are not conducive to the levels of activity required, for example at sub optimal temperatures, pH, or ionic strength or the process requires the use of solvents that are not compatible with the enzyme activity. Often the wild type enzyme has only a low level of activity necessary for the process and so mutant or engineered enzymes are sought that better meet the requirements of the process.

[0010] Polyamides, are only poorly susceptible to enzymatic hydrolysis due to their high crystallinity and lack of solubility under conditions supporting enzyme activity. Low n number oligomeric products of the chemical hydrolysis of polyamides, on the other hand, are water soluble at temperatures that support enzyme activity, and enzymes able to hydrolyse these oligomers (colloquially known as nylonases, nylon hydrolases or polyamide hydrolases) have been identified. These include NylA (EC3.5.2.12: 6- aminohexanoate cyclic dimer hydrolase originally known as El), which is capable of hydrolysing cyclic polyamide 6 dimers, to provide the 6-amino hexanoate (AHA) monomer. NylB (EC3.5.1.46: 6- aminohexanoate dimer hydrolase, originally known as Eli) is capable of hydrolysing low n number polyamide 6 oligomers. It has been suggested that NylB of Arthrobacter K172 (SEQ ID NO: 1) arose by gene duplication and subsequent mutation of an ancestral esterase (Eli’ or NylB2 - SEQ ID NO: 4) found on the same plasmid. NylC is an endo-type 6-aminohexanoate oligomer hydrolase (originally known as EIII) and is capable of hydrolysing linear and cyclic polyamide 6 oligomers. Mixtures of these enzymes have also been used, for example combinations of NylC and NylB or combinations of a hydrolase, PAM1, with NylB or NylC (see for example US6214592 and WO2023 / 149514).

[0011] Although NylB was originally identified as a polyamide 6 dimer hydrolase, the wild type enzyme from Arthrobacter (SEQ ID NO:1) has also been shown to hydrolyse certain polyamide 6,6 oligomers (e.g. US6214592, WO2023 / 149514).

[0012] The activity of NylB enzymes towards amino terminated polyamide oligomers (such as the odd numbered oligomers of polyamide 6,6 - trimers, pentamers etc.) can be significantly lower than towards carboxyl terminated oligomers leading to bottlenecks in the enzymatic hydrolysis of these oligomers.

[0013] Recently a large number of mutants of NylB have been identified, as a result of a computational study to derive putative ancestral variants of NylB followed by subsequent rounds of mutation. Some of these enzymes are said to provide a variety of improved properties, including improved activity against certain polyamide 6,6 oligomers (WO2024 / 156025).

[0014] There remains a need however, for improved enzymes for use in industrial processes for the depolymerisation of polyamides such as polyamide 6 and polyamide 6,6. For example, it would be advantageous to provide approaches for more effective hydrolysis of bottleneck oligomers.

[0015] SUMMARY OF THE INVENTION The present inventors have identified amino acid substitutions in the amino acid sequence of amidases such as polyamide hydrolases, which lead to an increase in hydrolytic activity towards polyamides such as polyamide 6 and polyamide 6,6, particularly towards their oligomers and an increased hydrolytic activity towards amino terminated oligomers compared to the wild type (SEQ ID NO: 1). These mutants carry a 181G, 266H double mutation with respect to SEQ ID NO: 1 ; or a 181R, 266H, or a 181 A 266H double mutation with respect to SEQ ID NO: 1.

[0016] The inventors have also identified that substitution of the arginine residue at position 187 with respect to SEQ ID NO:1 or SEQ ID NO:2, with alanine (A), cysteine (C), aspartate (D) (otherwise known as aspartic acid), glutamate (E) (otherwise known as glutamic acid), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), leucine (L), glutamine (Q), threonine (T), or valine (V) provides hydrolases with an improved spectrum of activity.

[0017] In one aspect the disclosure provides polypeptides capable of hydrolysing an amide bond in a linear amide polymer wherein the polypeptide comprises a glycine residue (G) (e.g. SEQ ID NO:2) or an arginine residue (R) (e.g. SEQ ID NOG), at position 181 and a histidine residue (H) at position 266, with respect to SEQ ID NO:1; optionally with the exclusion of SEQ ID NOs:4 to 13.

[0018] In one embodiment the disclosure provides a polypeptide capable of hydrolysing an amide bond in a linear amide polymer wherein the polypeptide comprises a glycine residue (G) at position 181 and a histidine residue (H) at position 266 (e.g. SEQ ID NOG) with respect to SEQ ID NO:1 optionally with the exclusion of SEQ ID NOs: 4 to 13.

[0019] In one embodiment the disclosure provides polypeptides capable of hydrolysing an amide bond in a linear amide polymer wherein the polypeptide comprises an arginine residue (R) at position 181 and an histidine residue (H) at position 266 (e.g. SEQ ID NOG).

[0020] In some embodiments the polypeptide comprises an amino acid selected from the group consisting of alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine at position 187 with respect to SEQ ID NO:1.

[0021] In some embodiments the polypeptide is a polyamide hydrolase. In some embodiments the polypeptide is a polyamide 6 (PA6) or polyamide 6,6 (PA66) hydrolase. In some embodiments the polypeptide is a polyamide 6,6 (PA66) hydrolase. In some embodiments the polypeptide hydrolyses one or more of a polyamide 6,6 dimer, N-trimer, C-trimer, tetramer, N-pentamer, C-pentamer and / or hexamer and in some embodiments the polypeptide hydrolyses insoluble or sparingly water soluble oligomers. In some embodiments the polypeptide provides an improved rate of hydrolysis of these units compared to the wild type (WT) enzyme, without the amino acid substitutions.

[0022] In addition to the sequence substitutions described above, in some embodiments the polypeptide comprises one or more of the following features: (i) at least 50% amino acid sequence identity to SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO: 117; and / or

[0023] (ii) a catalytic triad comprising a serine residue at position 112, a lysine residue at position 115 and a tyrosine or phenylalanine, optionally a tyrosine residue, at position 215 with respect to SEQ ID NO: 1; and / or

[0024] (iii) a nitrogen inverting residue at position 170 with respect to SEQ ID NO: 1; optionally wherein the nitrogen inverting residue is a tyrosine residue;

[0025] (iv) the polypeptide has 6-aminohexanoate dimer hydrolase activity or polyamide 6,6 dimer hydrolase activity.

[0026] In some embodiments, the polypeptide may comprise at least (iv); at least (i) and (iv); at least (i), (ii) and (iv); at least (i), (ii) and (iii); at least (ii) and (iii); or (i), (ii), (iii) and (iv).

[0027] In some embodiments a mutant polypeptide is derived from a wild type polypeptide having 6- aminoheaxanoate dimer hydrolase activity by replacing the amino acid at position 181with a glycine residue and / or replacing the amino acid at position 266 with a histidine residue to provide a polypeptide whose sequence comprises a glycine residue at position 181 and a histidine residue at position 266 with respect to SEQ ID NO: 1.

[0028] In some embodiments a mutant polypeptide is derived from a wild type polypeptide having 6- aminoheaxanoate dimer hydrolase activity by replacing the amino acid at position 181with an arginine residue and / or replacing the amino acid at position 266 with a histidine residue to provide a polypeptide whose sequence comprises an arginine residue at position 181 and a histidine residue at position 266 with respect to SEQ ID NO: 1.

[0029] In some embodiments a mutant polypeptide is derived from a wild type polypeptide having 6- aminoheaxanoate dimer hydrolase activity by replacing the amino acid at position 181with an alanine residue and / or replacing the amino acid at position 266 with a histidine residue to provide a polypeptide whose sequence comprises an alanine residue at position 181 and a histidine residue at position 266 with respect to SEQ ID NO: 1.

[0030] In some embodiments a polypeptide as disclosed herein has at least 60% or at least 70% sequence identity to SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:281 or SEQ ID NO: 117.

[0031] In some embodiments a polypeptide as disclosed herein, comprises amino acids 2 to 392 of SEQ ID NO: 2 or an amino acid sequence having at least 60% sequence identity thereto. In some embodiments a polypeptide as disclosed herein, comprises amino acids 2 to 392 of SEQ ID NO: 3 or an amino acid sequence having at least 60% sequence identity thereto.

[0032] In some embodiments the polypeptide comprises the amino acid sequence of amino acids 2 to 392 of SEQ ID NO:2; or an amino acid sequence having at least 96% sequence identity thereto.

[0033] In some embodiments the disclosure provides a polypeptide capable of hydrolysing an amide bond in a linear amide polymer as disclosed herein, wherein the polypeptide comprises the amino acid sequence of amino acids 2 to 412 of SEQ ID NO: 29; or an amino acid having 70% sequence identity thereto.

[0034] In some embodiments the disclosure provides a polypeptide capable of hydrolysing an amide bond in a linear amide polymer as disclosed herein, wherein the amino acid sequence of any of SEQ ID NO:s 117 to 199 or a polypeptide having at least 60 % or at least 70% sequence identity thereto.

[0035] In a further aspect, the disclosure provides a polypeptide capable of hydrolysing an amide bond in a linear amide polymer wherein the polypeptide comprises a residue selected from alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine residue at position 187 with reference to SEQ ID NO:1.

[0036] In some embodiments the polypeptide as disclosed herein is a polypeptide having polyamide hydrolase activity comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:33 or a polypeptide having at least 60% sequence identity to either SEQ ID NO:1 or SEQ ID NO:33 wherein the amino acid at position 187 with reference to SEQ ID NO:1 is substituted by an amino acid selected from the group consisting of alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine.

[0037] In some embodiments, the polypeptide comprises a residue selected from alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine residue at position 187 with the proviso that if the amino acid at position 187 with respect to SEQ ID NO:1 is A, K, E or L, then the amino acid at position 181 is A, R, G, or N; and / or the amino acid at position 266 is H, G or M.

[0038] In some embodiments the polypeptide comprises a residue selected from alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine residue at position 187 with the proviso that if the amino acid at position 187 with respect to SEQ ID NO:1 is A, then the amino acid at position 181 is A, R, G, or N; and / or the amino acid at position 266 is H, G or M.

[0039] Such polypeptides may be derived from any of the other sequences disclosed herein by substituting the amino acid at position 187 with reference to SEQ ID NO:1 with an amino acid selected from alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine.

[0040] In some embodiments the polypeptides of the disclosure are capable of hydrolysing a polyamide polymer or a polyamide oligomer. In some embodiments the polyamide polymer is a polyamide 6 (PA6) polymer, or a polyamide 6,6 (PA66) polymer and / or the polyamide oligomer is a polyamide 6 or polyamide 6,6 oligomer. In some embodiments a polypeptide is capable of hydrolysing a water-soluble polyamide 6 oligomer or polyamide 6,6 oligomer. In some embodiments a polyamide oligomer is terminated by at least one amino group. In some embodiments the polyamide oligomer has two amino termini. In some embodiments a polyamide oligomer is terminated by at least one carboxylate group. In some embodiments the polyamide oligomer has two carboxylate termini.

[0041] In a further aspect, the disclosure provides a polypeptide as described herein which is immobilized on a solid support.

[0042] In a further aspect, the disclosure provides polynucleotides comprising a nucleic acid sequence encoding the polypeptides disclosed herein. In a further aspect the disclosure provides an expression vector comprising the aforementioned polynucleotide, and in a further aspect the disclosure provides host cells comprising the aforementioned expression vectors or polynucleotides.

[0043] In a further aspect the disclosure provides a process for the preparation of a polypeptide disclosed herein, comprising expressing the polypeptide from a polynucleotide as disclosed herein and recovering the polypeptide.

[0044] In a further aspect, the disclosure provides a composition comprising the polypeptide as disclosed herein, or a polynucleotide as disclosed herein, or an expression vector as disclosed herein, or a host cell as disclosed herein.

[0045] In a further aspect the disclosure provides a process for hydrolysing a polyamide polymer or polyamide oligomer comprising contacting the polyamide polymer or polyamide oligomer with a polypeptide as described herein. In some embodiments, the polyamide polymer is partially chemically hydrolysed to provide polyamide oligomers and the polyamide oligomers are contacted with a polypeptide. In some embodiments the partial chemical hydrolysis provides a composition comprising water soluble polyamide oligomers and in some embodiments the chemical hydrolysis provides a mixture of oligomers comprising at least dimers and trimers, or at least dimers, trimers, tetramers and pentamers. In some embodiments, the partial chemical hydrolysis provides a composition additionally comprising insoluble oligomers. In some embodiments the partial chemical hydrolysis is a partial acid or partial base hydrolysis. In some embodiments a polyamide polymer or polyamide oligomer is a polyamide 6,6 polymer or oligomer. In some embodiments the products of the hydrolysis comprises adipic acid and hexamethylene diamine. In a further aspect the disclosure provides a process comprising hydrolysing a polyamide polymer or polyamide oligomer by a process comprising contacting the polyamide polymer or polyamide oligomer with a polypeptide as described herein, a composition as described herein, a host cell or composition comprising a host cell as described herein. The process may include carrying out the process to provide adipic acid and / or hexamethylene diamine monomers. The process may include recovering the adipic acid and / or the hexamethylene diamine monomers resulting from the process.

[0046] In a further aspect the disclosure provides a process for the preparation of polyamide 6,6 comprising carrying out a process for the hydrolysis of a polyamide polymer as described above, recovering adipic acid and / or hexamethylene diamine monomers resulting from the process and synthesising a polyamide 6,6 polymer from the recovered adipic acid and / or the hexamethylene diamine monomers.

[0047] In a further aspect the disclosure provides a method for increasing the polyamide 6,6 hydrolase activity of an amidase enzyme comprising:

[0048] (a) identifying the amino acid sequence of a polypeptide having either polyamide 6,6 hydrolase activity, or 6-aminohexanoate dimer hydrolase activity, wherein the amidase lacks either a glycine, arginine or alanine residue at position 181 or a histidine residue at position 266; or lacks both a glycine, arginine or alanine residue at position 181 and a histidine residue at position 266, with reference to SEQ ID NO:1;

[0049] (b) substituting the amino acid at position 181 with a glycine residue, an arginine residue or an alanine residue and / or substituting the amino acid at position 266 with a histidine residue; wherein positions are relative to the amino acid sequence of SEQ ID NO: 1 and optionally if the amidase sequence has other than an alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine or valine at position 187 in relation to SEQ ID NO:1; then

[0050] (c) substituting the amino acid at position 187 with respect to SEQ ID NO: 1 with an amino acid selected from the group consisting of alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine.

[0051] In a further aspect the disclosure provides a method for increasing the polyamide 6,6 hydrolase activity of an amidase enzyme comprising:

[0052] (a) identifying the amino acid sequence of a polypeptide having either polyamide 6,6 hydrolase activity, or 6-aminohexanoate dimer hydrolase activity, wherein the amidase sequence has an amino acid other than an alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine at position 187 in relation to SEQ ID NO:1; and (b) replacing the amino acid at position 187 with respect to SEQ ID NO:1 with an amino acid selected from the group consisting of alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine; and optionally

[0053] (i) if the sequence does not comprise a glycine, arginine or alanine residue at position 181 with respect to SEQ ID NO:1, then replacing the amino acid with a glycine, arginine or alanine residue and / or

[0054] (ii) if the sequence does not comprise a histidine residue at position 266 with respect to SEQ ID NO:1, replacing the amino acid at position 266 with a histidine residue;

[0055] In some embodiments the identified sequence of the polypeptide having either polyamide 6,6 hydrolase activity, or 6-aminohexanoate dimer hydrolase activity has at least one of the following additional features:

[0056] (i) an amino acid sequence of at least 50% identity to SEQ ID NO:1;

[0057] (ii) a catalytic triad comprising a serine residue at position 112, a lysine residue at a position 115 and a tyrosine or phenylalanine residue at a position 215;

[0058] (iii) a nitrogen inverting residue at position 170; optionally wherein the nitrogen inverting residue is a tyrosine residue.

[0059] In some embodiments the methods described above provide a method for increasing the polyamide 6,6 hydrolase activity of an amidase enzyme, particularly towards dimers of polyamide 6,6.

[0060] A further aspect of the disclosure provides polypeptides prepared according to any of the above processes.

[0061] In some embodiments the resulting polypeptide prepared according to the methods have increased hydrolytic activity towards polyamide 6,6 oligomers having terminal amino groups and / or increased hydrolytic activity towards polyamide 6,6 oligomers having terminal carboxylate groups. In some embodiments the resulting polypeptide has increased hydrolytic activity towards polyamide 6,6 dimers.

[0062] In a further aspect the disclosure provides an assay for indicating a hydrolytic activity of an amidase enzyme towards a polyamide oligomer, wherein the hydrolytic activity releases at least one diamine monomer; the process comprising: contacting the amidase enzyme with the polyamide oligomer; contacting a diamine monomer released, with a putrescine oxidase in the presence of oxygen, to produce an oxidised diamine monomer and a peroxide; contacting the peroxide with a peroxidase in the presence of peroxidase substrate; and detecting the activity of the peroxidase on the peroxidase substrate to indicate the activity of the amidase enzyme towards a polyamide oligomer. In some embodiments the peroxidase substrate is a colorogenic substrate and detection of the coloured product indicates activity of the polypeptide towards the polyamide oligomer. In a further aspect the disclosure provides a method for monitoring the progress of a polyamide hydrolysis to produce at least one diamine product, the method comprising detecting the presence or the quantity of the diamine product using an assay as described herein. In some embodiments, at least one step in the hydrolysis is carried out using an enzyme. In some embodiments the enzyme is a polypeptide as described herein

[0063] DETAILED DESCRIPTION

[0064] The present disclosure relates to newly identified mutants of the enzyme NyLB (SEQ ID NO:1), and inter alia, to a process for recycling polyamide polymers using these enzymes. The disclosure particularly provides mutants of the NylB enzyme carrying 181G and 266H mutations or 181R and 266H mutations and mutations that substitute the amino acid at the 187 position, which is typically an arginine in the wild type, to an amino acid selected from alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine which provide additional advantages, but may also be used in the absence of mutations at the 181 position and / or the 266 position. This disclosure also provides methods for using the new variants.

[0065] These mutant enzymes have improved properties compared to the wild type including improved activity against polyamide 6,6 oligomers, the ability to hydrolyse amine terminated oligomers and / or carboxy terminated oligomers (odd number oligomers) and the ability to process compositions comprising sparingly solubility or higher n-number oligomers. The disclosure also relates to new assays for assaying the activity of the enzyme.

[0066] Unless otherwise defined, technical and scientific terms used herein have the meaning commonly understood by those of ordinary skill in the art to which the invention pertains.

[0067] Any embodiment of the invention may be applied to any aspect of the invention unless otherwise expressly stated.

[0068] In one aspect the disclosure provides a polypeptide capable of hydrolysing an amide bond in a linear amide polymer wherein the polypeptide comprises a glycine residue at position 181 and a histidine residue at position 266 with respect to SEQ ID NO:1 (e.g. SEQ ID NO:2). In some embodiments the disclosure provides a polypeptide capable of hydrolysing an amide bond in a linear amide polymer wherein the polypeptide comprises a glycine residue at position 181 and a histidine residue at position 266 with respect to SEQ ID NO:1, with the exclusion of SEQ ID NOs: 4 to 13.

[0069] In one aspect the disclosure provides a polypeptide capable of hydrolysing an amide bond in a linear amide polymer wherein the polypeptide comprises an arginine residue at position 181 and a histidine residue at position 266 with respect to SEQ ID NO:1 (e,g, SEQ ID NO:3). The polypeptide capable of hydrolysing an amide bond in a linear amide polymer, maybe a polyamide hydrolase for example the polypeptide maybe a polyamide 6 (PA6) or polyamide 6,6 (PA66) hydrolase. In some embodiments the polypeptide is a polyamide 6,6 (PA66) hydrolase.

[0070] The polypeptide typically comprises one or more of the following features: (i) at least 50% amino acid sequence identity to SEQ ID NO:2; (ii) a catalytic triad comprising a serine at position 112, a lysine residue at a position 115 and a tyrosine or phenylalanine, optionally a tyrosine residue, at a position 215 with respect to SEQ ID NO:2; (iii) a nitrogen inverting residue at position 170 with respect to SEQ ID NO:2. In some embodiments the nitrogen inverting residue is a tyrosine residue; (iv) the polypeptide has 6-aminohexanoate dimer hydrolase activity. 6-aminohexanoate dimer hydrolase activity is the ability to hydrolyse the amide bond between two polyamide 6 monomers in a polyamide 6 dimer to provide the individual monomers.

[0071] In some embodiments, the polypeptide may comprise at least (iv); at least (i) and (iv); at least (i), (ii), and (iv); at least (i), (ii) and (iii); at least (ii) and (iii) or (i), (ii), (iii) and (iv).

[0072] In some embodiments the catalytic triad comprises a serine residue at position 112, a lysine residue at a position 115 and a tyrosine residue, at a position 215 with respect to SEQ ID NO:2.

[0073] In one aspect the disclosure provides a polypeptide capable of hydrolysing an amide bond in a linear amide polymer as disclosed herein, which is derived from a wild type polypeptide having 6- aminoheaxanoate dimer hydrolase activity by replacing the amino acid at position 181with a glycine residue or an arginine residue and / or replacing the amino acid at position 266 with a histidine residue to provide a polypeptide whose sequence comprises a glycine residue or arginine residue at position 181 and a histidine residue at position 266 with respect to SEQ ID NO:1.

[0074] 6-aminoheaxanoate dimer hydrolases may be readily identified using key word searching of Genbank (https: / / www.ncbi.nlm.nih.gov / genbank / ) and / or by selecting polypeptides that have a sequence identity of at least 50% or 60% with SEQ ID NO:1 (UniProt accession no: P07061) using a BLAST search (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) and extracting those having the catalytic triad referred to above. Such polypeptides can readily be expressed and tested according to methods disclosed elsewhere herein to verify activity prior to carrying out the necessary amino acid substitutions.

[0075] The term “derived from” as used herein, should be taken to mean that the polypeptide is a mutant polypeptide of a wild type polypeptide wherein, if the amino acid at position 181 is other than glycine, then it is replaced with a glycine residue or an arginine residue and if the amino acid at position 266 is other than a histidine residue, then it is replaced with a histidine residue. In some embodiments no other amino acid changes are made. In some embodiments further amino acid changes may be made such as those described elsewhere herein. Particularly, the amino acid at position 187 may be substituted to provide a polypeptide that that has amino acid selected from alanine, cysteine, glutamate, aspartate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine at position 187.

[0076] In some embodiments the mutant polypeptides have an increased hydrolytic activity towards a polyamide 6,6 polymer or oligomer. In some embodiments such mutants have an increased activity towards amino terminated (N-terminated) oligomers. Amino terminated oligomers (N-terminated oligomers) are oligomers having at least one amino terminus, particularly two amino termini. Typically such oligomers are derived from polyamides, such as polyamides comprising at least one diamide monomer, e.g. polyamide 6,6.

[0077] In some embodiments such mutant has an increased activity towards carboxy-terminated (C-terminated) oligomers, e.g. having one or two carboxy termini.

[0078] An increased activity means an increased ability of the polypeptide to hydrolyse a polyamide polymer or polyamide oligomer in comparison to wild-type NylB enzymes (such as SEQ ID NO:1). In an embodiment, the activity of the polypeptide may be increased by at least 1, 2, 5, 10, 20, 30 50, 100, 300, 500, or 1000% compared to the wild type polypeptide.

[0079] In some embodiments the disclosure provides a polypeptide capable of hydrolysing an amide bond in a linear amide polymer as disclosed herein, which comprises amino acids 2 to 392 of SEQ ID NO:2 or an amino acid sequence having at least 60% sequence identity thereto.

[0080] In some embodiments polypeptides disclosed herein have at least 60% amino acid sequence identity to SEQ ID NO: 2; or to amino acids 2 to 392 of SEQ ID NO:2; at least 65% amino acid sequence identity to SEQ ID NO:2 or to amino acids 2 to 392 of SEQ ID NO:2; at least 70% amino acid sequence identity, at least 75% amino acid sequence identity; at least 80% amino acid sequence identity; at least 85% amino acid sequence identity; at least 90% amino acid sequence identity; or at least 95% amino acid sequence identity to SEQ ID NO:2 or to amino acids 2 to 392 of SEQ ID NO:2. In some embodiments polypeptides have at least 96, 97, 98 or 99% sequence identity to SEQ ID NO:2 or to amino acids 2 to 392 of SEQ ID NO:2. In some embodiments polypeptides have no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid changes with respect to SEQ ID NO:2 or amino acids 2 to 392 of SEQ ID NO:2.

[0081] In some embodiments a polypeptide comprises the amino acid sequence of amino acids 2 to 392 of SEQ ID NO:2; or an amino acid sequence having at least 96% sequence identity thereto. In some embodiments the polypeptide comprises the amino acid sequence of amino acids 2 to 392 of SEQ ID NO:2; or an amino acid sequence having at least 97%, 98% or 99% sequence identity thereto. In some embodiments the polypeptide comprises the amino acid sequence of amino acids 2 to 392 of SEQ ID NO:2 having no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions with respect to SEQ ID NO:2. In some embodiments the polypeptide comprises amino acids 2-392 of SEQ ID NO:2. In some embodiments a polypeptide consists of amino acids 1-392 of SEQ ID NO:2.

[0082] In each case, where a peptide is said to have a sequence identity of less than 100% to another, active peptide, such as SEQ ID NO:1 or SEQ ID NO:2, non-identical amino acids do not occur at substituted positions, nor do they alter key functional amino acids of the peptide such as the catalytic triad or nitrogen inverting residue as described elsewhere herein.

[0083] In one group of identified mutants the polypeptide is selected from SEQ ID NO:22 to SEQ ID NO:29 and polypeptides having at least 70% sequence identity thereto.

[0084] In some embodiments the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:28 or SEQ ID NO:29 or polypeptides having at least 70% sequence identity thereto.

[0085] In some embodiments the polypeptide comprises the amino acid sequence of amino acids 2 to 419 of SEQ ID NO:29 or an amino acid having 70% sequence identity thereto.

[0086] In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:30 wherein:

[0087] X at position 5 is T or A;

[0088] X at position 9 is H, D or Y

[0089] X at position 12 is Y or F;

[0090] X at position 16 is present or absent;

[0091] X at position 31 is N or S;

[0092] X at position 34 is T or A;

[0093] X at position 36 is A or S;

[0094] X at position 37 is H or N;

[0095] X at position 39 is G or A;

[0096] X at position 41 is I, V or L;

[0097] X at position 42 is V, L or M;

[0098] X at position 46 is V, A or T;

[0099] X at position 47 is V or I;

[0100] X at position 63 is D, N or G;

[0101] X at position 65 is V or L;

[0102] X at position 66 is present or absent;

[0103] X at position 67 is present or absent;

[0104] X at position 69 is L or M;

[0105] X at position 71 is G or D;

[0106] X at position 77 is E or D;

[0107] X at position 80 is Y, F or H; X at position 81 is T or A;

[0108] X at position 86 is V or I;

[0109] X at position 87 is V or L;

[0110] X at position 90 is N, D or G;

[0111] X at position 91 is Q, D, E or K;

[0112] X at position 93 is V or I;

[0113] X at position 94 is G or A;

[0114] X at position 96 is Y or H;

[0115] X at position 102 is A or G;

[0116] X at position 105 is D or Q;

[0117] X at position 109 V or L;

[0118] X at position 112 is V or I;

[0119] X at position 116 is M or L;

[0120] X at position 120 is L or V;

[0121] X at position 121 is V, M or I;

[0122] X at position 123 is A or S;

[0123] X at position 124 is L or V;

[0124] X at position 125 is I or V;

[0125] X at position 126 is D or E;

[0126] X at position 128 is G or A;

[0127] X at position 130 is I or V;

[0128] X at position 136 is I or V;

[0129] X at position 140 is I, V or L;

[0130] X at position 147 is A or V;

[0131] X at position 148 is Y or F;

[0132] X at position 153 is I or V;

[0133] X at position 155 is H, N, D or Q;

[0134] X at position 156 is V or L;

[0135] X at position 161 is I or V;

[0136] X at position 163 is I, V or L;

[0137] X at position 168 is D, N or E;

[0138] X at position 174 is S or A;

[0139] X at position 180 is G, R, or A

[0140] X at position 182 is S or A;

[0141] X at position 183 is A, S or G;

[0142] X at position 186 is R or is A, C D, E, F, G, H, 1, 1, L, Q, T or V.

[0143] X at position 195 is D or G; X at position 201 is T or A;

[0144] X at position 202 is T or S ;

[0145] X at positions 208 is N or absent;

[0146] X at positions 209 is present or absent;

[0147] X at positions 210 is present or absent;

[0148] X at positions 211 is present or absent;

[0149] X at position 215 is E, N, Q or K;

[0150] X at position 222 is N or H;

[0151] X at position 225 is V or I;

[0152] X at position 227 is A or G;

[0153] X at position 229 is I or V;

[0154] X at position 234 is T or S ;

[0155] X at position 241 is A or V;

[0156] X at position 248 is S or A;

[0157] X at position 249 is K or R;

[0158] X at position 250 is L or I;

[0159] X at position 251 is D or G;

[0160] X at position 255 is D or E;

[0161] X at position 257 is T or A;

[0162] X at position 258 is I or V;

[0163] X at position 263 is A, T or S;

[0164] X at position 270 is G or A;

[0165] X at position 272 is V or I;

[0166] X at position 284 is Q or R;

[0167] X at position 285 is L, I or M;

[0168] X at position 286 is M, L or V;

[0169] X at position 288 is D, N or E;

[0170] X at position 292 is G or A;

[0171] X at position 296 is R or K;

[0172] X at position 307 is L or F;

[0173] X at position 311 is D or S ;

[0174] X at position 313 is E, D or N;

[0175] X at position 319 is S or G;

[0176] X at position 322 is A or S ;

[0177] X at position 324 is H or F;

[0178] X at position 338 is N or H;

[0179] X at position 343 is V or I; X at position 344 is T or S ;

[0180] X at position 345 is G or A;

[0181] X at position 349 is Y or H;

[0182] X at position 355 is I, L or V;

[0183] X at position 359 is T, S or A;

[0184] X at position 360 is D, Q or N;

[0185] X at position 361 is T or S;

[0186] X at position 362 is V or A;

[0187] X at position 366 is M, L or F;

[0188] X at position 368 is T, A or S;

[0189] X at position 371 is E or D;

[0190] X at position 373 is E or D;

[0191] X at position 376 is H, E or Q;

[0192] X at position 378 is H or N;

[0193] X at position 381 is Q or D;

[0194] X at position 382 is N, T or S;

[0195] X at position 384 is L, I or V;

[0196] X at position 386 is L or I;

[0197] X at position 391 is S, A or T; and / or

[0198] X at position 392 is L or M; wherein X at other positions is any amino acid.

[0199] In some embodiments X at position 16 is absent. If X at amino acid 16 is present it is Q, E or A.

[0200] In some embodiments X at position 66 is absent. If X at amino acid 66 is present it is T or A. In some embodiments X at position 67 is absent. If X at position 67 is present it is E or S. In some embodiments X at position 208, 209, 210 and 211 are absent. If X at position 208, 209, 210 and 211 are present each is any amino acid. In some embodiments if X at position 208, 209, 210 and 211 are present they are N, S, T and G respectively (SEQ ID NO:47).

[0201] In some embodiments X at position 1 is M; X at position 2 is N, T, V or P; X at position 3 is A, S, T, or N; X at position 4 is P, S, G, A or T; X at position 6 is G or A; X at position 9 is H, T, A, P or G; X at position 10 is S, D, A or P; X at position 13 is P or A; X at position 16 is missing or is Q, E or A; X at position 17 is T, A or P; X at position 18 is G, S or V; X at position 19 is Q, P, A or H; X at position 21 is T, G or L; X at position 22 is L or Q; X at position 24 is T, N, S or L; X at position 27 is E, V or T; X at position 28 is G, A or P; X at position 29 is P or T; X at position 30 is D,Y, N or H; X at position 31 is N or S; X at position 39 is G or A; X at position 44 is T or M; X at position 46 is T, V or A; X at position 48 is S, A, T or R; X at position 50 is R or S; X at position 51 is L, R, F or H; X at position 52 is P or R; X at position 53 is D, A or P; X at position 54 is A, N, S or E; X at position 55 is S, T, R or P; X at position 56 is A, V or D; X at position 57 is A, V, E or S; X at position 58 is A, V, P or G; X at position 59 is A, T, V or F; X at position 60 is G, D, E, V or S; X at position 61 is S, R or N; X at position 63 is N, D or G; X at position 64 is G, A or H; X at position 66 is absent or is T or A; X at position 67 is absent or is S or E; X at position 68 is G, D or R; X at position 71 is D or G; X at position 73 is L, K, T, R, Q, E or A; X at position 74 is H, R, Q or A; X at position 78 is E, G or A; X at position 79 is T, S or V; X at position 80 is Y, F or H; X at position 88 is H, Q, T or R; X at position 89 is H, G or R; X at position 90 is N, D or K; X at position 91 is Q, D, E or K; X at position 94 is A or G; X at position 102 is A or G; X at position 106 is L or R; X at position 127 is S, A, E or D; X at position 128 is G or A; X at position 129 is E, G, S, A or L; X at position 131 is V, D or N; X at position 133 is T, S or R; X at position 134 is A, H or R; X at position 135 is R, K or P; X at position 137 is V, F or T; X at position 138 is D or R; X at position 144 is A, K or S; X at position 145 is G, T or D; X at position 147 is A or V; X at position 155 is N, H d or Q; X at position 160 is A, V, S or Q; X at position 162 is H, E, A or S; X at position 164 is D, N or I; X at position 166 is S, N or R; X at position 170 is V, G, L, E or T; X at position 173 is A, D or T; X at position 178 is T, A or R; X at position 183 is A, S or G; X at position 187 is T, G A or E; X at position 188 is R or S; X at position 190 is P, D, E or A; X at position 191 is G or E; X at position 194 is Q, E, A, D, Y or V; X at position 195 is D or G; X at position 196 is T, M or D; X at position 197 is Y or T, X at position 198 is A or D; X at position 204 is T or R; X at position 206 is N, G, S or P; X at position 208 is absent or N, X at position 209 is absent or S; X at position 210 is absent or T; X at position 211 is absent or G; X at position 212 is Q, T, E or R; X at position 213 is A,V,L or T; X at position 214 is S, N, P or G; X at position 227 is A or G; X at position 236 is L, R or E; X at position 239 is S or P; X at position 240 is E , D or A; X at position 241 is A or V; X at position 244 is T, K, E or R; X at position 245 is Y, H or R; X at position 251 is D or G; X at position 254 is R, S or A; X at position 262 is S, P, E or R; X at position 268 is A or P; X at position 270 is G or A; X at position 273 is S, L or C; X at position 274 is C, A or T; X at position 289 is R, G, H or D; X at position 291 is E, K A or V; X at position 292 is A or G; X at position 293 is H or P; X at position 294 is L or G; X at position 300 is P, A, K or R; X at position 301 is E, D, H or A; X at position 303 is V, I or T; X at position 304 is G, Q or E; X at position 305 is N, S , G or A; X at position 308 is N or A; X at position 311 is D or S; X at position 312 is R, K, Q or T; X at position 316 is V, K or T; X at position 317 is G, D or Y; X at position 318 is S, A or E; X at position 319 is S or G; X at position 323 is I, A, G, T or R; X at position 324 is H or F; X at position 325 is P or H; X at position 326 is Q, G E H, N or D; X at position 336 is T or M; X at position 339 is E, G or D; X at position 342 is N or I; X at position 345 is G or A; X at position 352 is Y or N; X at position 358 is P, A T or R; X at position 362 is V or A; X at position 363 is M, I or T; X at position 365 is K, Q or A; X at position 367 is S, P or C; X at position 374 is N, S, A or T; X at position 375 is L, G, D or A; X at position 376 is H, E or Q; X at position 377 is L, R or W; X at position 379 is Q, T, A or E; X at position 380 is L, R, A or V; X at position 382 is N, T or S; X at position 383 is Q, E, S, K or G; X at position 389 is S, C or G; X at position 390 is R, H, Y, E or A; X at position 394 is S, H, E, D or A; X at position 395 is R, N, I, A or V; X at position 396 is T, I, G, E or V; X at position 397 is A, L, T or N; X at position 398 is A, S, T, K or R; X at position 399 is D, E, G, T, K or R; X at position 400 is A, G T or K; X at position 401 is A, S, K or N; X at position 402 is A, T E, S or L; X at position 403 is absent or is T, Q or D; X at position 404 is absent or is D, E or V; X at position 405 is absent or is E, P, H or N; X at position 406 is absent or is A, E, T or R; X at position 407 is absent or is A or G; X at position 408 is absent or is D, A or S; X at position 409 is absent or is E or R; X at position 410 is absent or is A or T; X at position 411 is absent or is A or T; X at position 412 is absent or is P; X at position 413 is absent or is K or E; X at position 414 is absent or is S; X at position 415 is absent or is K; X at position 416 is absent or is D or E; X at position 417 is absent or is K, N or T; X at position 418 is absent or is A; and X at position 419 is absent or is A.

[0202] In some embodiments the polypeptide comprises the amino acid sequence of amino acids 2 to 412 of SEQ ID NO: 31 wherein:

[0203] X at position 2 is T or N;

[0204] X at position 10 is D or S;

[0205] X at position 45 is V or T;

[0206] X at position 47 is A or S;

[0207] X at position 50 is any amino acid, particularly R or L;

[0208] X at position 51 is any amino acid particularly R or P;

[0209] X at position 53 is any amino acid particularly N or A;

[0210] X at position 54 is any amino acid particularly R, S or T;

[0211] X at position 55 is V or A;

[0212] X at position 59 is D or G;

[0213] X at position 62 is D or N;

[0214] X at position 68 is D or G;

[0215] X at position 70 is any amino acid particularly K or L;

[0216] X at position 71 is R or H;

[0217] X at position 84 is L or V;

[0218] X at position 91 is G or A;

[0219] X at position 130 is S or T;

[0220] X at position 132 is any amino acid, particularly R or P;

[0221] X at position 137 is V or I;

[0222] X at position 152 is H or N;

[0223] X at position 161 N or D;

[0224] X at position 177 is G, A or R.

[0225] X at position 183 is R or is A, C D, E, F, G, H, 1, 1, L, Q, T or V. X at position 188 is any amino acid, particularly E or G;

[0226] X at position 206 is V or A;

[0227] X at position 222 is I or V;

[0228] X at position 244 is D or G;

[0229] X at position 284 is K or E;

[0230] X at position 285 is G or A;

[0231] X at position 286 is any amino acid, particularly P or H;

[0232] X at position 298 is S or N;

[0233] X at position 309 is A or V;

[0234] X at position 319 is any amino acid, particularly Q or G;

[0235] X at position 332 is any amino acid, particularly G or E;

[0236] X at position 336 is V or I;

[0237] X at position 351 is any amino acid, particularly A or P;

[0238] X at position 353 is D or Q;

[0239] X at position 356 is I or M;

[0240] X at position 359 is M or L;

[0241] X at position 367 is S or N;

[0242] X at position 376 is E or Q;

[0243] X at position 383 is H or R;

[0244] X at position 389 is any amino acid, particularly I or T;

[0245] X at position 392 is E or D;

[0246] X at position 396 is any amino acid, particularly T or D;

[0247] X at position 397 is present or absent if present it is any amino acid, particularly a D;

[0248] X at position 398 is present or absent if present it is any amino acid, particularly an E;

[0249] X at position 399 is present or absent if present it is any amino acid, particularly an A;

[0250] X at position 400 is present or absent if present it is any amino acid, particularly an A;

[0251] X at position 401 is present or absent if present it is any amino acid, particularly an A;

[0252] X at position 403 is T or A;

[0253] X at position 404 is T or A;

[0254] X at position 406 is E or K; and / or

[0255] X at position 410 is N or K; optionally X at positions 397, 398, 399, 400 and 401 are present; optionally they are D, E, A, A and A respectively.

[0256] In some embodiments X at position 2 is T or N; X at position 10 is S or D; X at position 45 is T or V; X at position 50 is L or R; X at position 51 is P or R; X at position 53 is A or N; X at position 54 is T, S or R; X at position 55 is A or V; X at position 59 is G or D; X at position 68 is G or D; X at position 91 is G or A; X at position 188 is E or G; X at position 106 is V or A; X at position 244 is D or G; X at position 285 is G or A; X at position 298 is N or S; X at position 310 is V or A; X at position 319 is G or Q; X at position 332 is E or G; X at position 251 is P or A; X at position 367 is N or S; X at position 389 is I or T; X at position 396 is D or T; X at position 397 is absent or D; X at position 398 is absent or E; X at position 399 is absent or A; X at position 400 is absent or A; and X at position 401 is absent or A.

[0257] In some embodiments the polypeptide comprises an amino acid sequence selected from SEQ ID NO:s 22-29 In some embodiments the polypeptide sequence lacks the initiating methionine. In some embodiments the polypeptide consists of an amino acid sequence selected from SEQ ID NO:s 22 to 29.

[0258] In some embodiments the polypeptide as disclosed herein is a polypeptide comprising the amino acid sequence of SEQ ID NO:22 to SEQ ID NO:29; or an amino acid having 70% sequence identity to any of SEQ ID NO:22 to SEQ ID NO:29; and optionally wherein the polypeptide comprises an amino acid selected from the group consisting of alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine at position 187 with respect to SEQ ID NO:1.

[0259] A further group of NylB homologue polypeptides according to the disclosure were identified from the UNIPROT database (SEQ ID NOs:33 to 116), by sequence homology to SEQ ID NO:1 and including a catalytic triad as laid out herein and including a nitrogen inverting residue as laid out herein. 181G, 266H mutants of these polypeptides are SEQ ID NOs:117 to 199, which are a further group of peptides of the disclosure. These mutants are derived from wild type sequences, SEQ ID NOs:33 to 116 by replacing the amino acid at position 181 with respect to SEQ ID NO:1 with a glycine residue and / or replacing the amino acid at position 266 with respect to SEQ ID NO:1, with a histidine residue.

[0260] In a further embodiment, a polypeptide selected from the group consisting of SEQ ID NOs:117 to 199 or a polypeptide having at least 70% sequence identity thereto are provided. In some embodiments the polypeptide may comprise or consist of polypeptides comprising the amino acid sequence of any of SEQ ID NOs:117 to 199 or a polypeptide having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments the polypeptide may comprise or consist of a polypeptide having no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino substitution(s) with respect to the amino acid sequence of any of SEQ ID NOs:117 to 199.

[0261] As for other polypeptides disclosed herein, polypeptides in which the amino acid at position 181 is G, R or A may also be provided. In some embodiments of these polypeptides, the amino acid at position 187 with reference to SEQ ID NO:2 may be selected from A, C, D, E, F, G, H, I, L, Q, T or V. Polypeptides of this disclosure having the 181G, 266H mutation and having at least 60% sequence identity to SEQ ID NO:2 include SEQ ID NOs:6 to 13; SEQ ID NOs:22 to 29; SEQ ID NOs:117, 118, 122, 128, 131, 133 and 149; SEQ ID NOs:151 to 154; SEQ ID NO:157, SEQ ID NO:160, SEQ ID NOs : 162 to 164; SEQ ID NOs : 167 to 169 ; SEQ ID NO: 173 ; SEQ ID NOs : 175 to 177 ; SEQ ID NOs : 179, 181, 184, 187, 192, 193, and SEQ ID NO: 196. These polypeptides provide another embodiment of the disclosure.

[0262] Polypeptides having the 181G, 266H mutation and having at least 70% sequence identity to SEQ ID NO:2 include SEQ ID NOs:6 to 13, SEQ ID Nos:23, 117, 118, 122, 128, 131, 152, 153, 154, 157, 162, 163, 168, 169, 175, 176, 179, 181, 184 and SEQ ID NO:192. These polypeptides provide another embodiment of the disclosure. SEQ ID NO:373 provides a consensus of these sequences.

[0263] Polypeptides having the 181G, 266H mutation and having at least 80% sequence identity to SEQ ID NO:2 include SEQ ID NOs:6 to 13, SEQ ID NOs:117, 118, 153, 162, 163, 162, 163 and 179. These polypeptides provide another embodiment of the disclosure. SEQ ID NO:272 provides a consensus of these sequences.

[0264] Polypeptides having the 181G, 266H mutation and having at least 90% sequence identity to SEQ ID NO:2 include SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO: 117 these sequences provide another embodiment of the disclosure.

[0265] As for other polypeptides disclosed herein, polypeptides of the above groups in which the amino acid at position 181 is G, R or A may also be provided. In some embodiments of these polypeptides, the amino acid at position 187 with reference to SEQ ID NO:2 may be selected from A, C, D, E, F, G, H, I, L, Q, T or V.

[0266] In some embodiments the polypeptide is a polypeptide comprising SEQ ID NO:373 wherein:

[0267] X at position 2 is S, T or absent

[0268] X at position 3 is A, D, V or absent

[0269] X at position 4 is A or absent

[0270] X at position 5 is L, D, T or absent

[0271] X at position 6 is G, A, or absent

[0272] X at position 7 is S, A or absent

[0273] X at position 8 is G, D or absent

[0274] X at position 9 is D, H or absent

[0275] X at position 10 is N, L, P or absent

[0276] X at position 11 is T, P, D or absent X at position 12 is S, E, A R or absent

[0277] X at position 13 is H, E, P, A, G or absent

[0278] X at position 14 is D, R, V, N, E, P or absent

[0279] X at position 15 is T, R, P, G, I or absent

[0280] X at position 16 is D, A, K, S or absent

[0281] X at position 17 is S, R, I, A, P or absent

[0282] X at position 18 is R, P, Q, H, or absent

[0283] X at position 19 is R, H, N, A, T, D or absent

[0284] X at position 20 is R, G, A, T, D, S, P or absent

[0285] X at position 21 is R, S, P, G, A, T or absent

[0286] X at position 22 is T, P, S, A, L, G, V, D or absent

[0287] X at position 23 is T or absent

[0288] X at position 24 is G or absent

[0289] X at position 25 is P, Q, S, A, T or absent

[0290] X at position 26 is H or absent

[0291] X at position 27 is P or absent

[0292] X at position 28 is A, G, D, P, S or absent

[0293] X at position 29 is R or absent

[0294] X at position 30 is Y, F or absent

[0295] X at position 31 is P, A or absent

[0296] X at position 32 is G, D or absent

[0297] X at position 33 is G, A or absent

[0298] X at position 34 is A, E, G, P or absent

[0299] X at position 35 is P, A, R or absent

[0300] X at position 36 is G, S, E, N or absent

[0301] X at position 37 is E, Q, R, V, A, H or absent

[0302] X at position 39 is R, N, D or S

[0303] X at position 41 is E or D

[0304] X at position 42 is S, G or T

[0305] X at position 45 is E, D, Q or T

[0306] X at position 46 is A, P or G

[0307] X at position 48 is Q, H, D, Y or G

[0308] X at position 52 is A or T

[0309] X at position 54 is S, A or G

[0310] X at position 55 is H, R, G or N

[0311] X at position 56 is L or M

[0312] X at position 57 is G, N or A X at position 59 is I, L or M

[0313] X at position 60 is L, V, M or T

[0314] X at position 61 is P or H

[0315] X at position 62 is T, S, V, M

[0316] X at position 64 is V, T, A or S

[0317] X at position 65 is I or V

[0318] X at position 66 is P, S or A

[0319] X at position 68 is R, H, N or T

[0320] X at position 69 is S, D, P, R, V, A, D or absent

[0321] X at position 70 is P, V or E

[0322] X at position 71 is A, N, E, H, S, T or G

[0323] X at position 72 is A, T, V, S, D or absent

[0324] X at position 73 is P, R or S

[0325] X at position 74 is S, V, A, G, L, Y, P, T or absent

[0326] X at position 75 is G, A or absent

[0327] X at position 76 is A or absent

[0328] X at position 77 is A or absent

[0329] X at position 78 is E or absent

[0330] X at position 79 is E, H, A, S, G, D, E or T

[0331] X at position 80 is A, P, G

[0332] X at position 81 is T, V, L, A, F

[0333] X at position 82 is V, A, S, D

[0334] X at position 83 is R, H, N

[0335] X at position 85 is D, G, E

[0336] X at position 86 is S, A, V, P, L or absent

[0337] X at position 87 is L, I, V, F or absent

[0338] X at position 88 is T, A, E, L, G or absent

[0339] X at position 89 is P, S, T, A or absent

[0340] X at position 90 is R, Q, A, H or G

[0341] X at position 91 is L, M or V

[0342] X at position 92 is P or D

[0343] X at position 93 is D, G, E, A or T

[0344] X at position 95 is Q, E, V, L, R or G

[0345] X at position 96 is Q, R, A or D

[0346] X at position 97 is R or Q

[0347] X at position 99 is E or D

[0348] X at position 100 is Q, E, R or A X at position 101 is S or T

[0349] X at position 102 is Y, F or C

[0350] X at position 104 is D or N

[0351] X at position 106 is F or L

[0352] X at position 110 is R or H

[0353] X at position 111 is G or R

[0354] X at position 112 is G, D, T, S, A, D, E or N

[0355] X at position 113 E, R, D, H or Q

[0356] X at position 114 is V or L

[0357] X at position 115 is L, V or I

[0358] X at position 117 is E or D

[0359] X at position 118 is Y, F or H

[0360] X at position 119 is Y, S or A

[0361] X at position 120 is R, A, G or K

[0362] X at position 121 is A, S, P or D

[0363] X at position 124 is A or G

[0364] X at position 125 is P or A

[0365] X at position 126 is D or H

[0366] X at position 127 is D, Q, A or G

[0367] X at position 128 is R, L or P

[0368] X at position 131 is L, V or I

[0369] X at position 133 is S or A

[0370] X at position 134 is V or I

[0371] X at position 138 is L, M or I

[0372] X at position 140 is G or A

[0373] X at position 141 is T, I or L

[0374] X at position 142 is V, T or absent

[0375] X at position 143 is V, F, A or M

[0376] X at position 145 is A, V or S

[0377] X at position 146 is L, F or V

[0378] X at position 147 is V, I or A

[0379] X at position 148 is D, E or G

[0380] X at position 149 is E, G, D, Q, A, R or S

[0381] X at position 150 is G, A, S or D

[0382] X at position 151 is R, L, A, S or E

[0383] X at position 152 is I, L or V

[0384] X at position 153 is D or I X at position 154 is P or A

[0385] X at position 155 is A, S, E, R or D

[0386] X at position 156 is R, Q, G or A

[0387] X at position 157 is P, R or A

[0388] X at position 158 V or I

[0389] X at position 159 is T or V

[0390] X at position 160 E, A, H, T, R or D

[0391] X at position 161 is Y or H

[0392] X at position 162 is V or I

[0393] X at position 163 is P or R

[0394] X at position 166 is E, A or G

[0395] X at position 167 is G or D

[0396] X at position 169 is V or A

[0397] X at position 174 is S, A, Q or T

[0398] X at position 175 is D or G or absent

[0399] X at position 176 is D or absent

[0400] X at position 177 is A, P or absent

[0401] X at position 178 is A, N or absent

[0402] X at position 179 is A or absent

[0403] X at position 180 is P or absent

[0404] X at position 181 is R or absent

[0405] X at position 182 is V or absent

[0406] X at position 183 is T or absent

[0407] X at position 184 is I, L or V

[0408] X at position 185 is Q, L or R

[0409] X at position 186 is Q, H or R

[0410] X at position 187 is V, L or A

[0411] X at position 191 is E, Q, V, A or R

[0412] X at position 192 is V or I

[0413] X at position 193 is A, S, G or H

[0414] X at position 194 is I, V or L

[0415] X at position 195 is N or D

[0416] X at position 197 is S, N, D or R

[0417] X at position 199 is D, N or E

[0418] X at position 201 is V, I, L or T

[0419] X at position 202 is D or N

[0420] X at position 204 is A, G, D or T X at position 205 is S or A

[0421] X at position 209 is T, A or R

[0422] X at position 211 is G, A or R; particularly G

[0423] X at position 213 is S or A

[0424] X at position 217 is R, A, S, C, D, E, F, G, H, 1, 1, L, Q, T or V

[0425] X at position 218 is S, T, P, A, D, V, R or E

[0426] X at position 219 is R or A

[0427] X at position 220 is R, A, L, C or G

[0428] X at position 221 is D, E, H, T, D, A, P or E

[0429] X at position 222 is G, D or S

[0430] X at position 224 is P or A

[0431] X at position 225 is A, E, P, V, D, E or I

[0432] X at position 226 is D, N or S

[0433] X at position 227 is T, D or N

[0434] X at position 228 is Y or A

[0435] X at position 229 is E, A or S

[0436] X at position 232 is T, A or V

[0437] X at position 233 is T, S or G

[0438] X at position 235 is R or T

[0439] X at position 236 is G or T

[0440] X at position 237 is R or absent

[0441] X at position 238 is P or absent

[0442] X at position 239 is D or absent

[0443] X at position 240 is A or absent

[0444] X at position 241 is D or absent

[0445] X at position 242 is A or absent

[0446] X at position 243 is S, D, N or P

[0447] X at position 245 is A, S, G, R or T

[0448] X at position 246 is T, V, L or G

[0449] X at position 248 is E or A

[0450] X at position 255 is N, D or H

[0451] X at position 258 is V or I

[0452] X at position 260 is A or G

[0453] X at position 262 is I or V

[0454] X at position 263 is I or V

[0455] X at position 267 is T or S

[0456] X at position 269 is L, M or S X at position 270 is R or P

[0457] X at position 272 is A, S, G, V, P or H

[0458] X at position 273 is E, D or A

[0459] X at position 274 is A, L, V or I

[0460] X at position 275 is L or V

[0461] X at position 277 is I, V, T, R, A or E

[0462] X at position 278 is H, Y, F or R

[0463] X at position 279 is L or V

[0464] X at position 281 is A or S

[0465] X at position 282 is K or R

[0466] X at position 283 is L or I

[0467] X at position 284 is D or G

[0468] X at position 286 is D or G

[0469] X at position 287 is R, L, H or Q

[0470] X at position 290 is T, I or A

[0471] X at position 291 is I or V

[0472] X at position 295 is T, Q, S, R or A

[0473] X at position 296 is T, A or S

[0474] X at position 300 is F, C or Y

[0475] X at position 301 is A or P

[0476] X at position 303 is G or A

[0477] X at position 304 is G or A

[0478] X at position 305 is V or I

[0479] X at position 306 is S or C

[0480] X at position 307 is C, S, A or T

[0481] X at position 317 is R or Q

[0482] X at position 318 is M, V, L or A

[0483] X at position 319 is M, I, V or L

[0484] X at position 322 is G, S or R

[0485] X at position 324 is H, A, V, I, S, L, T or K

[0486] X at position 325 is A or G

[0487] X at position 326 is P or A

[0488] X at position 327 is G or R

[0489] X at position 332 is S or P

[0490] X at position 333 is E, Q, G or A

[0491] X at position 334 is T, A, D, G or E

[0492] X at position 336 is V, T or A X at position 337 is R, S, D or H

[0493] X at position 338 is S, R, G or A

[0494] X at position 339 is I, V or T

[0495] X at position 340 is M or L

[0496] X at position 341 is E, A, S or D

[0497] X at position 344 is S or D

[0498] X at position 345 is R, H, Q, T or P

[0499] X at position 346 is E, A, T or Q

[0500] X at position 349 is T, P, D or M

[0501] X at position 350 is Y, D, P or absent

[0502] X at position 351 is E, K, P, A, S or D

[0503] X at position 352 is G, D, H or S

[0504] X at position 354 is T or P

[0505] X at position 355 is G, N, S, D, E, T or A

[0506] X at position 356 is T, A, E, V, D, I or R

[0507] X at position 357 is F or H

[0508] X at position 359 is D, N, E, G or S

[0509] X at position 364 is R or S

[0510] X at position 369 is T or M

[0511] X at position 371 is N or D

[0512] X at position 372 is E or D

[0513] X at position 374 is G, A or S

[0514] X at position 377 is S or T

[0515] X at position 378 is G or A

[0516] X at position 379 is I or V

[0517] X at position 385 is N, S or Y

[0518] X at position 388 is L or I

[0519] X at position 389 is D or E

[0520] X at position 391 is P, L, R, Q, M or A

[0521] X at position 392 is T or S

[0522] X at position 394 is S, A or T

[0523] X at position 396 is I or V

[0524] X at position 399 is L or F

[0525] X at position 401 is S, T, A or V

[0526] X at position 404 is E, D or G

[0527] X at position 406 is D, Y or E

[0528] X at position 407 is T, G or S X at position 408 is D, E, R, A or P

[0529] X at position 409 is H, D, E, F, Y, Q or S

[0530] X at position 410 is W, G, L or R

[0531] X at position 411 is H, N or D

[0532] X at position 412 is G, D, R, A or E

[0533] X at position 413 is V, L, R, K or A

[0534] X at position 415 is N, S, Q, T or G

[0535] X at position 416 is D, G, A, E, S, R, Q or T

[0536] X at position 417 is V, I or L

[0537] X at position 419 is L or V

[0538] X at position 420 is D or E

[0539] X at position 421 is V, I or L

[0540] X at position 422 is S or C

[0541] X at position 423 is R, H, E or T

[0542] X at position 424 is A or S

[0543] X at position 427 is A, G, T, R, S, P, Q or absent

[0544] X at position 428 is M, A, V, L, F, I, P, V or absent

[0545] X at position 429 is A, P, R, G, S or absent

[0546] X at position 430 is Y, A or absent

[0547] X at position 431 is A or absent

[0548] X at position 432 is A or absent

[0549] X at position 433 is E or absent

[0550] X at position 434 is T or absent

[0551] X at position 444 is A or absent

[0552] X at position 445 is P or absent

[0553] X at position 446 is D or absent

[0554] X at position 447 is S or absent

[0555] X at position 448 is K or absent

[0556] X at position 449 is E or absent

[0557] X at position 450 is N or absent

[0558] X at position 451 is A or absent

[0559] X at position 452 is A or absent.

[0560] In some embodiments the polypeptide is selected from; SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:23, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:128, SEQ ID NO:131, SEQ ID NO:152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 157, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:179, SEQ ID NO:181, SEQ ID NO: 184 and SEQ ID NO: 192 or a sequence having at least 80% sequence identity to any of these; and wherein the said sequence is optionally substituted at position 187 with an amino acid selected from the group consisting of alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine.

[0561] In some embodiments the polypeptide is a polypeptide according to SEQ ID NO:372 wherein:

[0562] X at position 5 is S, A, T or L

[0563] X at position 8 is Q, P or A

[0564] X at position 11 is A, P or D

[0565] X at position 13 is Y or F

[0566] X at position 16 is A or G

[0567] X at position 17 is A, E or G

[0568] X at position 18 is A, P or R

[0569] X at position 20 is E, Q or R

[0570] X at position 22 is T, N or H

[0571] X at position 25 is S or G

[0572] X at position 29 is P or A

[0573] X at position 31 is H, Q or D

[0574] X at position 37 is A or S

[0575] X at position 38 is H or R

[0576] X at position 40 is G or S

[0577] X at position 42 is M, I or L

[0578] X at position 43 is V or L

[0579] X at position 45 is S or T

[0580] X at position 47 is A, V or T

[0581] X at position 48 is V or I

[0582] X at position 49 is S or P

[0583] X at position 52 is P, D or S

[0584] X at position 53 is V or P

[0585] X at position 54 is N or A

[0586] X at position 55 is A or T

[0587] X at position 57 is G, A, S, L or Y

[0588] X at position 58 is H, E, A or S

[0589] X at position 59 is A or P

[0590] X at position 60 is L, V or T

[0591] X at position 61 is A or V X at position 64 is D or G

[0592] X at position 65 is A, S or V

[0593] X at position 66 is I or L

[0594] X at position 67 is A, T or E

[0595] X at position 68 is A, T or P

[0596] X at position 69 is Q or R

[0597] X at position 70 is L or M

[0598] X at position 72 is D or G

[0599] X at position 74 is E or Q

[0600] X at position 75 is Q, R or A

[0601] X at position 79 is Q or E

[0602] X at position 80 is T or S

[0603] X at position 81 is Y or C

[0604] X at position 83 is D or N

[0605] X at position 85 is F or L

[0606] X at position 91 is T, S, G or A

[0607] X at position 94 is V, L or I

[0608] X at position 99 is R or A

[0609] X at position 100 is A or P

[0610] X at position 104 is A or P

[0611] X at position 112 is A or S

[0612] X at position 122 is V or F

[0613] X at position 126 is V or I

[0614] X at position 127 is D or E

[0615] X at position 128 is E, D or Q

[0616] X at position 130 is R or L

[0617] X at position 134 is A or E

[0618] X at position 135 is Q or R

[0619] X at position 137 is V or I

[0620] X at position 139 is E, H, T or R

[0621] X at position 140 is Y or H

[0622] X at position 145 is A, G or E

[0623] X at position 148 is V or A

[0624] X at position 153 is S, T or Q

[0625] X at position 154 is G or absent

[0626] X at position 155 is D or absent

[0627] X at position 156 is P or absent X at position 157 is N or absent

[0628] X at position 158 is A or absent

[0629] X at position 159 is P or absent

[0630] X at position 160 is R or absent

[0631] X at position 161 is V or absent

[0632] X at position 162 is T or absent

[0633] X at position 163 is V or L

[0634] X at position 164 is L or Q

[0635] X at position 166 is V or L

[0636] X at position 170 is Q or E

[0637] X at position 171 is I or V

[0638] X at position 172 is S or A

[0639] X at position 173 is I or V

[0640] X at position 176 is N or S

[0641] X at position 183 is A or G

[0642] X at position 190 is G, R or A; particularly G;

[0643] X at position 196 is R, A, S, C, D, E, F, G, H, 1, 1, L, Q, T or V

[0644] X at position 197 is T or S

[0645] X at position 200 is H, D, T or A

[0646] X at position 204 is A or E

[0647] X at position 205 is D or N

[0648] X at position 206 is T or D

[0649] X at position 208 is S, R or A

[0650] X at position 211 is T or A

[0651] X at position 212 is T or S

[0652] X at position 216 is D, S or N

[0653] X at position 218 is S, G, R or A

[0654] X at position 235 is I or V

[0655] X at position 236 is V or I

[0656] X at position 240 is T or S

[0657] X at position 242 is L or M

[0658] X at position 243 is R or P

[0659] X at position 245 is V or A

[0660] X at position 250 is T, I or V

[0661] X at position 251 is Y or H

[0662] X at position 254 is A or S

[0663] X at position 260 is R or L X at position 268 is T, Q, S or R X at position 269 is T or A X at position 276 is G or A X at position 280 is C or S X at position 297 is V, I or H X at position 306 is E, Q or G X at position 307 is D, G, T or A X at position 310 is R or E X at position 311 is R, S or G X at position 312 is V or I X at position 313 is L or M X at position 314 is A, S or E X at position 318 is H or R X at position 319 is E or A X at position 323 is D or Y X at position 324 is K, E or P X at position 325 is G or D X at position 328 is N, S or G X at position 329 is T or A X at position 332 is D, E, G or N X at position 344 is N or D X at position 362 is D or E X at position 364 is L, R, Q or P X at position 369 is I or V X at position 374 is S or T X at position 377 is D or E X at position 379 is Y or D X at position 381 is E, R, D or A X at position 385 is R or G X at position 386 is L or V X at position 388 is N, S or Q X at position 389 is G, A or D X at position 390 is I or V X at position 394 is V or I X at position 396 is R or H X at position 400 is A or T X at position 401 is V or M X at position 402 A or absent

[0664] In some embodiments the polypeptide is a polypeptide selected from the group consisting of SEQ ID SEQ ID NOs:6 to 13, SEQ ID NOs:117, 118, 153, 162, 163, 162, 163 and 179 or a polypeptide having at least 70% sequence identity thereto.

[0665] In any of these peptides the amino acid at position 187 with respect to SEQ ID NO:1 may be selected from the group consisting of C, D, E, F, I, L, Q, or V; particularly I or L. In any of these peptides the amino acid at position 187 with respect to SEQ ID NO:1 may be selected from the group consisting of A, C, D, G, H, I, L, M, Q, S, T or V; optionally A, C, D, H, I, L, Q, or T; further optionally C, D, H, I, L or Q.

[0666] In some embodiments polypeptides have at least 60% amino acid sequence identity to SEQ ID NO: 2; at least 65% amino acid sequence identity to SEQ ID NO:2; at least 70% amino acid sequence identity to SEQ ID NO:2; at least 75% amino acid sequence identity to SEQ ID NO:2; at least 80% amino acid sequence identity to SEQ ID NO:2; at least 85% amino acid sequence identity to SEQ ID NO:2; at least 90% amino acid sequence identity to SEQ ID NO:2; or at least 95% amino acid sequence identity to SEQ ID NO:2. In some embodiments polypeptides have at least 96, 97, 98 or 99% sequence identity to SEQ ID NO:2. In some embodiments such sequences have no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid changes with respect to SEQ ID NO:2.

[0667] In a further aspect, the invention provides polypeptides capable of hydrolysing an amide bond in a linear amide polymer wherein the polypeptide comprises an amino acid selected from alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine residues at position 187 with reference to SEQ ID NO:1.

[0668] Such polypeptides demonstrate improved polyamide 6,6 hydrolase activity, particularly improved polyamide 6,6 oligomerase activity when compared to NylB (SEQ ID NO:1) or to their wild type counterparts. These polypeptides may have at least 30% 40%, 50%, 60%, 70%, 80% 90% or more sequence identity to SEQ ID NO:1 or SEQ ID NO:33.

[0669] In some embodiments the polypeptide comprises a residue selected from alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine residue at position 187 with the proviso that if the amino acid at position 187 with respect to SEQ ID NO:1 is A, N, L, K or E then the amino acid at position 181 is A, R or G; particularly R or G and / or the amino acid at position 266 is H, G or M; particularly H. In some embodiments, if the polypeptide comprises an alanine residue at position 187 with respect to SEQ ID NO:1 then the amino acid at position 181 is A, R or G; particularly R or G and / or the amino acid at position 266 is H, G or M; particularly H; in some embodiments if the polypeptide has sequence identity of at least 60% with SEQ ID NO:2 and comprises an alanine residue at position 187 with respect to SEQ ID NO: 1 then the amino acid at position 181 is A, R or , G; particularly R or G and / or the amino acid at position 266 is H, G or M; particularly H.

[0670] In some embodiments (particularly if the peptide has a sequence identity of at least 60% with respect to SEQ ID NO:1) the polypeptide comprises a residue selected from alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine residue at position 187 with the proviso that if the amino acid at position 187 with respect to SEQ ID NO:1 is alanine then the polypeptide is not a polypeptide comprising the sequence of SEQ ID NO:34 or 67.

[0671] In some embodiments (particularly if the peptide has a sequence identity of less than 60% with respect to SEQ ID NO:1) the polypeptide comprises a residue selected from alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine residue at position 187 with the proviso that if the amino acid at position 187 with respect to SEQ ID NO:1 is N then the polypeptide is not a polypeptide comprising the sequence of SEQ ID NO:78.

[0672] In some embodiments (particularly if the peptide has a sequence identity of less than 60% with respect to SEQ ID NO:1) the polypeptide comprises a residue selected from alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine residue at position 187 with the proviso that if the amino acid at position 187 with respect to SEQ ID NO:1 is L then the polypeptide is not a polypeptide comprising the sequence of 62 or 373.

[0673] In some embodiments (particularly if the peptide has a sequence identity of less than 60% with respect to SEQ ID NO:1) the polypeptide comprises a residue selected from alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine residue at position 187 with the proviso that if the amino acid at position 187 with respect to SEQ ID NO:1 is K then the polypeptide is not a polypeptide comprising the sequence of SEQ ID NOs: 374, 375 or 376.

[0674] In some embodiments (particularly if the peptide has a sequence identity of less than 60% with respect to SEQ ID NO:1) the polypeptide comprises a residue selected from alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine residue at position 187 with the proviso that if the amino acid at position 187 with respect to SEQ ID NO:1 is E then the polypeptide is not a polypeptide comprising the sequence of SEQ ID NO:377. Such polypeptides may be derived from any of the other sequences disclosed herein by substituting the amino acid at position 187 with reference to SEQ ID NO:1 with an amino acid selected from alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine.

[0675] The polypeptide may additionally comprise (i) a catalytic triad comprising a serine residue at position 112, a lysine residue at a position 115 and a tyrosine or phenylalanine, optionally a tyrosine residue, at a position 215 with respect to SEQ ID NO:1;

[0676] (ii) a nitrogen inverting residue at position 170 with respect to SEQ ID NO:1; optionally wherein the nitrogen inverting residue is a tyrosine residue.

[0677] Such peptides have improved polyamide hydrolase activity with respect to their wild type sequences and / or with respect to SEQ ID NO:1 in the absence of a substitution at the 181 or 266 position. Typically, such peptides have M, C, N or D at position 181 and D, S, M, C, A or N at position 266.

[0678] Polypeptides having a sequence identity of at least 60% to SEQ ID NO:1 include: SEQ ID NO:33, SEQ ID NOs: 14-21; SEQ ID NO:38, SEQ ID NO:44, SEQ ID NOs:48-50, SEQ ID NOs:68-71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:79-81, SEQ ID NOs:84-86, SEQ ID NO:90, SEQ ID NOs:92-94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO: 101, SEQ ID NO: 104, SEQ ID NO: 109, SEQ ID NO: 110 and SEQ ID NO: 115.

[0679] Polypeptides having a sequence identity of at least 70% to SEQ ID NO:1 include: SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NOs:69-71, SEQ ID NO:74, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO: 101 and SEQ ID NO: 109.

[0680] Polypeptides having a sequence identity of at least 80% to SEQ ID NO:1 include: SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:70, SEQ ID NO:79, SEQ ID NO:80 and SEQ ID NO: 96.

[0681] Suitable peptides include those comprising the sequence of SEQ ID NO:379 wherein:

[0682] X at position 2 is S, T or absent

[0683] X at position 3 is A, D, V or absent

[0684] X at position 4 is A or absent

[0685] X at position 5 is L, D, T or absent

[0686] X at position 6 is G, A, or absent

[0687] X at position 7 is S, A or absent

[0688] X at position 8 is G, D or absent

[0689] X at position 9 is D, H or absent

[0690] X at position 10 is N, L, P or absent

[0691] X at position 11 is T, P, D or absent X at position 12 is S, E, A R or absent

[0692] X at position 13 is H, E, P, A, G or absent

[0693] X at position 14 is D, R, V, N, E, P or absent

[0694] X at position 15 is T, R, P, G, I or absent

[0695] X at position 16 is D, A, K, S or absent

[0696] X at position 17 is S, R, I, A, P or absent

[0697] X at position 18 is R, P, Q, H, or absent

[0698] X at position 19 is R, H, N, A, T, D or absent

[0699] X at position 20 is R, G, A, T, D, S, P or absent

[0700] X at position 21 is R, S, P, G, A, T or absent

[0701] X at position 22 is T, P, S, A, L, G, V, D or absent

[0702] X at position 23 is T or absent

[0703] X at position 24 is G or absent

[0704] X at position 25 is P, Q, S, A, T or absent

[0705] X at position 26 is H or absent

[0706] X at position 27 is P or absent

[0707] X at position 28 is A, G, D, P, S or absent

[0708] X at position 29 is R or absent

[0709] X at position 30 is Y, F or absent

[0710] X at position 31 is P, A or absent

[0711] X at position 32 is G, D or absent

[0712] X at position 33 is G, A or absent

[0713] X at position 34 is A, E, G, P or absent

[0714] X at position 35 is P, A, R or absent

[0715] X at position 36 is G, S, E, N or absent

[0716] X at position 37 is E, Q, R, V, A, H or absent

[0717] X at position 39 is R, N, D or S

[0718] X at position 41 is E or D

[0719] X at position 42 is S, G or T

[0720] X at position 45 is E, D, Q or T

[0721] X at position 46 is A, P or G

[0722] X at position 48 is Q, H, D, Y or G

[0723] X at position 52 is A or T

[0724] X at position 54 is S, A or G

[0725] X at position 55 is H, R, G or N

[0726] X at position 56 is L or M

[0727] X at position 57 is G, N or A X at position 59 is I, L or M

[0728] X at position 60 is L, V, M or T

[0729] X at position 61 is P or H

[0730] X at position 62 is T, S, V, M

[0731] X at position 64 is V, T, A or S

[0732] X at position 65 is I or V

[0733] X at position 66 is P, S or A

[0734] X at position 68 is R, H, N or T

[0735] X at position 69 is S, D, P, R, V, A, D or absent

[0736] X at position 70 is P, V or E

[0737] X at position 71 is A, N, E, H, S, T or G

[0738] X at position 72 is A, T, V, S, D or absent

[0739] X at position 73 is P, R or S

[0740] X at position 74 is S, V, A, G, L, Y, P, T or absent

[0741] X at position 75 is G, A or absent

[0742] X at position 76 is A or absent

[0743] X at position 77 is A or absent

[0744] X at position 78 is E or absent

[0745] X at position 79 is E, H, A, S, G, D, E or T

[0746] X at position 80 is A, P, G

[0747] X at position 81 is T, V, L, A, F

[0748] X at position 82 is V, A, S, D

[0749] X at position 83 is R, H, N

[0750] X at position 85 is D, G, E

[0751] X at position 86 is S, A, V, P, L or absent

[0752] X at position 87 is L, I, V, F or absent

[0753] X at position 88 is T, A, E, L, G or absent

[0754] X at position 89 is P, S, T, A or absent

[0755] X at position 90 is R, Q, A, H or G

[0756] X at position 91 is L, M or V

[0757] X at position 92 is P or D

[0758] X at position 93 is D, G, E, A or T

[0759] X at position 95 is Q, E, V, L, R or G

[0760] X at position 96 is Q, R, A or D

[0761] X at position 97 is R or Q

[0762] X at position 99 is E or D

[0763] X at position 100 is Q, E, R or A X at position 101 is S or T

[0764] X at position 102 is Y, F or C

[0765] X at position 104 is D or N

[0766] X at position 106 is F or L

[0767] X at position 110 is R or H

[0768] X at position 111 is G or R

[0769] X at position 112 is G, D, T, S, A, D, E or N

[0770] X at position 113 E, R, D, H or Q

[0771] X at position 114 is V or L

[0772] X at position 115 is L, V or I

[0773] X at position 117 is E or D

[0774] X at position 118 is Y, F or H

[0775] X at position 119 is Y, S or A

[0776] X at position 120 is R, A, G or K

[0777] X at position 121 is A, S, P or D

[0778] X at position 124 is A or G

[0779] X at position 125 is P or A

[0780] X at position 126 is D or H

[0781] X at position 127 is D, Q, A or G

[0782] X at position 128 is R, L or P

[0783] X at position 131 is L, V or I

[0784] X at position 133 is S or A

[0785] X at position 134 is V or I

[0786] X at position 138 is L, M or I

[0787] X at position 140 is G or A

[0788] X at position 141 is T, I or L

[0789] X at position 142 is V, T or absent

[0790] X at position 143 is V, F, A or M

[0791] X at position 145 is A, V or S

[0792] X at position 146 is L, F or V

[0793] X at position 147 is V, I or A

[0794] X at position 148 is D, E or G

[0795] X at position 149 is E, G, D, Q, A, R or S

[0796] X at position 150 is G, A, S or D

[0797] X at position 151 is R, L, A, S or E

[0798] X at position 152 is I, L or V

[0799] X at position 153 is D or I X at position 154 is P or A

[0800] X at position 155 is A, S, E, R or D

[0801] X at position 156 is R, Q, G or A

[0802] X at position 157 is P, R or A

[0803] X at position 158 V or I

[0804] X at position 159 is T or V

[0805] X at position 160 E, A, H, T, R or D

[0806] X at position 161 is Y or H

[0807] X at position 162 is V or I

[0808] X at position 163 is P or R

[0809] X at position 166 is E, A or G

[0810] X at position 167 is G or D

[0811] X at position 169 is V or A

[0812] X at position 174 is S, A, Q or T

[0813] X at position 175 is D or G or absent

[0814] X at position 176 is D or absent

[0815] X at position 177 is A, P or absent

[0816] X at position 178 is A, N or absent

[0817] X at position 179 is A or absent

[0818] X at position 180 is P or absent

[0819] X at position 181 is R or absent

[0820] X at position 182 is V or absent

[0821] X at position 183 is T or absent

[0822] X at position 184 is I, L or V

[0823] X at position 185 is Q, L or R

[0824] X at position 186 is Q, H or R

[0825] X at position 187 is V, L or A

[0826] X at position 191 is E, Q, V, A or R

[0827] X at position 192 is V or I

[0828] X at position 193 is A, S, G or H

[0829] X at position 194 is I, V or L

[0830] X at position 195 is N or D

[0831] X at position 197 is S, N, D or R

[0832] X at position 199 is D, N or E

[0833] X at position 201 is V, I, L or T

[0834] X at position 202 is D or N

[0835] X at position 204 is A, G, D or T X at position 205 is S or A

[0836] X at position 209 is T, A or R

[0837] X at position 211 is E, D or S; particularly D

[0838] X at position 213 is S or A

[0839] X at position 217 is R, A, S, C, D, E, F, G, H, 1, 1, L, Q, T or V

[0840] X at position 218 is S, T, P, A, D, V, R or E

[0841] X at position 219 is R or A

[0842] X at position 220 is R, A, L, C or G

[0843] X at position 221 is D, E, H, T, D, A, P or E

[0844] X at position 222 is G, D or S

[0845] X at position 224 is P or A

[0846] X at position 225 is A, E, P, V, D, E or I

[0847] X at position 226 is D, N or S

[0848] X at position 227 is T, D or N

[0849] X at position 228 is Y or A

[0850] X at position 229 is E, A or S

[0851] X at position 232 is T, A or V

[0852] X at position 233 is T, S or G

[0853] X at position 235 is R or T

[0854] X at position 236 is G or T

[0855] X at position 237 is R or absent

[0856] X at position 238 is P or absent

[0857] X at position 239 is D or absent

[0858] X at position 240 is A or absent

[0859] X at position 241 is D or absent

[0860] X at position 242 is A or absent

[0861] X at position 243 is S, D, N or P

[0862] X at position 245 is A, S, G, R or T

[0863] X at position 246 is T, V, L or G

[0864] X at position 248 is E or A

[0865] X at position 255 is N, D or H

[0866] X at position 258 is V or I

[0867] X at position 260 is A or G

[0868] X at position 262 is I or V

[0869] X at position 263 is I or V

[0870] X at position 267 is T or S

[0871] X at position 269 is L, M or S X at position 270 is R or P

[0872] X at position 272 is A, S, G, V, P or H

[0873] X at position 273 is E, D or A

[0874] X at position 274 is A, L, V or I

[0875] X at position 275 is L or V

[0876] X at position 277 is I, V, T, R, A or E

[0877] X at position 278 is H, Y, F or R

[0878] X at position 279 is L or V

[0879] X at position 281 is A or S

[0880] X at position 282 is K or R

[0881] X at position 283 is L or I

[0882] X at position 284 is D or G

[0883] X at position 286 is D or G

[0884] X at position 287 is R, L, H or Q

[0885] X at position 290 is T, I or A

[0886] X at position 291 is I or V

[0887] X at position 295 is T, Q, S, R or A

[0888] X at position 296 is T, A or S

[0889] X at position 300 is F, C or Y

[0890] X at position 301 is A or P

[0891] X at position 302 is N, A, D or S particularly N

[0892] X at position 303 is G or A

[0893] X at position 304 is G or A

[0894] X at position 305 is V or I

[0895] X at position 306 is S or C

[0896] X at position 307 is C, S, A or T

[0897] X at position 317 is R or Q

[0898] X at position 318 is M, V, L or A

[0899] X at position 319 is M, I, V or L

[0900] X at position 322 is G, S or R

[0901] X at position 324 is H, A, V, I, S, L, T or K

[0902] X at position 325 is A or G

[0903] X at position 326 is P or A

[0904] X at position 327 is G or R

[0905] X at position 332 is S or P

[0906] X at position 333 is E, Q, G or A

[0907] X at position 334 is T, A, D, G or E X at position 336 is V, T or A

[0908] X at position 337 is R, S, D or H

[0909] X at position 338 is S, R, G or A

[0910] X at position 339 is I, V or T

[0911] X at position 340 is M or L

[0912] X at position 341 is E, A, S or D

[0913] X at position 344 is S or D

[0914] X at position 345 is R, H, Q, T or P

[0915] X at position 346 is E, A, T or Q

[0916] X at position 349 is T, P, D or M

[0917] X at position 350 is Y, D, P or absent

[0918] X at position 351 is E, K, P, A, S or D

[0919] X at position 352 is G, D, H or S

[0920] X at position 354 is T or P

[0921] X at position 355 is G, N, S, D, E, T or A

[0922] X at position 356 is T, A, E, V, D, I or R

[0923] X at position 357 is F or H

[0924] X at position 359 is D, N, E, G or S

[0925] X at position 364 is R or S

[0926] X at position 369 is T or M

[0927] X at position 371 is N or D

[0928] X at position 372 is E or D

[0929] X at position 374 is G, A or S

[0930] X at position 377 is S or T

[0931] X at position 378 is G or A

[0932] X at position 379 is I or V

[0933] X at position 385 is N, S or Y

[0934] X at position 388 is L or I

[0935] X at position 389 is D or E

[0936] X at position 391 is P, L, R, Q, M or A

[0937] X at position 392 is T or S

[0938] X at position 394 is S, A or T

[0939] X at position 396 is I or V

[0940] X at position 399 is L or F

[0941] X at position 401 is S, T, A or V

[0942] X at position 404 is E, D or G

[0943] X at position 406 is D, Y or E X at position 407 is T, G or S

[0944] X at position 408 is D, E, R, A or P

[0945] X at position 409 is H, D, E, F, Y, Q or S

[0946] X at position 410 is W, G, L or R

[0947] X at position 411 is H, N or D

[0948] X at position 412 is G, D, R, A or E

[0949] X at position 413 is V, L, R, K or A

[0950] X at position 415 is N, S, Q, T or G

[0951] X at position 416 is D, G, A, E, S, R, Q or T

[0952] X at position 417 is V, I or L

[0953] X at position 419 is L or V

[0954] X at position 420 is D or E

[0955] X at position 421 is V, I or L

[0956] X at position 422 is S or C

[0957] X at position 423 is R, H, E or T

[0958] X at position 424 is A or S

[0959] X at position 427 is A, G, T, R, S, P, Q or absent

[0960] X at position 428 is M, A, V, L, F, I, P, V or absent

[0961] X at position 429 is A, P, R, G, S or absent

[0962] X at position 430 is Y, A or absent

[0963] X at position 431 is A or absent

[0964] X at position 432 is A or absent

[0965] X at position 433 is E or absent

[0966] X at position 434 is T or absent

[0967] X at position 444 is A or absent

[0968] X at position 445 is P or absent

[0969] X at position 446 is D or absent

[0970] X at position 447 is S or absent

[0971] X at position 448 is K or absent

[0972] X at position 449 is E or absent

[0973] X at position 450 is N or absent

[0974] X at position 451 is A or absent

[0975] X at position 452 is A or absent

[0976] Further suitable peptides include those of SEQ ID NO:380 wherein:

[0977] X at position 2 is S, T or absent

[0978] X at position 3 is A, D, V or absent X at position 4 is A or absent

[0979] X at position 5 is L, D, T or absent

[0980] X at position 6 is G, A, or absent

[0981] X at position 7 is S, A or absent

[0982] X at position 8 is G, D or absent

[0983] X at position 9 is D, H or absent

[0984] X at position 10 is N, L, P or absent

[0985] X at position 11 is T, P, D or absent

[0986] X at position 12 is S, E, A R or absent

[0987] X at position 13 is H, E, P, A, G or absent

[0988] X at position 14 is D, R, V, N, E, P or absent

[0989] X at position 15 is T, R, P, G, I or absent

[0990] X at position 16 is D, A, K, S or absent

[0991] X at position 17 is S, R, I, A, P or absent

[0992] X at position 18 is R, P, Q, H, or absent

[0993] X at position 19 is R, H, N, A, T, D or absent

[0994] X at position 20 is R, G, A, T, D, S, P or absent

[0995] X at position 21 is R, S, P, G, A, T or absent

[0996] X at position 22 is T, P, S, A, L, G, V, D or absent

[0997] X at position 23 is T or absent

[0998] X at position 24 is G or absent

[0999] X at position 25 is P, Q, S, A, T or absent

[1000] X at position 26 is H or absent

[1001] X at position 27 is P or absent

[1002] X at position 28 is A, G, D, P, S or absent

[1003] X at position 29 is R or absent

[1004] X at position 30 is Y, F or absent

[1005] X at position 31 is P, A or absent

[1006] X at position 32 is G, D or absent

[1007] X at position 33 is G, A or absent

[1008] X at position 34 is A, E, G, P or absent

[1009] X at position 35 is P, A, R or absent

[1010] X at position 36 is G, S, E, N or absent

[1011] X at position 37 is E, Q, R, V, A, H or absent

[1012] X at position 39 is R, N, D or S

[1013] X at position 41 is E or D

[1014] X at position 42 is S, G or T X at position 45 is E, D, Q or T

[1015] X at position 46 is A, P or G

[1016] X at position 48 is Q, H, D, Y or G

[1017] X at position 52 is A or T

[1018] X at position 54 is S, A or G

[1019] X at position 55 is H, R, G or N

[1020] X at position 56 is L or M

[1021] X at position 57 is G, N or A

[1022] X at position 59 is I, L or M

[1023] X at position 60 is L, V, M or T

[1024] X at position 61 is P or H

[1025] X at position 62 is T, S, V, M

[1026] X at position 64 is V, T, A or S

[1027] X at position 65 is I or V

[1028] X at position 66 is P, S or A

[1029] X at position 68 is R, H, N or T

[1030] X at position 69 is S, D, P, R, V, A, D or absent

[1031] X at position 70 is P, V or E

[1032] X at position 71 is A, N, E, H, S, T or G

[1033] X at position 72 is A, T, V, S, D or absent

[1034] X at position 73 is P, R or S

[1035] X at position 74 is S, V, A, G, L, Y, P, T or absent

[1036] X at position 75 is G, A or absent

[1037] X at position 76 is A or absent

[1038] X at position 77 is A or absent

[1039] X at position 78 is E or absent

[1040] X at position 79 is E, H, A, S, G, D, E or T

[1041] X at position 80 is A, P, G

[1042] X at position 81 is T, V, L, A, F

[1043] X at position 82 is V, A, S, D

[1044] X at position 83 is R, H, N

[1045] X at position 85 is D, G, E

[1046] X at position 86 is S, A, V, P, L or absent

[1047] X at position 87 is L, I, V, F or absent

[1048] X at position 88 is T, A, E, L, G or absent

[1049] X at position 89 is P, S, T, A or absent

[1050] X at position 90 is R, Q, A, H or G X at position 91 is L, M or V

[1051] X at position 92 is P or D

[1052] X at position 93 is D, G, E, A or T

[1053] X at position 95 is Q, E, V, L, R or G

[1054] X at position 96 is Q, R, A or D

[1055] X at position 97 is R or Q

[1056] X at position 99 is E or D

[1057] X at position 100 is Q, E, R or A

[1058] X at position 101 is S or T

[1059] X at position 102 is Y, F or C

[1060] X at position 104 is D or N

[1061] X at position 106 is F or L

[1062] X at position 110 is R or H

[1063] X at position 111 is G or R

[1064] X at position 112 is G, D, T, S, A, D, E or N

[1065] X at position 113 E, R, D, H or Q

[1066] X at position 114 is V or L

[1067] X at position 115 is L, V or I

[1068] X at position 117 is E or D

[1069] X at position 118 is Y, F or H

[1070] X at position 119 is Y, S or A

[1071] X at position 120 is R, A, G or K

[1072] X at position 121 is A, S, P or D

[1073] X at position 124 is A or G

[1074] X at position 125 is P or A

[1075] X at position 126 is D or H

[1076] X at position 127 is D, Q, A or G

[1077] X at position 128 is R, L or P

[1078] X at position 131 is L, V or I

[1079] X at position 133 is S or A

[1080] X at position 134 is V or I

[1081] X at position 138 is L, M or I

[1082] X at position 140 is G or A

[1083] X at position 141 is T, I or L

[1084] X at position 142 is V, T or absent

[1085] X at position 143 is V, F, A or M

[1086] X at position 145 is A, V or S X at position 146 is L, F or V

[1087] X at position 147 is V, I or A

[1088] X at position 148 is D, E or G

[1089] X at position 149 is E, G, D, Q, A, R or S

[1090] X at position 150 is G, A, S or D

[1091] X at position 151 is R, L, A, S or E

[1092] X at position 152 is I, L or V

[1093] X at position 153 is D or I

[1094] X at position 154 is P or A

[1095] X at position 155 is A, S, E, R or D

[1096] X at position 156 is R, Q, G or A

[1097] X at position 157 is P, R or A

[1098] X at position 158 V or I

[1099] X at position 159 is T or V

[1100] X at position 160 E, A, H, T, R or D

[1101] X at position 161 is Y or H

[1102] X at position 162 is V or I

[1103] X at position 163 is P or R

[1104] X at position 166 is E, A or G

[1105] X at position 167 is G or D

[1106] X at position 169 is V or A

[1107] X at position 174 is S, A, Q or T

[1108] X at position 175 is D or G or absent

[1109] X at position 176 is D or absent

[1110] X at position 177 is A, P or absent

[1111] X at position 178 is A, N or absent

[1112] X at position 179 is A or absent

[1113] X at position 180 is P or absent

[1114] X at position 181 is R or absent

[1115] X at position 182 is V or absent

[1116] X at position 183 is T or absent

[1117] X at position 184 is I, L or V

[1118] X at position 185 is Q, L or R

[1119] X at position 186 is Q, H or R

[1120] X at position 187 is V, L or A

[1121] X at position 191 is E, Q, V, A or R

[1122] X at position 192 is V or I X at position 193 is A, S, G or H

[1123] X at position 194 is I, V or L

[1124] X at position 195 is N or D

[1125] X at position 197 is S, N, D or R

[1126] X at position 199 is D, N or E

[1127] X at position 201 is V, I, L or T

[1128] X at position 202 is D or N

[1129] X at position 204 is A, G, D or T

[1130] X at position 205 is S or A

[1131] X at position 209 is T, A or R

[1132] X at position 211 is M, C, or D; particularly D

[1133] X at position 213 is S or A

[1134] X at position 217 is R, A, S, C, D, E, F, G, H, 1, 1, L, Q, T or V

[1135] X at position 218 is S, T, P, A, D, V, R or E

[1136] X at position 219 is R or A

[1137] X at position 220 is R, A, L, C or G

[1138] X at position 221 is D, E, H, T, D, A, P or E

[1139] X at position 222 is G, D or S

[1140] X at position 224 is P or A

[1141] X at position 225 is A, E, P, V, D, E or I

[1142] X at position 226 is D, N or S

[1143] X at position 227 is T, D or N

[1144] X at position 228 is Y or A

[1145] X at position 229 is E, A or S

[1146] X at position 232 is T, A or V

[1147] X at position 233 is T, S or G

[1148] X at position 235 is R or T

[1149] X at position 236 is G or T

[1150] X at position 237 is R or absent

[1151] X at position 238 is P or absent

[1152] X at position 239 is D or absent

[1153] X at position 240 is A or absent

[1154] X at position 241 is D or absent

[1155] X at position 242 is A or absent

[1156] X at position 243 is S, D, N or P

[1157] X at position 245 is A, S, G, R or T

[1158] X at position 246 is T, V, L or G X at position 248 is E or A

[1159] X at position 255 is N, D or H

[1160] X at position 258 is V or I

[1161] X at position 260 is A or G

[1162] X at position 262 is I or V

[1163] X at position 263 is I or V

[1164] X at position 267 is T or S

[1165] X at position 269 is L, M or S

[1166] X at position 270 is R or P

[1167] X at position 272 is A, S, G, V, P or H

[1168] X at position 273 is E, D or A

[1169] X at position 274 is A, L, V or I

[1170] X at position 275 is L or V

[1171] X at position 276 is N, A, D or S particularly N

[1172] X at position 277 is I, V, T, R, A or E

[1173] X at position 278 is H, Y, F or R

[1174] X at position 279 is L or V

[1175] X at position 281 is A or S

[1176] X at position 282 is K or R

[1177] X at position 283 is L or I

[1178] X at position 284 is D or G

[1179] X at position 286 is D or G

[1180] X at position 287 is R, L, H or Q

[1181] X at position 290 is T, I or A

[1182] X at position 291 is I or V

[1183] X at position 295 is T, Q, S, R or A

[1184] X at position 296 is T, A or S

[1185] X at position 300 is F, C or Y

[1186] X at position 301 is A or P

[1187] X at position 303 is G or A

[1188] X at position 304 is G or A

[1189] X at position 305 is V or I

[1190] X at position 306 is S or C

[1191] X at position 307 is C, S, A or T

[1192] X at position 317 is R or Q

[1193] X at position 318 is M, V, L or A

[1194] X at position 319 is M, I, V or L X at position 322 is G, S or R

[1195] X at position 324 is H, A, V, I, S, L, T or K

[1196] X at position 325 is A or G

[1197] X at position 326 is P or A

[1198] X at position 327 is G or R

[1199] X at position 332 is S or P

[1200] X at position 333 is E, Q, G or A

[1201] X at position 334 is T, A, D, G or E

[1202] X at position 336 is V, T or A

[1203] X at position 337 is R, S, D or H

[1204] X at position 338 is S, R, G or A

[1205] X at position 339 is I, V or T

[1206] X at position 340 is M or L

[1207] X at position 341 is E, A, S or D

[1208] X at position 344 is S or D

[1209] X at position 345 is R, H, Q, T or P

[1210] X at position 346 is E, A, T or Q

[1211] X at position 349 is T, P, D or M

[1212] X at position 350 is Y, D, P or absent

[1213] X at position 351 is E, K, P, A, S or D

[1214] X at position 352 is G, D, H or S

[1215] X at position 354 is T or P

[1216] X at position 355 is G, N, S, D, E, T or A

[1217] X at position 356 is T, A, E, V, D, I or R

[1218] X at position 357 is F or H

[1219] X at position 359 is D, N, E, G or S

[1220] X at position 364 is R or S

[1221] X at position 369 is T or M

[1222] X at position 371 is N or D

[1223] X at position 372 is E or D

[1224] X at position 374 is G, A or S

[1225] X at position 377 is S or T

[1226] X at position 378 is G or A

[1227] X at position 379 is I or V

[1228] X at position 385 is N, S or Y

[1229] X at position 388 is L or I

[1230] X at position 389 is D or E X at position 391 is P, L, R, Q, M or A

[1231] X at position 392 is T or S

[1232] X at position 394 is S, A or T

[1233] X at position 396 is I or V

[1234] X at position 399 is L or F

[1235] X at position 401 is S, T, A or V

[1236] X at position 404 is E, D or G

[1237] X at position 406 is D, Y or E

[1238] X at position 407 is T, G or S

[1239] X at position 408 is D, E, R, A or P

[1240] X at position 409 is H, D, E, F, Y, Q or S

[1241] X at position 410 is W, G, L or R

[1242] X at position 411 is H, N or D

[1243] X at position 412 is G, D, R, A or E

[1244] X at position 413 is V, L, R, K or A

[1245] X at position 415 is N, S, Q, T or G

[1246] X at position 416 is D, G, A, E, S, R, Q or T

[1247] X at position 417 is V, I or L

[1248] X at position 419 is L or V

[1249] X at position 420 is D or E

[1250] X at position 421 is V, I or L

[1251] X at position 422 is S or C

[1252] X at position 423 is R, H, E or T

[1253] X at position 424 is A or S

[1254] X at position 427 is A, G, T, R, S, P, Q or absent

[1255] X at position 428 is M, A, V, L, F, I, P, V or absent

[1256] X at position 429 is A, P, R, G, S or absent

[1257] X at position 430 is Y, A or absent

[1258] X at position 431 is A or absent

[1259] X at position 432 is A or absent

[1260] X at position 433 is E or absent

[1261] X at position 434 is T or absent

[1262] X at position 444 is A or absent

[1263] X at position 445 is P or absent

[1264] X at position 446 is D or absent

[1265] X at position 447 is S or absent

[1266] X at position 448 is K or absent X at position 449 is E or absent

[1267] X at position 450 is N or absent

[1268] X at position 451 is A or absent

[1269] X at position 452 is A or absent

[1270] In some embodiments the polypeptides are selected from SEQ ID NO:33, SEQ ID NOs: 14-21; SEQ ID NO:38, SEQ ID NO:44, SEQ ID NOs:48-50, SEQ ID NOs:68-71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NOs:79-81, SEQ ID NOs:84-86, SEQ ID NO:90, SEQ ID NOs:92-94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO: 101, SEQ ID NO: 104, SEQ ID NO: 109, SEQ ID NO: 110 and SEQ ID NO: 115 or a polypeptide having at least 80% sequence identity to any of these polypeptides, in each case wherein the amino acid at position 187 with respect to SEQ ID NO:1 is selected from alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine; optionally with the proviso that if the amino acid at position 196 is A, then the amino acid at position 190 is A, R or G,; and / or the amino acid at position 285 is H, G or M

[1271] In some embodiments the polypeptides is selected from: SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NOs:69-71, SEQ ID NO:74, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO: 101 and SEQ ID NO: 109; or a polypeptide having at least 80% sequence identity to any of these poly peptides in each case wherein the amino acid at position 187 with respect to SEQ ID NO:1 is selected from alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine.

[1272] In some embodiments the polypeptides is selected from: SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:70, SEQ ID NO:79, SEQ ID NO:80 and SEQ ID NO: 96; or a polypeptide having at least 80% sequence identity thereto; in each case wherein the amino acid at position 187 with respect to SEQ ID NO:1 is selected from alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine.

[1273] In some embodiments the amino acid at position 187 with respect to SEQ ID NO:1 is selected from A, C, D, G, H, I, L, M, Q, S, T or V; optionally A, C, D, H, I, L, Q or T; further optionally C, D, H, I, L or Q.

[1274] In some embodiments, the amino acid at position 181 with respect to SEQ ID NO:1 is selected from G A or R; particularly G and / or the amino acid at position 266 with respect to SEQ ID NO:1 is N or H particularly H.

[1275] In peptides disclosed herein, the amino acid at position 187 with respect to SEQ ID NO:1 may be selected from the group consisting of C, D, E, F, I, L, Q, or V; particularly I or L. In any of the peptides disclosed herein, the amino acid at position 187 with respect to SEQ ID NO: 1 may be selected from the group consisting of C, D, E, F, I, L, Q, or V; particularly I or L.

[1276] In peptides disclosed herein, the amino acid at position 187 with respect to SEQ ID NO:1 may be selected from the group consisting of C, D, E, F, I, L, Q, or V; particularly I or L. In any of the peptides disclosed herein, the amino acid at position 187 with respect to SEQ ID NO:1 may be selected from the group consisting of A, C, D, G, H, I, L, M, Q, S, T or V; optionally A, C, D, H, I, L, Q, or T; further optionally C, D, H, I, L or Q.

[1277] In some embodiments the polypeptides as disclosed herein are capable of hydrolysing a polyamide polymer or a polyamide oligomer. In some embodiments the polyamide polymer is a polyamide 6 (PA6) polymer, or a polyamide 6,6 (PA66) polymer and / or the polyamide oligomer is a polyamide 6 or polyamide 6,6 oligomer.

[1278] Polyamide oligomers are low n-number polyamide polymers comprising a small number of monomer units. Polyamide 6,6 oligomers comprise hexamethylene diamine monomers and adipic acid monomers. Odd numbered polyamide oligomers form polyamides comprising diamine monomers and diacid monomers, such as polyamide 6,6, are terminated by either bis -carboxylate groups or bis-amino groups.

[1279] Individual oligomers as referred to herein may consist of up to 40-mers of polyamide, for example up to 30-mers or up to 20-mers. In some cases, the polyamide oligomer may consist of up to 20-mers, 19- mers, 18-mers, 17-mers, 16-mers, 15-mers, 14-mers, 13-mers, 12-mers, 11-mers or 10-mers. In some cases, the polyamide oligomer may consist of 10-mers, 9-mers, 8-mers, 7-mers or 6-mers, of polyamide. In some cases, the polyamide oligomers may consist of 6-mers, 5-mers, 4-mers, 3-mers or dimers. From a formal perspective dimers are not considered oligomers, however, for convenience herein the term oligomer includes dimers unless the context clearly indicates otherwise.

[1280] Low n-number oligomers (including dimers), such as those of polyamide 6,6, are soluble in water to varying degrees depending on the molecular weight. Partially hydrolysed compositions as described further herein are typically a mixture of at least dimers, trimers, tetramers, pentamers and hexamers, but may also comprise higher n-number oligomers some of which may be soluble to a small degree, sparingly soluble or, if not completely soluble, become accessible to the activity of the polyamide. In some embodiments, polypeptides disclosed herein have improved activity towards bis-amino oligomers. In some embodiments, polypeptides disclosed herein have improved activity towards bis- carboxy oligomers. In some embodiments polypeptides disclosed herein have improved activity towards dimers. In some embodiments polypeptides disclosed herein have improved activity towards insoluble or sparingly soluble oligomers.

[1281] In some embodiments a polypeptide is capable of hydrolysing a water soluble polyamide 6 oligomer or polyamide 6,6 oligomer. In some embodiments a polyamide oligomer has at least one -NH2 terminus. In some embodiments an oligomer is terminated by bis-amino groups or bis-carboxylate groups, i.e. it is terminated at each end by such groups.

[1282] In a further aspect, the disclosure provides a polypeptide as described herein which is immobilized on a solid support. Solid supports include beads or membranes of a variety of insoluble materials including polymers and inorganic materials such as ceramics. In some embodiments the polypeptide is immobilized through a covalent bond to the solid support. In some embodiments the polypeptide may be coupled to the solid support through non-covalent interactions such as through a chelate e.g. a histidine tag-nickel chelate.

[1283] In a further aspect, the disclosure provides a polynucleotide comprising a nucleic acid sequence encoding the polypeptide as disclosed herein. The polynucleotide may be DNA or RNA and may be codon optimised for expression in a given host cell in a manner that is well understood by a person of skill in the art. The polynucleotide may form part of an expression cassette, comprising a promoter driving expression of the polypeptide operably linked to the polynucleotide sequence, and a stop as well as various other control sequences well known in the art for modifying expression of polypeptides from expression cassettes in host cells. The promoter may be a constitutive promoter or an inducible promoter, in which expression of the polypeptide occurs in response to a stimulus such as the presence in the medium of a particular compound, light, heat etc. In a further aspect the disclosure provides an expression vector comprising the polynucleotide or expression cassette. The choice of expression vector is determined by multiple factors including the host organism or cell chosen for expression, whether expression is required to be induced or constitutive and the ability of the host cell to express the polypeptide is sufficient.

[1284] In a further aspect the disclosure provides a host cell comprising the polynucleotides or the expression vectors described herein. In an embodiment the disclosure provides compositions comprising the host cells. Hosts cells may be prokaryotic or eukaryotic and include bacteria, yeast, fungi, mammalian, insect or plant cells, although prokaryotic cells such as Escherichia coli are more typically used.

[1285] The term "host cell" should be taken to include progeny of the original transformed cell, whether or not the progeny carry further mutations not present in the original cell, for example as a result of mutations arising as a result of growth division or passage of the cells through various media.

[1286] In a further aspect, the disclosure provides a composition comprising the polypeptide as disclosed herein, the polynucleotides disclosed herein the expression vector disclosed herein or the host cells disclosed herein. In some embodiments the composition is an aqueous composition. In some embodiments the composition is a dried composition such as a freeze-dried composition. In some embodiments the composition comprises a preservative. In a further aspect the disclosure provides a process for the preparation of a polypeptide disclosed herein comprising expressing the polypeptide from a polynucleotide as disclosed herein and recovering the polypeptide. In some embodiments expressing the polypeptide comprises expressing the polypeptide from a nucleic acid within a host cell. In some embodiments expressing the polypeptide comprises propagating a host cell as described herein expressing the polypeptide from the nucleic acid and recovering the polypeptide. In some embodiments the host cell is propagated in a medium and the polypeptide is recovered from the host cell and / or from the medium.

[1287] A further aspect of the disclosure provides a process for hydrolysing a polyamide polymer or oligomer (e.g a polyamide polymer or polyamide oligomer) comprising contacting the polymer or polyamide oligomer with a polypeptide as described herein. In some embodiments, a polyamide polymer is partially chemically hydrolysed to provide polyamide oligomers and the polyamide oligomers are contacted with the polypeptide. In some embodiments the partial chemical hydrolysis provides a composition comprising water soluble polyamide oligomers and in some embodiments the chemical hydrolysis provides a mixture of oligomers comprising at least dimers and trimers, or at least dimers, trimers, tetramers and pentamers. In some embodiments, the partial chemical hydrolysis provides a composition additionally comprising insoluble oligomers. In some embodiments the partial chemical hydrolysis is a partial acid catalysed or base catalysed hydrolysis. In some embodiments a polyamide polymer or polyamide oligomer is a polyamide 6,6 polymer or oligomer. In some embodiments the composition can be an aqueous composition.

[1288] Compositions comprising insoluble oligomers comprise suspensions of particulate material comprising the insoluble or partially soluble oligomeric material. Treatment of the composition with a polypeptide as disclosed herein leads to a reduction in the optical density of the aqueous composition as the polymeric material is reduced in molecular weight due to the hydrolysis. The progress of the reaction can therefore be followed spectrophotometrically.

[1289] In some embodiments the polyamide is comprised in a complex product comprising one or more other materials, including one or more additional polymers, metals, fabrics and so on. In some embodiments the polyamide (such as polyamide 6 or polyamide 6,6 is found in combination with one or more of polyethylene terephthalate (PET), polylactic acid (PL A), poly trimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polyethylene terephthalate (PET), polyhydroxy alkanoate (PH A), polybutylene succinate (PBS), polybutylene succinate adipate (PBS A), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), polycaprolactone (PCL), and poly(ethylene adipate) (PEA) or any combination of two or more of these. In some embodiments the adipic acid and / or the hexamethylene diamine monomers resulting from the process maybe recovered and recycled for use as a starting material in the preparation of further compounds or in the preparation of polyamide polymer. Thus, in a further aspect the disclosure provides a process comprising hydrolysing a polyamide 6,6 polymer or oligomer by a process comprising contacting the polyamide polymer or polyamide oligomer with a polypeptide as described herein, to provide adipic acid and / or hexamethylene diamine monomers; recovering the adipic acid and / or the hexamethylene diamine monomers resulting from the process and synthesising a polyamide 6,6 polymer from the recovered adipic acid and / or the hexamethylene diamine monomers.

[1290] The monomers may be recovered by any suitable process known in the art for example by column chromatography, distillation, solvent extraction, electrodialysis, adsorption onto anion exchange media, selective crystallization or by derivatisation and recovery and de-derivatisation of the derivative.

[1291] The polypeptides disclosed herein may be used alone or in combination with another enzyme in order to take advantage of the properties of both enzymes. For example, a poly amidase (or nylonase) may be used in combination with a PETase or an esterase. In some embodiments a polypeptide capable of hydrolysing polyamide 6 polymers or oligomers may be combined with a polypeptide capable of hydrolysing polyamide 6,6 polymers or oligomers.

[1292] In a further embodiment a first amidase polypeptide capable of hydrolysing certain polyamide 6 or 6,6 oligomers but which hydrolyses other polyamide 6,6 oligomers poorly or not at all may be combined with a second amidase polypeptide capable of hydrolysing at least one of the oligomers that the first amidase hydrolyses poorly or not at all. In one embodiment for example a polypeptide having 181G 266H (e.g. SEQ ID NO:2) can be combined with a polypeptide having 181R 266H (e.g. SEQ ID NO:323) which hydrolyses odd number oligomers poorly but hydrolyses dimers well (see the experimental examples below). In a further embodiment, a polypeptide that metabolises bis-carboxylate oligomers well, but bis-amino oligomers less well, can be combined with a polypeptide that metabolises bis-amino oligomers well, but bis-carboxylate oligomers less well; for example a polyamidase carrying a 181G 266H sequence, which does not metabolise polyamide 6,6 N-trimers well, can be combined with 181G 187L, which does.

[1293] A further aspect of the disclosure provides a method for increasing the polyamide 6,6 hydrolase activity of an amidase enzyme comprising:

[1294] (a) identifying the amino acid sequence of a polypeptide having either polyamide 6,6 hydrolase activity, or 6-aminohexanoate dimer hydrolase activity, wherein the amidase lacks:

[1295] (i) a glycine residue at position 181 or a histidine residue at position 266, or lacks both a glycine residue at position 181 and a histidine residue at position 266 with reference to SEQ ID NO:1;

[1296] (ii) an arginine residue at position 181 or a histidine residue at position 266, or lacks both an arginine residue at position 181 and a histidine residue at position 266 with reference to SEQ ID NO:l;or (iii) an alanine residue at position 181 or a histidine residue at position 266, or lacks both an alanine residue at position 181 and a histidine residue at position 266 with reference to SEQ ID NO:1

[1297] (b) substituting the amino acid at position 181 with a glycine residue and / or substituting the amino acid at position 266 with a histidine residue; or substituting the amino acid at position 181 with an arginine residue and / or substituting the amino acid at position 266 with a histidine residue; or substituting the amino acid at position 181 with an alanine residue and / or substituting the amino acid at position 266 with a histidine residue; wherein positions are relative to the amino acid sequence of SEQ ID NO: 1 ; and optionally if the amidase sequence has other than an alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine or valine at position 187 in relation to SEQ ID NO:1; then

[1298] (c) replacing the amino acid at position 187 with respect to SEQ ID NO:1 with an amino acid selected from the group consisting of alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine.

[1299] A further aspect the disclosure provides a method for increasing the polyamide 6,6 hydrolase activity of an amidase enzyme comprising:

[1300] (a) identifying the amino acid sequence of a polypeptide having either polyamide 6,6 hydrolase activity, or 6-aminohexanoate dimer hydrolase activity, wherein the amidase sequence has an amino acid other than an alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine at position 187 in relation to SEQ ID NO:1; and

[1301] (b) replacing the amino acid at position 187 with respect to SEQ ID NO:1 with an amino acid selected from the group consisting of alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine; and optionally

[1302] (i) if the sequence does not comprise a glycine residue at position 181 with respect to SEQ ID NO:1, then replacing the amino acid with a glycine;

[1303] (ii) if the sequence does not comprise an alanine residue at position 181 with respect to SEQ ID NO:1, then replacing the amino acid with an alanine; or

[1304] (iii) if the sequence does not comprise an arginine residue at position 181 with respect to SEQ ID NO:1, then replacing the amino acid with an arginine; and

[1305] (iv) if the sequence does not comprise a histidine residue at position 266 with respect to SEQ ID NO:1, replacing the amino acid at position 266 with a histidine residue.

[1306] In some embodiments the resulting polypeptide has increased hydrolytic activity towards polyamide 6,6 oligomers having terminal amino groups. In some embodiments the resulting polypeptide has increased hydrolytic activity towards polyamide 6,6 oligomers having terminal carboxylate groups. In some embodiments the resulting polypeptide has increased hydrolytic activity towards polyamide 6,6 dimers.

[1307] The identified sequence of the polypeptide having either polyamide 6,6 hydrolase activity, or 6- aminohexanoate dimer hydrolase activity has at least one of the following additional features:

[1308] (i) an amino acid sequence of at least 50% identity to SEQ ID NO:1;

[1309] (ii) a catalytic triad comprising a serine residue at position 112, a lysine residue at a position 115 and a tyrosine or phenylalanine residue at a position 215

[1310] (iii) a nitrogen inverting residue at position 170; optionally wherein the nitrogen inverting residue is a tyrosine residue.

[1311] The polyamide 6,6 hydrolase activity increased may be one or more of polyamide 6,6 hexamer hydrolase, polyamide 6,6 N-pentamer hydrolase, polyamide 6,6 C-pentamer hydrolase, polyamide 6,6 tetramer hydrolase, polyamide 6,6 N-trimer hydrolase, polyamide 6,6 C-trimer hydrolase, or polyamide 6,6 dimer hydrolase activity.

[1312] The polypeptide having either polyamide 6,6 hydrolase activity, or 6-aminohexanoate dimer hydrolase activity identified in step (a) may have at least 30% at least 40% at least 50%, at least 60% at least 70% at least 80% or higher sequence identity to a sequence selected from SEQ ID NO:1, SEQ ID NOs:14 to SEQ ID NO:21, SEQ ID NO:33 or SEQ ID NO:381.

[1313] The polypeptide may have increased hydrolytic activity towards polyamide 6,6 oligomers, including those having terminal amino groups, terminal carboxylate groups or dimers

[1314] The disclose also provide polypeptides prepared according to the above methods..

[1315] A yet further aspect of the disclosure provides methods for assaying the activity of polyamide amidases such as those described herein. Thus an assay for indicating a hydrolytic activity of an amidase enzyme towards a polyamide oligomer (or to one or more oligomers), is disclosed, wherein the hydrolytic activity releases at least one diamine monomer; the process comprising: contacting the amidase enzyme with the polyamide oligomer, for example the dimer 6-[(6-aminohexyl)amino]-6-oxo-hexanoic acid; contacting a diamine monomer released, with a putrescine oxidase in the presence of oxygen, to produce an oxidised diamine monomer and a peroxide (typically H2O2); contacting the peroxide with a peroxidase in the presence of peroxidase substrate; and detecting the activity of the peroxidase on the peroxidase substrate to indicate the activity of the amidase enzyme towards a polyamide oligomer.

[1316] Assays of this type are biocompatible and so can be used for continuous assays in which the amidase, one or more oligomers, putrescine oxidase, the peroxidase and the peroxidase substrate can be brought together in one composition comprising oxygen, wherein the hydrolytic action of the polypeptide on the one or more oligomers can be followed by measuring the activity of the peroxidase on the peroxidase substrate. Conveniently the putrescene oxidase is that obtainable from Rhodococcus erythropolis having the sequence of SEQ ID NO:32.

[1317] Conveniently the peroxidase substrate is a colourogenic substrate of a type widely known in the art, for example 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and the activity of the peroxidase on the peroxidase substrate may be detected by detecting the colour of the product. The peroxidase is conveniently horseradish peroxidase (HRP).

[1318] Also disclosed herein is a method for monitoring the progress of a polyamide hydrolysis to produce at least one diamine product, the method comprising detecting the presence or the quantity of the diamine product using an assay as disclosed herein. In some embodiments the polyamide is polyamide 6,6. In some embodiments at least one step in the hydrolysis is carried out using an enzyme, optionally wherein the enzyme is an amidase, such as those described herein.

[1319] DEFINITIONS

[1320] As used herein, the articles "a" and "an" refer to one (or to at least one) of the object or objects of the article, unless explicitly stated otherwise. As used herein, the term "about" refers to an approximate value that may vary by up to 10%, for example up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10%. As used herein the words "has", "have", "having" "comprise", "comprises" and "comprising" in relation to an element or elements should be understood to mean the inclusion of that element or elements but not the exclusion of any other element(s). The use of the term “with respect to” can be used interchangeably with terms, such as, “according to” or “with reference to”.

[1321] The terms “protein”, “polypeptide” and “peptide” refer to a molecule formed from a chain of amino acids linked by peptide bonds irrespective of the number of amino acids in the chain. The terms are used interchangeably unless the context implies otherwise.

[1322] The term “mutant” when used in relation to a protein, polypeptide or peptide herein is to be understood to mean a variant of that protein, polypeptide or peptide in which one or more amino acids have been inserted, deleted or exchanged or another amino acid. The term is typically used in relation to a variant of a wild type protein, polypeptide or peptide in which one or more amino acids have been inserted, deleted or exchanged or another amino acid through the use of a laboratory manipulation of the wild type sequence. As used herein the term “variant” in relation to polypeptide sequence means a polypeptide having a similar, but non identical amino acid sequence to the reference polypeptide. Sequence variants typically refer to sequences having the same or similar enzyme activity. The term “wild type” refers to a polypeptide as it occurs in nature, or as it was originally identified in nature. The term wild type may also refer to a polypeptide that has the amino acid sequence present before any amino acid changes are made to provide a mutant.

[1323] When one or more amino acids are substituted with one or more other amino acids, substitution may be with naturally occurring amino acids or non-naturally occurring amino acids. Preferably with naturally occurring amino acids. Naturally occurring amino acids are alanine (A) Arginine (R), Asparagine (N) Aspartate (D), Cysteine, (C) Glutamine (Q) Glutamate (E), Glycine (G) Histidine (H), Isoleucine (I) Leucine (L), Lysine (K) Methionine (M), Phenylalanine (F), Proline (P), Serine (S) Threonine (T), Tryptophan (W) Tyrosine (Y) and Valine (V). Substitutions may be either conservative or nonconservative substitution. Conservative substitution is the substitution of an amino acid for an amino acid from the same class of amino acids. Non conservative substitution is substitution with an amino acid of a different type or class. Amino acid classes are well known to the skilled person and includes the groups given in Table 1:

[1324] Table 1

[1325] Mutations of a particular polypeptide are shown by identifiers in which the non-mutant amino acid is followed by its position and then its substituted amino acid; hence a substitution of an aspartate at position 266 with a histidine is represented as D266H.

[1326] Unless stated otherwise amino acid positions of disclosed variants are in relation to SEQ ID NO: 1 (which is the naturally occurring NylB of Flavobacterium with accession number P07061) or SEQ ID NO:2. In this context “in relation to” means the equivalent position in that polypeptide when optimally aligned with the reference polypeptide (typically SEQ ID NO:1 or SEQ ID NO:2). Such alignments may be carried out using “Clustal Omega” (available at https: / / www.ebi.ac.uk / jdispatcher / msa / clustalo) for multiple sequence alignments or “Needle” (available at https: / / www.ebi.ac.uk / jdispatcher / psa / emboss_needle) for single alignments, using the following parameters: matrix- EBlossum62; gap opening penalty - 10, gap extending penalty - 0.5; end opening penalty -10; end extending penalty 0.5.

[1327] The term "sequence identity" typically as percentage identity (%ID) refers to the number of identical amino acid matches between two polypeptide sequences following an optimal alignment (i.e. aligned so as to maximize overlap and % identity but minimizing gaps in the sequence). Sequence identity between two sequences of approximately the same size may conveniently be determined using various programs making use of the Needleman -Wunsch algorithm; (Needleman and Wunsch (1970) using the following parameters: matrix- EBlossum62; gap opening penalty - 10, gap extending penalty - 0.5; end opening penalty -10; end extending penalty 0.5.

[1328] Where a polypeptide is referred to as having a minimum sequence identity to a reference polypeptide, polypeptides may have at least 60% amino acid sequence identity to the reference; at least 65% amino acid sequence identity; at least 70% amino acid sequence identity; at least 75% amino acid sequence identity; at least 80% amino acid sequence identity; at least 85% amino acid sequence identity; at least 90% amino acid sequence identity; or at least 95% amino acid sequence identity to the reference sequence. In some embodiments the polypeptides may have at least 96, 97, 98 or 99% sequence identity to the reference. In some embodiments the polypeptide may have no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid changes with respect to the reference. Such sequence changes are made without the loss of the catalytic triad or the nitrogen inverting residue referred to elsewhere herein.

[1329] BRIEF DESCRIPTION OF THE DRAWINGS

[1330] Figure 1: illustrates polyamide 6,6 dimer hydrolase activities of a collection of wild type polypeptides derived by sequence similarity to wild type NylB (SEQ ID NO:1) against polyamide 6 (A) and polyamide 6,6 (B) dimers.

[1331] Figure 2: illustrates the effect of substitution mutants at the 181 position of NylB wild type (SEQ ID NO:1) on its polyamide 6,6 dimer hydrolyse activity.

[1332] Figure 3: illustrates the polyamide 6,6 dimer hydrolase activity of a collection of single and double NylB substitution mutants.

[1333] Figure 4: shows an alignment of SEQ ID NO: 1 (NylB) and SEQ ID NO:2 (NylB_GH) carried out using the Needleman Wunch Algorithm. Residues of a catalytic triad having a serine at position 112, a lysine at a position 115 and a tyrosine at a position 215 are shown (<D) and the position of a nitrogen inverting residue at position 170 (y) is identified. SEQ IDN0:2 carries a 181G266H double mutation, highlighted (A). Figure 5: compares the spectrum of polyamide 6,6 oligomer hydrolase activity of NylB WT (SEQ ID NO:1) to that of two double mutants of NylB, NylB D181G_N266H (SEQ ID NO:2) and NylB D181R_N266H (SEQ ID NO:3). Results are compared to Microbacterium sp polypeptide identified by sequence similarity to NylB WT (SEQ ID NO:1) and found to have NylB-like activity (UNIPROT accession No. WP300593461, SEQ ID NO:9).

[1334] Figure 5A: provides the polyamide 6,6 oligomer hydrolase activity data from three replicate digestions of partially acid hydrolysed polyamide 6,6. Figures are peak area integrations from LC-MS runs representing oligomers from dimers to hexamers. Lower figures compared to the no-enzyme control indicate activity against that oligomer.

[1335] Figure 5B: illustrates the data in Figure 5 A. Data for each oligomer has been averaged and normalised against no-enzyme controls. Values below 100% represent polyamide 6,6 hydrolase activity against that monomer, whilst higher values suggest that the enzyme has a reduced capacity to hydrolyse that oligomer.

[1336] Figure 6: shows an alignment of 8 mutant polypeptides carrying the 181G 266H double mutation. Sequences are aligned using CLUSTAL Omega. Residues at 181 and 266, the catalytic triad and nitrogen inverting residue are highlighted.

[1337] Figure 7: illustrates the spectrum of polyamide 6,6 oligomer hydrolase activity of a collection of polypeptides having sequence similarity to the wild type NylB enzyme (SEQ ID NO: 1). Figures 7A and 7C represent results from two separate experiments on different collections of polypeptides. Activity without (WT) and with (GH) the 181G 266H double mutation is compared. Each table shows the results of three replicate digestions of partially acid hydrolysed polyamide 6,6. Data are peak area integrations from LC-MS runs representing each oligomer. Lower figures compared to the no-enzyme control indicate activity against that oligomer.

[1338] Figures 7B and 7D: illustrate the data of the tables of Figures 7A and 7C in which data for each oligomer is averaged and normalised against no-enzyme controls for each experiment. Values below 100% indicate activity against that monomer, whilst higher values suggest that the enzyme has a reduced capacity to hydrolyse that oligomer.

[1339] Figure 8: illustrates the polyamide 6,6 N-trimer hydrolase activity of 19 substitution mutants at the R187 position of the NylB homologue 1WYC_A (Seq ID NO:33), the wild type of which carries 18 ID 266N. WT=wild type; NEC= no-enzyme control.

[1340] Figures 9A, 9B and 9C: illustrate the spectrum of polyamide 6,6 oligomer hydrolase activity of the 1WYC_A 181N, 266H (SEQ ID NO:117) mutant peptide in comparison to the nineteen R187 amino acid substitution mutants of this peptide. Each bar represents the value of the integrated peaks of each oligomer from an LC-MS separation of the reaction mixture at the end of the incubation period. Values are normalised against the no-enzyme control values for each oligomer.

[1341] Figure 10: illustrates, the spectrum of polyamide 6,6 oligomer hydrolase activity of substitution mutants at positions 181, 187 and / or 266 of the 1WYC_A peptide (SEQ ID NO:33).

[1342] Figure 11: is a schematic diagram of the peroxidase linked putrescine oxidase assay for polyamide 6,6 dimer or trimer hydrolase. ABTS = 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)

[1343] Figures 12A to 12H: illustrate the polyamide 6,6 dimer hydrolase activity of 8 NylB homologue polypeptides as measured using the coupled putrescene oxidase / HRP detection assay set out in Example 8. PRO305 is NylB_GH (SEQ ID NO:2).

[1344] Figures 13: illustrates the effect of wild type NylB (SEQ ID NO:1), a NylC thermostable mutant (SEQ ID NO:200), NylB RH (SEQ ID NO:323) and NylB GH (SEQ ID NO:2) against a partial acid hydrolysate of polyamide 6,6 comprising n=2 to n=6 oligomers as well as insoluble and sparingly soluble oligomers; as measured by optical density at 600nm. Both NylB_GH (SEQ ID NO:2) and NylC (SEQ ID NO: 200) are capable of reducing the OD of the mixture indicating the clearance of insoluble or sparingly soluble polyamide 6,6 oligomers and that they are both capable of hydrolysing these, sparingly soluble and / or insoluble polyamide 6,6 oligomers.

[1345] EXAMPLES

[1346] Example 1: Identification of NylB Wild Type Homologues

[1347] Putative NylB enzymes in the UNIPROT database were identified by sequence homology with SEQ ID NO: 1 (Table 2). A selection of their coding sequences were cloned into the pET-28(a)+ vector to express His-tagged enzymes and over expressed in E. coli. The individual enzymes were purified on nickel columns and tested for activity against PA6 and PA6,6 dimers.

[1348] Table 2

[1349]

[1350] DNA inserts for each polypeptide were cloned into the pET-28(a)+ vector using the Ncol and Xhol restriction sites, leading to expression of each sequence with a C-terminal polyhistidine tag. Plasmids were transformed into competent BL21 (DE3) (New England Biolabs) using the standard heat shock protocol and plated with kanamycin as the selection antibiotic. A single colony was used to inoculate a starter culture of LB broth which was incubated for 16 hours at 37°C. The starter culture was then used to inoculate TB-AIM (TB auto induction medium) at a 1:100 dilution, which was incubated at 23°C for 72 hours. The resulting cell culture was pelleted with centrifugation at 3220 g for thirty minutes. Cells were resuspended in buffer A (50 mM sodium phosphate buffer containing 300 mM NaCl and 20 mM imidazole) and lysed through ultrasonication. The lysed cells were then centrifuged at 12,000 g for one hour and the lysate (supernatant) was separated and filtered. The polyhistidine tagged protein was then purified using an AKTA start instrument (Cytiva) equipped with a 5 mL HisTrap nickel column using an imidazole gradient increasing to 500 mM. The purified proteins were then desalted using a PD-10 column (Cytiva) and their concentrations standardised prior to use using a spectrophotometer and computationally calculated extinction coefficients.

[1351] Polyamide 6 and polyamide 6,6 dimer and trimer hydrolase assays

[1352] Polyamide 6 (6-(6-aminohexanamido)hexanoic acid) and 6,6 (6-((6-aminohexyl)amino)-6- oxohexanoic acid) dimers prepared according to Johnson (1995) were dissolved in distilled water to a final concentration of 100 mM. They were combined with equal volumes of pM purified enzyme solution in 50 mM pH 8 sodium phosphate buffer to give a final concentration of 50 mM dimer and 2.5 pM of enzyme. Samples containing polyamide 6 dimer were incubated at 37°C for one hour before the reaction was quenched with the addition of an equal volume of 10% formic acid, followed by quantification of Polyamide 6 monomer (6-aminohexanoic acid) with LC-MS relative to a calibration curve performed with a commercial standard. Samples containing polyamide 6,6 dimer were incubated at 37°C for three hours before the reaction was quenched with the addition of an equal volume of 10% formic acid, followed by quantification of adipic acid with LC-MS relative to a calibration curve performed with a commercial standard. Analysis was performed with a gradient of A (Water with 0.1% formic acid) versus B (Acetonitrile with 0.1% formic acid). 0 - 0.5 mins 5% B, 0.5 - 6.5 mins 5 - 95% B, 6.5 - 7.5 mins 95% B, 7.5 - 8.5 mins 95 - 5% B, 8.5 - 10 mins 95% B. Run with a flow rate of 0.3 mL / min using a Phenomenex Cl 8 Peptide XB-C18 column. The MS was analysed with an Agilent SQ- MS with the capillary voltage set to 4 kV, gas temperature of 300 degrees at 7 L / min and the nebuliser set to 15 psi.

[1353] Polyamide 6,6 oligomer hydrolase assays

[1354] Polyamide 6,6 (12% w / v) was dissolved in 6 molar hydrochloric acid and heated to 98°C. The reaction was held at this temperature with stirring for approximately 3 hours before being cooled. An equal amount of 6 molar sodium hydroxide was added to adjust the pH to 7.8 and then the solution was diluted 2x to give a final starting polyamide 6,6 concentration of 3%. The resulting oligomer solution contained at least monomers, dimers, amine-terminated trimers (n-trimers), carboxy terminated trimers (C- trimers), tetramers, N-pentamers, C-pentamers and hexamers.

[1355] 25 pL of the polyamide 6,6 oligomeric solution was combined with 25 pL of a 20 pM solution of the polyamide 6,6 hydrolase being assayed in 200 mM pH 8 sodium phosphate buffer, in the well of a 96 deep well plate. The plate was sealed and incubated at 37°C with shaking at 200 rpm for four hours. The reaction was quenched through the addition of 50 pL of formic acid, before the addition of 900 pL methanol to precipitate the buffer. The plate was centrifuged and the supernatant transferred to a fresh 96 well plate for analysis. The reactions were analysed using reverse phase LC-MS with a mobile phase of acetonitrile (containing formic acid) and water.

[1356] Analysis was performed with a gradient of A (Water with 0.1% formic acid) versus B (Acetonitrile with 0.1% formic acid). 0 - 0.5 mins 5% B, 0.5 - 6.5 mins 5 - 95% B, 6.5 - 7.5 mins 95% B, 7.5 - 8.5 mins 95 - 5% B, 8.5 - 10 mins 95% B. Run with a flow rate of 0.3 mL / min using a Phenomenex C18 Peptide XB-C18 column. The MS was analysed with an Agilent SQ-MS with the capillary voltage set to 4 kV, gas temperature of 300 degrees at 7 L / min and the nebuliser set to 15 psi. Integrations were calculated of extracted ion chromatograms of the molecular ion species in the positive mode (hexamethylenediamine, N-terminal trimer, N-terminal pentamer) and negative mode (adipic acid, dimer, C-terminal trimer, tetramer, C-terminal pentamer, hexamer).

[1357] Results are illustrated in Figure 1.

[1358] Example 2: Initial mutant screen at D181 and N266

[1359] DNA inserts coding for substitution mutants at position 181 of SEQ ID NO:1 were cloned into PET- 28a+ and over-expressed in E coli as described in Example 1. Hydrolytic activity against polyamide 6,6 dimer was determined according to Example 1. The results, shown in Figure 2, revealed that mutants D181G (SEQ ID NO:327) and D181N (SEQ ID NO:328) had beneficial activity. D181R (SEQ ID NO:326) also showed some residual activity. Other substitutions lead to large reductions in activity against the dimer.

[1360] The results of a screen of double mutants at positions 181 and 266 are illustrated in Figure 3. NylB D181G N266H (NylB_GH; SEQ ID NO:2) and NylB D181R N266H (NylB_RH; SEQ ID NO:323) both give activity that is greater than D181R (SEQ ID NO:326) or D181G (SEQ ID NO:327) alone.

[1361] Double mutants D181R N266G (NylB_RG; SEQ ID NO:324) and D181N N266M (NylB_NM; SEQ ID NO:325) gave activity that was higher than the wild type (SEQ ID NO:1), but only D181R N266H (NylB_RH; SEQ ID NO:323) and D181G N266H (NylB_GH; SEQ ID NO:2) showed improved activity over the single D181R (SEQ ID NO:326) or D181G (SEQ ID NO:327) mutations.

[1362] Example 3: Oligo Hydrolysis Spectrum of selected double mutants

[1363] Wild type NylB (SEQ ID NO:1), NylB_GH (SEQ ID NO:2), NylB_RH (SEQ ID NO:323) and a naturally occurring hydrolase from Microbacterium sp. (UNIPROT accession number WP300593461; SEQ ID NO:9) were over expressed in E. coli and purified according to Examples 1 and 2. The Microbacterium hydrolase was identified from protein similarity searches as having similarity to SEQ ID NO:1, but also carrying the 181G 266H amino acid substitution. Peptides were over expressed and purified according to Example 1. Mixed polyamide 6,6 oligomers were prepared according to Example 1 and the individual peptides were assayed against this mixture in triplicate as detailed above. Following incubation, HDMA, adipate, polyamide 6,6 dimer, N-trimer, C-trimer, tetramer, N-pentamer, C- pentamer and hexamer in the reaction mix were identified by LC-MS and the area of peaks corresponding to each oligomer was calculated and compared against a no-enzyme control for each oligomer from the mix.

[1364] Figure 5 illustrates the spectrum of activity of the two double mutants. Figure 5 A provides a table of raw data corresponding to the area under each peak, for each replicate. Figure 5B illustrates this data by calculating the mean of the three replicates and normalising this figure against the mean no -enzyme control for each oligomer. Values below 100% indicate that the oligomer has been depleted by comparison with the no-enzyme control, suggesting that the enzyme has polyamide 6,6 oligomer hydrolase active against that oligomer.

[1365] Of note, the wild type NylB (SEQ ID NO:1) is able to hydrolyse dimers, trimers, tetramers pentamers and hexamers, but has a preference for C-terminated trimers and pentamers. NylB_RH (SEQ ID NO:323) has a similar spectrum of activity but appears to be very poor at hydrolysing N-terminated trimers and pentamers. However, NylB_GH (SEQ ID NO:2) hydrolyses all oligomers including N- terminated species. The Microbacterium hydrolase having the GH substitution (SEQ ID NO:9) has a very similar spectrum of oligomer hydrolase activity to NylB_GH (SEQ ID NO:2). This data shows an improved activity over the wild type (SEQ ID NO:1). Whist the NylB_RH (SEQ ID NO:323) mutant hydrolyses the N-terminated oligomers poorly, it appears to hydrolyse the dimer better than wild type NylB (SEQ ID NO:1) NylB_GH (SQ ID NO:2) and Microbacterium hydrolase (SEQ ID NO:9). Example 4: Polyamide 6,6 oligomer hydrolase activity of selected wild type peptides and their respective GH mutants

[1366] Wild type enzymes with sequence similarity to SEQ ID NO:1 were identified from the UNIPROT database (see Table 3) and NH mutants were prepared following alignment and identification of the equivalent positions. The sequences of each selected GH double mutant is compared in Figure 6. Wild type and GH mutants were assayed for polyamide 6,6 oligomer hydrolase activity according to Example 3 in two batches (Figures 7A&7B and Figures 7C&7D). In each case results were compared to wild type (SEQ ID NO:1) and NylB GH (SEQ ID NO:2). The results of integrations were recorded for each of the polyamide 6,6 (PA66) oligomers within the mass range of the instrument.

[1367] Table 3 In each case GH mutants had an improved polyamide 6,6 hydrolase activity against dimers, tetramers and C- trimers, and in most cases C-pentamers compared to their wild type counterpart (Figures 7B, 7D). Although there was a trend towards an improvement in N- pentamers, this was not apparent across all peptides.

[1368] Example 5: Mutant screen at R187

[1369] A homologue of NylB, 1WYC_A (SEQ ID NO:33) was identified by sequence homology to SEQ ID NO:1. A saturated library of R187 substitution mutants was prepared in this homologue against a background of 1WYC_A D181G N266H (SEQ ID NO:117). The saturation library was transformed into BL21 (DE3) E. coli and the transformants were used to inoculate a 96 deep well plate of TB-AIM culture containing kanamycin and glycerol in triplicate. The proteins were expressed by incubating the plate with shaking at 700 rpm at 20°C for 72 hours. After this period, the plate was centrifuged at 3,000 g for thirty minutes to pellet the cells. The medium was removed and the cells resuspended in BugBuster™ HT lysis buffer (Millipore). The suspension was incubated at 37°C for 30 minutes with shaking at 700 rpm. The plate was again centrifuged at 3,000g, and the lysate was transferred to two new 96 deep well plates, one containing polyamide 6,6 oligomers prepared by incomplete hydrolysis according to Example 1, and the other was incubated in the presence of polyamide 6,6 bis N-terminal trimer.

[1370] The oligomer plate was incubated at 37°C for 30 minutes and the trimer plate at 37°C for 90 minutes. Both reactions were quenched through the addition of equal volumes of 10% formic acid at the end of the incubation period and the reactions were diluted in water before analysis via LC-MS according to Example 3. Concentrations of trimer in the trimer screen at the end of the incubation period were calculated by comparing LC-MS peak area against a calibration curve.

[1371] Figure 8 illustrates the results of the N-trimer hydrolase screen. Almost all mutants appear to show some improvement in hydrolysis of N-trimer compared to the wild type, R187L (SEQ ID 338) and R187I (SEQ ID NO:336) having the highest apparent activity.

[1372] Figures 9A, 9B and 9C illustrate the results of the oligomer screen.

[1373] 1WC_A_NH has good polyamide 6,6 dimer C-trimers, tetramers and C-pentamer , hydrolase activity, similar to other NH mutants. R187A (SEQ ID NO:329), R187C (SEQ ID NO:330), R187D (SEQ ID NO:331), R187E (SEQ ID NO:332), R187H (SEQ ID NO:335), R187I (SEQ ID NO:336), R187L (SEQ ID NO:338), R187Q (SEQ ID NO:342), R187T (SEQ ID NO:345) and R187V (SEQ ID NO:346) have a similar spectrum of polyamide 6,6 oligomer hydrolase activities. They each have good activity against dimers, N and C-trimers (although there is a tendency for C-trimers to be less favoured), tetramers, both N and C-pentamers and hexamers. Among these R187Q (SEQ ID NO:342) and R187D (SEQ ID NO: 331) appear to have particularly good activity towards hexamers. Whilst they still represent an improvement over the NH mutant alone, R187F (SEQ ID NO:333) has slightly poorer overall activity towards all oligomers, but particularly towards the hexamers.

[1374] Whilst they still represent an improvement over the NH mutant alone, R187G (SEQ ID NO:334) appears to disfavour C-terminated trimers compared to the more generally active mutants.

[1375] Whilst they still represent an improvement over the NH mutant alone, R187K (SEQ ID NO:337) appears to have a poorer ability to hydrolyse N-trimers and N-pentamers, whilst R187S (SEQ ID NO:344), R187W (SEQ ID NO:347) and R187Y (SEQ ID NO:348) appear to have a similar spectrum to R187K (SEQ ID NO:337), but poorer overall performance.

[1376] Whilst they still represent an improvement over the NH mutant alone R187M (SEQ ID NO:339), R187N (SEQ ID NO:340) and R187S (SEQ ID NO:344) have good activity towards N-trimers but not towards C-trimers. The difference is less obvious for pentamers.

[1377] R187P (SEQ ID NO:341) does not appear to have activity that is distinguishable from the no-enzyme control.

[1378] Example 6: R187 substitution in 1WYC_A backgrounds

[1379] The following substitution mutants were constructed and tested as described above. The results are illustrated in Figure 10.

[1380] A single R187L mutation (SEQ ID NO:347) in the wild type 1WYC_A (SEQ ID NO:33) background may have a small positive effect on hydrolysis of N-trimers, but was otherwise poorly active. The D181G N266H double mutation (SEQ ID NO:117), in a 1WYC_A background, improves activity on C-trimers and C-pentamers, (as it does in the NylB WT (SEQ ID NO:1) background). In contrast, when the N266H in SEQ ID NO: 117 is replaced with an R187L substitution (SEQ ID NO:350), the effect is to improve activity on the N- trimers, and N-pentamers, rather than the C-trimers and C-pentamers. Example 7: Peroxidase linked putrescine oxidase assay

[1381] A method was specifically developed for assaying activity of enzymes towards polyamide 6,6 dimers and N-terminal trimers employing a putrescine oxidase / peroxidase couple to detect the presence of diamine monomers. A reaction schematic is shown in Figure 11. The reaction is biocompatible and so the hydrolysis of the polyamide 6,6 (PA66) dimer can be continuously assayed and an approximate rate for polyamide 6,6 hydrolysing enzymes determined. а. Polyamide 6,6 dimer hydrolase activity

[1382] A stock solution is made up containing 5 mM polyamide 6,6 dimer (6-[(6-Aminohexyl)amino]-6-oxo- hexanoic Acid), 5 mM ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) and 0.3 mg / mL horseradish peroxidase in pH 8 100 mM sodium phosphate buffer. 90 pL is added to the well of a 96 well plate, followed by 10 pL of a combined purified enzyme stock containing 20 pM of the polyamide б,6 hydrolase being assayed and 200 pM of the putrescine oxidase from Rhodococcus erythropolis (SEQ ID NO:32. The plate is inserted into a plate reader and the temperature is set to 37°C. The absorbance at 414 nm is measured at 20 second intervals. Three separate batches of mutants were tested (see Tables 4, 5 and 6 below).

[1383] Figure 12, A to H, illustrate the polyamide 6,6 dimer hydrolase activity of 8 sample peptides in comparison to NylB (SEQ ID NO:1) and NylB_NH (SEQ ID NO2 - also labelled PRO305 in some experiments) using the assay.

[1384] Tables 4, 5 and 6 show the results from Figure 13 converted to rates and expressed as % of wild type NylB (SEQ ID NO: 1) polyamide 6,6 dimer hydrolase activity for each batch.

[1385] Table 4

[1386] Table 5

[1387] Table 6 b. N-Trimer hydrolase activity

[1388] A stock solution is made up containing 5 mM polyamide 6,6 N-trimer i / V' , / V^-bis(6- aminohexyl)adipamide) prepared according to Johnson (1995), 5 mM ABTS (2,2'-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid)) and 0.3 mg / mL horseradish peroxidase in pH 8 100 mM sodium phosphate buffer. 90 pL is added to the well of a 96 well plate, followed by 10 pL of a combined purified enzyme stock containing 20 pM of the hydrolase being assayed and 200 pM of the putrescine oxidase.

[1389] A partial acid hydrosylate of polyamide 6,6 comprising long chain, insoluble and sparingly solubility oligomers as well as the shorter chain material (e.g. n=2 to n=6) was treated with NylB (SEQ ID NO: 1), NylC (SEQ ID NO:200), NylB RH (SEQ ID NO:323) and NylB GH (SEQ ID NO:2). Progress of digestion was followed using optical density at 600nm. NylC (SEQ ID NO:200), which has excellent oligomer clearing activity, but stalls at the dimer and C-trimer, was included as a positive control. NylB RH (SEQ ID NO:323) has poor activity towards the N-terminated oligomers (data not shown) but NylB GH (SEQ ID NO:2) has good activity against insoluble or sparingly soluble oligomeric material as illustrated by the reduction in OD at 600nm, see Figure 13.

[1390] The disclosure provides, inter alia, the following embodiments:

[1391] Eml. A polypeptide capable of hydrolysing an amide bond in a linear amide polymer wherein the polypeptide comprises a glycine, alanine or arginine residue at position 181 and a histidine residue at position 266 with respect to SEQ ID NO:1; optionally with the exclusion of SEQ ID NOs:4 to 13. Em2. A polypeptide according to Em , comprising an amino acid selected from the group consisting of alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine at position 187 with respect to SEQ ID NO:1.

[1392] Em3. A polypeptide according to either of Ems 1 or 2, comprising one or more of the following features:(i) at least 50% amino acid sequence identity to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:381 or SEQ ID NO:117(ii) a catalytic triad comprising a serine residue at position 112, a lysine residue at a position 115 and a tyrosine or phenylalanine, optionally a tyrosine residue, at a position 215 with respect to SEQ ID NO: 1 ; (iii) a nitrogen inverting residue at position 170 with respect to SEQ ID NO: 1 ; optionally wherein the nitrogen inverting residue is a tyrosine residue ;(iv) the polypeptide has 6- aminohexanoate dimer hydrolase activity and / or polyamide 66 dimer hydrolase activity or polyamide 6,6 dimer hydrolase activity.

[1393] Em4. The polypeptide according to any of Ems 1 to 3, comprising either or both of:(i) a catalytic triad comprising a serine residue at position 112, a lysine residue at a position 115 and a tyrosine or phenylalanine, optionally a tyrosine residue, at a position 215 with respect to SEQ ID NOl;(ii) a nitrogen inverting residue at position 170 with respect to SEQ ID NO: 1 ; optionally wherein the nitrogen inverting residue is a tyrosine residue.

[1394] Em5. The polypeptide according to any of Ems 1 to 4, which has at least 70% sequence identity to SEQ ID NO: 2; SEQ ID NO:3 SEQ ID NO:381 or SEQ ID NO: 117.

[1395] Em6. The polypeptide according to any of Ems 1 to 5, comprising the amino acid sequence of amino acids 2 to 419 of SEQ ID NO:30 wherein: X at position 5 is T or A; X at position 9 is H, D or Y, X at position 12 is Y or F;X at position 16 is present or absent; X at position 31 is N or S;X at position 34 is T or A;X at position 36 is A or S;X at position 37 is H or N;X at position 39 is G or A;X at position 41 is I, V or L;X at position 42 is V, L or M;X at position 46 is V, A or T;X at position 47 is V or I;X at position 63 is D, N or G;X at position 65 is V or L;X at position 66 is present or absent; X at position 67 is present or absent; X at position 69 is L or M; X at position 71 is G or D;X at position 77 is E or D;X at position 80 is Y, F or H;X at position 81 is T or A;X at position 86 is V or I;X at position 87 is V or L;X at position 90 is N, D or G;X at position 91 is Q, D, E or K;X at position 93 is V or I;X at position 94 is G or A;X at position 96 is Y or H;X at position 102 is A or G;X at position 105 is D or Q;X at position 109 V or L;X at position 112 is V or I;X at position 116 is M or L;X at position 120 is L or V;X at position 121 is V, M or I;X at position 123 is A or S;X at position 124 is L or V;X at position 125 is I or V;X at position 126 is D or E;X at position 128 is G or A;X at position 130 is I or V;X at position 136 is I or V;X at position 140 is I, V or L;X at position 147 is A or V;X at position 148 is Y or F;X at position 153 is I or V;X at position 155 is H, N, D or Q;X at position 156 is V or L;X at position 161 is I or V;X at position 163 is I, V or L;X at position 168 is D, N or E;X at position 174 is S or A;X at position 180 is G, R, or A;X at position 182 is S or A; X at position 183 is A, S or G;X at position 186 is R or is A, C D, E, F, G, H, 1, 1, L, Q, T or V.X at position 195 is D or G;X at position 201 is T or A;X at position 202 is T or S;X at positions 208 is N or absent; X at positions 209 is present or absent; X at positions 210 is present or absent; X at positions 211 is present or absent; X at position 215 is E, N, Q or K;X at position 222 is N or H;X at position 225 is V or I;X at position 227 is A or G;X at position 229 is I or V;X at position 234 is T or S;X at position 241 is A or V;X at position 248 is S or A;X at position 249 is K or R;X at position 250 is L or I;X at position 251 is D or G;X at position 255 is D or E;X at position 257 is T or A;X at position 258 is I or V; at position 263 is A, T or S;X at position 270 is G or A;X at position 272 is V or I;X at position 284 is Q or R;X at position 285 is L, I or M;X at position 286 is M, L or V;X at position 288 is D, N or E;X at position 292 is G or A;X at position 296 is R or K;X at position 307 is L or F;X at position 311 is D or S;X at position 313 is E, D or N;X at position 319 is S or G;X at position 322 is A or S;X at position 324 is H or F;X at position 338 is N or H;X at position 343 is V or I;X at position 344 is T or S;X at position 345 is G or A;X at position 349 is Y or H;X at position 355 is I, L or V;X at position 359 is T, S or A;X at position 360 is D, Q or N;X at position 361 is T or S;X at position 362 is V or A;X at position 366 is M, L or F;X at position 368 is T, A or S;X at position 371 is E or D;X at position 373 is E or D;X at position 376 is H, E or Q;X at position 378 is H or N; X at position 381 is Q or D;X at position 382 is N, T or S;X at position 384 is L, I or V;X at position 386 is L or I;X at position 391 is S, A or T; and / or X at position 392 is L or M;wherein X at other positions is any amino acid.

[1396] Em7. The polypeptide according to Em5, having the amino acid sequence of amino acids 2 to 412 of SEQ ID NO: 31 wherein: X at position 2 is T or N;X at position 10 is D or S;X at position 45 is V or T;X at position 47 is A or S;X at position 50 is any amino acid, particularly R or L;X at position 51 is any amino acid particularly R or P;X at position 53 is any amino acid particularly N or A;X at position 54 is any amino acid particularly R, S or T;X at position 55 is V or A;X at position 59 is D or G;X at position 62 is D or N;X at position 68 is D or G;X at position 70 is any amino acid particularly K or L;X at position 71 is R or H;X at position 84 is L or V;X at position 91 is G or A;X at position 130 is S or T;X at position 132 is any amino acid, particularly R or P;X at position 137 is V or I;X at position 152 is H or N;X at position 161 N or D;X at position 177 is G, A or R.X at position 183 is R or is A, C D, E, F, G, H, 1, 1, L, Q, T or V.X at position 188 is any amino acid, particularly E or G;X at position 206 is V or A;X at position 222 is I or V;X at position 244 is D or G;X at position 284 is K or E;X at position 285 is G or A;X at position 286 is any amino acid, particularly P or H;X at position 298 is S or N;X at position 309 is A or V;X at position 319 is any amino acid, particularly Q or G;X at position 332 is any amino acid, particularly G or E;X at position 336 is V or I;X at position 351 is any amino acid, particularly A or P;X at position 353 is D or Q;X at position 356 is I or M;X at position 359 is M or L;X at position 367 is S or N;X at position 376 is E or Q;X at position 383 is H or R;X at position 389 is any amino acid, particularly I or T;X at position 392 is E or D;X at position 396 is any amino acid, particularly T or D;X at position 397 is present or absent if present it is any amino acid, particularly a D;X at position 398 is present or absent if present it is any amino acid, particularly an E;X at position 399 is present or absent if present it is any amino acid, particularly an A;X at position 400 is present or absent if present it is any amino acid, particularly an A;X at position 401 is present or absent if present it is any amino acid, particularly an A;X at position 403 is T or A; X at position 404 is T or A;X at position 406 is E or K; and X at position 410 is N or K; optionally X at positions 397, 398, 399, 400 and 401 are present; optionally they are D, E, A, A and A respectively.

[1397] Em8. A polypeptide according to any of Ems 1 to 4, comprising the amino acid sequence of SEQ ID NO:22 to SEQ ID NO:29; or an amino acid having 70% sequence identity to any of SEQ ID NO:22 to SEQ ID NO:29; and optionally wherein the polypeptide comprises an amino acid selected from the group consisting of alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine at position 187 with respect to SEQ ID NO:1.

[1398] Em9. A polypeptide according to any of claims 1 to 4, comprising an amino acid sequence of SEQ ID NO:373 wherein X at position 2 is S, T or absent; X at position 3 is A, D, V or absent; X at position 4 is A or absent; X at position 5 is L, D, T or absent; X at position 6 is G, A, or absent; X at position 7 is

[1399] S, A or absent; X at position 8 is G, D or absent; X at position 9 is D, H or absent; X at position 10 is N, L, P or absent; X at position 11 is T, P, D or absent; X at position 12 is S, E, A R or absent; X at position 13 is H, E, P, A, G or absent; X at position 14 is D, R, V, N, E, P or absent; X at position 15 is

[1400] T, R, P, G, I or absent; X at position 16 is D, A, K, S or absent; X at position 17 is S, R, I, A, P or absent; X at position 18 is R, P, Q, H, or absent; X at position 19 is R, H, N, A, T, D or absent; X at position 20 is R, G, A, T, D, S, P or absent; X at position 21 is R, S, P, G, A, T or absent; X at position 22 is T, P, S, A, L, G, V, D or absent; X at position 23 is T or absent; X at position 24 is G or absent; X at position 25 is P, Q, S, A, T or absent; X at position 26 is H or absent; X at position 27 is P or absent; X at position 28 is A, G, D, P, S or absent; X at position 29 is R or absent; X at position 30 is Y, F or absent; X at position 31 is P, A or absent; X at position 32 is G, D or absent; X at position 33 is G, A or absent; X at position 34 is A, E, G, P or absent; X at position 35 is P, A, R or absent; X at position 36 is G, S, E, N or absent; X at position 37 is E, Q, R, V, A, H or absent; X at position 39 is R, N, D or S;X at position 41 is E or D;X at position 42 is S, G or T;X at position 45 is E, D, Q or T;X at position 46 is A, P or G;X at position 48 is Q, H, D, Y or G;X at position 52 is A or T;X at position 54 is S, A or G;X at position 55 is H, R, or N;X at position 56 is L or M;X at position 57 is G, N or A;X at position 59 is I, L or M;X at position 60 is L, V, M or T;X at position 61 is P or H;X at position 62 is T, S, V, M;X at position 64 is V, T, A or S;X at position 65 is I or V;X at position 66 is P, S or A;X at position 68 is R, H, N or T;X at position 69 is S, D, P, R, V, A, D or absent; X at position 70 is P, V or E;X at position 71 is A, N, E, H, S, T or G;X at position 72 is A, T, V, S, D or absent; X at position 73 is P, R or S;X at position 74 is S, V, A, G, L, Y, P, T or absent; X at position 75 is G, A or absent; X at position 76 is A or absent; X at position 77 is A or absent; X at position 78 is E or absent; X at position 79 is E, H, A, S, G, D, E or T;X at position 80 is A, P, G;X at position 81 is T, V, L, A, F;X at position 82 is V, A, S, D;X at position 83 is R, H, N;X at position 85 is D, G, E;X at position 86 is S, A, V, P, L or absent; X at position 87 is L, I, V, F or absent; X at position 88 is T, A, E, L, G or absent; X at position 89 is P, S, T, A or absent; X at position 90 is R, Q, A, H or G;X at position 91 is L, M or V;X at position 92 is P or D;X at position 93 is D, G, E, A or T;X at position 95 is Q, E, V, L, R or G;X at position 96 is Q, R, A or D;X at position 97 is R or Q;X at position 99 is E or D;X at position 100 is Q, E, R or A;X at position 101 is S or T;X at position 102 is Y, F or C;X at position 104 is D or N;X at position 106 is F or L;X at position 110 is R or H;X at position 111 is G or R;X at position 112 is G, D, T, S, A, D, E or N;X at position 113 E, R, D, H or Q;X at position 114 is V or L;X at position 115 is L, V or I;X at position 117 is E or D;X at position 118 is Y, F or H;X at position 119 is Y, S or A;X at position 120 is

[1401] R, A, G or K;X at position 121 is A, S, P or D;X at position 124 is A or G;X at position 125 is P or A;X at position 126 is D or H;X at position 127 is D, Q, A or G;X at position 128 is R, L or P;X at position 131 is L, V or I;X at position 133 is S or A;X at position 134 is V or I;X at position 138 is L, M or I; X at position 140 is G or A;X at position 141 is T, I or L;X at position 142 is V, T or absent; X at position 143 is V, F, A or M;X at position 145 is A, V or S;X at position 146 is L, F or V;X at position 147 is V, I or A;X at position 148 is D, E or G;X at position 149 is E, G, D, Q, A, R or S;X at position 150 is G, A, S or D;X at position 151 is R, L, A, S or E;X at position 152 is I, L or V;X at position 153 is D or I;X at position 154 is P or A;X at position 155 is A, S, E, R or D;X at position 156 is R, Q, G or A;X at position 157 is P, R or A;X at position 158 V or I;X at position 159 is T or V; X at position 160 E, A, H, T, R or D;X at position 161 is Y or H;X at position 162 is V or I;X at position 163 is P or R;X at position 166 is E, A or G;X at position 167 is G or D;X at position 169 is V or A; X at position 174 is

[1402] S, A, Q or T;X at position 175 is D or G or absent; X at position 176 is D or absent; X at position 177 is A, P or absent; X at position 178 is A, N or absent; X at position 179 is A or absent; X at position 180 is P or absent; X at position 181 is R or absent; X at position 182 is V or absent; X at position 183 is T or absent; X at position 184 is I, L or V;X at position 185 is Q, L or R;X at position 186 is Q, H or R;X at position 187 is V, L or A;X at position 191 is E, Q, V, A or R;X at position 192 is V or I;X at position 193 is A, S, G or H;X at position 194 is I, V or L;X at position 195 is N or D;X at position 197 is S, N, D or R;X at position 199 is D, N or E;X at position 201 is V, I, L or T;X at position 202 is D or N;X at position 204 is A, G, D or T;X at position 205 is S or A;X at position 209 is T, A or R;X at position 211 is G, A or R; particularly G;X at position 213 is S or A;X at position 217 is R, A, S, C, D, E, F, G, H, 1, 1, L, Q, T or V;X at position 218 is S, T, P, A, D, V, R or E;X at position 219 is R or A;X at position 220 is R, A, L, C or G;X at position 221 is D, E, H, T, D, A, P or E;X at position 222 is G, D or S;X at position 224 is P or A; X at position 225 is A, E, P, V, D, E or I;X at position 226 is D, N or S;X at position 227 is T, D or N; X at position 228 is Y or A; X at position 229 is E, A or S; X at position 232 is T, A or V; X at position 233 is T, S or G; X at position 235 is R or T; X at position 236 is G or T; X at position 237 is R or absent; X at position 238 is P or absent; X at position 239 is D or absent; X at position 240 is A or absent; X at position 241 is D or absent; X at position 242 is A or absent; X at position 243 is S, D, N or P;X at position 245 is A, S, G, R or T;X at position 246 is T, V, L or G;X at position 248 is E or A;X at position 255 is N, D or H;X at position 258 is V or I;X at position 260 is A or G;X at position 262 is I or V;X at position 263 is I or V;X at position 267 is T or S;X at position 269 is L, M or S;X at position 270 is R or P;X at position 272 is A, S, G, V, P or H;X at position 273 is E, D or A;X at position 274 is A, L, V or I;X at position 275 is L or V;X at position 277 is I, V, T, R, A or E;X at position 278 is H, Y, F or R;X at position 279 is L or V;X at position 281 is A or S;X at position 282 is K or R;X at position 283 is L or I;X at position 284 is D or G;X at position 286 is D or G;X at position 287 is R, L, H or Q;X at position 290 is T, I or A;X at position 291 is I or V;X at position 295 is T, Q, S, R or A;X at position 296 is T, A or S;X at position 300 is F, C or Y;X at position 301 is A or P;X at position 303 is G or A;X at position 304 is G or A;X at position 305 is V or I;X at position 306 is S or C ;X at position 307 is C, S, A or T;X at position 317 is R or Q;X at position 318 is M, V, L or A;X at position 319 is M, I, V or L;X at position 322 is G, S or R;X at position 324 is H, A, V, I, S, L, T or K;X at position 325 is A or G;X at position 326 is P or A ;X at position 327 is G or R;X at position 332 is S or P;X at position 333 is E, Q, G or A;X at position 334 is T, A, D, G or E;X at position 336 is V, T or A;X at position 337 is R, S, D or H;X at position 338 is S, R, G or A;X at position 339 is I, V or T;X at position 340 is M or L;X at position 341 is E, A, S or D;X at position 344 is S or D;X at position 345 is R, H, Q, T or P;X at position 346 is E, A, T or Q;X at position 349 is T, P, D or M;X at position 350 is Y, D, P or absent; X at position 351 is E, K, P, A, S or D;X at position 352 is G, D, H or S;X at position 354 is T or P;X at position 355 is G, N, S, D, E, T or A;X at position 356 is T, A, E, V, D, I or R;X at position 357 is F or H ;X at position 359 is D, N, E, G or S;X at position 364 is R or S;X at position 369 is T or M;X at position 371 is N or D;X at position 372 is E or D;X at position 374 is G, A or S ;X at position 377 is S or T;X at position 378 is G or A;X at position 379 is I or V;X at position 385 is N, S or Y;X at position 388 is L or I;X at position 389 is D or E;X at position 391 is P, L, R, Q, M or A ;X at position 392 is T or S;X at position 394 is S, A or T;X at position 396 is I or V;X at position 399 is L or F;X at position 401 is S, T, A or V;X at position 404 is E, D or G;X at position 406 is D, Y or E; X at position 407 is T, G or S;X at position 408 is D, E, R, A or P;X at position 409 is H, D, E, F, Y, Q or S; X at position 410 is W, G, L or R;X at position 411 is H, N or D;X at position 412 is G, D, R, A or E;X at position 413 is V, L, R, K or A;X at position 415 is N, S, Q, T or G;X at position 416 is D, G, A, E, S, R, Q or T;X at position 417 is V, I or L;X at position 419 is L or V;X at position 420 is D or E;X at position 421 is V, I or L;X at position 422 is S or C;X at position 423 is R, H, E or T;X at position 424 is A or S;X at position 427 is A, G, T, R, S, P, Q or absent; X at position 428 is M, A, V, L, F, I, P, V or absent ; X at position 429 is A, P, R, G, S or absent; X at position 430 is Y, A or absent; X at position 431 is A or absent; X at position 432 is A or absent; X at position 433 is E or absent; X at position 434 is T or absent; X at position 444 is A or absent; X at position 445 is P or absent; X at position 446 is D or absent; X at position 447 is S or absent; X at position 448 is K or absent; X at position 449 is E or absent; X at position 450 is N or absent; X at position 451 is A or absent; X at position 452 is A or absent. EmlO. A polypeptide according to any of Ems 1 to 4, selected from; SEQ ID NO:6, SEQ ID NO:7 SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:23, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:128, SEQ ID NO:131, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:157, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 184 and SEQ ID NO: 192 or a sequence having at least 80% sequence identity to any of these; and wherein the said sequence is optionally substituted at position 187 with an amino acid selected from the group consisting of alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine.

[1403] Emi l. A polypeptide according to any of Ems 1 to 4, comprising the amino acid sequence of amino acids 2 to 392 of SEQ ID NO:2; amino acids 2 to 342 of SEQ ID NO: 117 or amino acids 2 to 392 of SEQ ID NO: 367 (NylBGLH) or an amino acid sequence having at least 96% sequence identity to any of these.

[1404] Eml2. A polypeptide according to any of Ems 1 to 4, comprising the amino acid sequence of any of SEQ ID NOs:117 to 119 or a polypeptide having at least 70% sequence identity thereto; and wherein the arginine at position 187 with reference to SEQ ID NO:1 is substituted with an amino acid selected from the group consisting of alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine.

[1405] Eml 3. A polypeptide having polyamide hydrolase activity comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:33 or a polypeptide having at least 50% sequence identity to either SEQ ID NO:1 or SEQ ID NO:33 wherein the amino acid at position 187 with reference to SEQ ID NO:1 is substituted by an amino acid selected from the group consisting of alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine; optionally with the proviso that if the amino acid at position 187 with respect to SEQ ID NO:1 is A, K, E or L, then the amino acid at position 181 is A, R, G, or N; and / or the amino acid at position 266 is H, G or M.

[1406] Eml4. The polypeptide according to Eml3, comprising :(i) a catalytic triad comprising a serine residue at position 112, a lysine residue at a position 115 and a tyrosine or phenylalanine, optionally a tyrosine residue, at a position 215 with respect to SEQ ID NO:l;(ii) a nitrogen inverting residue at position 170 with respect to SEQ ID NO:1; optionally wherein the nitrogen inverting residue is a tyrosine residue.

[1407] Eml5. The polypeptide according to Ems 13 or 14, comprising the amino acid sequence of SEQ ID NO:1, sequence ID NO:33, SEQ ID NOs: 14-21; SEQ ID NO:38, SEQ ID NO:44, SEQ ID NOs:48-50, SEQ ID NOs:68-71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:79-81, SEQ ID NOs:84-86, SEQ ID NO:90, SEQ ID NOs:92-94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO: 101, SEQ ID NO: 104, SEQ ID NO: 109, SEQ ID NO: 110 and SEQ ID NO: 115 or a sequence having at least 80% sequence identity to any of these; and wherein in each case the amino acid at position 187 with reference to SEQ ID NO: 1 is substituted with an amino acid selected from the group consisting of alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine; provided that if the amino acid at position 196 is A, then the amino acid at position 190 is A, R, G, or N; and / or the amino acid at position 285 is H, G or M.

[1408] Eml 6. The polypeptide according to any of Ems 13 to 15, comprising the sequence of amino acids 5 to 402 of SEQ ID NO:379, wherein: X at position 5 is S, A, T or L;X at position 8 is Q, P or A;X at position 11 is A, P or D;X at position 13 is Y or F;X at position 16 is A or G;X at position 17 is A, E or G;X at position 18 is A, P or R;X at position 20 is E, Q or R;X at position 22 is T, N or H;X at position 25 is S or G;X at position 29 is P or A;X at position 31 is H, Q or D;X at position 37 is A or S;X at position 38 is H or R;X at position 40 is G or S;X at position 42 is M, I or L;X at position 43 is V or L;X at position 45 is S or T;X at position 47 is A, V or T;X at position 48 is V or I;X at position 49 is S or P;X at position 52 is P, D or S;X at position 53 is V or P;X at position 54 is N or A;X at position 55 is A or T;X at position 57 is G, A, S, L or Y;X at position 58 is H, E, A or S;X at position 59 is A or P;X at position 60 is L, V or T;X at position 61 is A or V;X at position 64 is D or G;X at position 65 is A, S or V;X at position 66 is I or L;X at position 67 is A, T or E;X at position 68 is A, T or P;X at position 69 is Q or R;X at position 70 is L or M;X at position 72 is D or G;X at position 74 is E or Q;X at position 75 is Q, R or A;X at position 79 is Q or E;X at position 80 is T or S;;X at position 81 is Y or C;X at position 83 is D or N;X at position 85 is F or L;X at position 91 is T, S, G or A;X at position 94 is V, L or I;X at position 99 is R or A;X at position 100 is A or P;X at position 104 is A or P;X at position 112 is A or S;X at position 122 is V or F;X at position 126 is V or I;X at position 127 is D or E;X at position 128 is E, D or Q;X at position 130 is R or L;X at position 134 is A or EX at position 135 is Q or RX at position 137 is V or IX at position 139 is E, H, T or RX at position 140 is Y or HX at position 145 is A, G or EX at position 148 is V or AX at position 153 is S, T or QX at position 154 is G or absentX at position 155 is D or absentX at position 156 is P or absentX at position 157 is N or absentX at position 158 is A or absentX at position 159 is P or absentX at position 160 is R or absentX at position 161 is V or absent X at position 162 is T or absentX at position 163 is V or LX at position 164 is L or QX at position 166 is V or LX at position 170 is Q or EX at position 171 is I or VX at position 172 is S or AX at position 173 is I or VX at position 176 is N or SX at position 183 is A or GX at position 190 is D, A, R, G or NX at position 196 is A, C, D, E, F, G, H, I, L, Q, T or VX at position 197 is T or SX at position 200 is H, D or AX at position 204 is A or EX at position 205 is D or NX at position 206 is T or DX at position 208 is S, R or AX at position 211 is T or AX at position 212 is T or SX at position 216 is D, S or NX at position 218 is S, G, R or AX at position 235 is I or VX at position 236 is V or IX at position 240 is T or SX at position 242 is L or MX at position 243 is R or PX at position 245 is V or AX at position 250 is T, I or VX at position 251 is Y or HX at position 254 is A or SX at position 260 is R or LX at position 268 is T, Q, S or RX at position 269 is T or AX at position 276 is G or AX at position 280 is C or SX at position 285 is N, H, G or MX at position 297 is V, I or HX at position 306 is E, Q or GX at position 307 is D, G, T or AX at position 310 is R or EX at position 311 is R, S or GX at position 312 is V or IX at position 313 is L or MX at position 314 is A, S or EX at position 318 is H or RX at position 319 is E or AX at position 323 is D or YX at position 324 is K, E or PX at position 325 is G or DX at position 328 is N, S or GX at position 329 is T or AX at position 332 is D, E, G or NX at position 344 is N or DX at position 362 is D or EX at position 364 is L, R, Q or PX at position 369 is I or VX at position 374 is S or TX at position 377 is D or EX at position 379 is Y or DX at position 381 is E, R, D or AX at position 385 is R or GX at position 386 is L or VX at position 388 is N, S or QX at position 389 is G, A or DX at position 390 is I or VX at position 394 is V or IX at position 396 is R or HX at position 400 is A or TX at position 401 is V or MX at position 402 A or absent.

[1409] The Em as above wherein if the amino acid at position 196 is A, then the amino acid at position 190 is A, R, G, or N; and / or the amino acid at position 285 is H, G or M.

[1410] The Em as above wherein polypeptide wherein X at position 190 is G or R; particularly G.

[1411] Eml7. The polypeptide according to any of Ems 13 to 16, wherein X at position 285 is H, G or M; particularly H.

[1412] Eml8. The polypeptide according to any of Ems 13 to 17, wherein X at position 196 is, C, D, E, F, I, L, Q, or V; particularly I or L.

[1413] Eml9. The polypeptide according to any Ems 13 to 17, wherein X at position 196 is A, C, D, G, H, I, L, M, Q, S, T or V; optionally A, C, D, H, I, L, Q, or T; further optionally C, D, H, I, L or Q.

[1414] Em20. The polypeptide of any preceding Em, which is capable of hydrolysing a polyamide polymer or a polyamide oligomer.

[1415] Em21. The polypeptide according to Em 20, wherein the polyamide is polyamide 6 or polyamide 6,6.

[1416] Em22. The polypeptide according to Ems 20 or 21, which is capable of hydrolysing a water soluble polyamide 6 oligomer or polyamide 6,6 oligomer.

[1417] Em23. The polypeptide according to any of Ems 20 to 22, which hydrolyses a polyamide 6,6 dimer, N- trimer, C-trimer, tetramer, N-pentamer, C-pentamer and / or hexamer or mixtures comprising any two or more of the foregoing.

[1418] Em24. A polynucleotide comprising a polynucleotide sequence encoding the polypeptide of any of Ems 1 to 23.

[1419] Em25. An expression vector comprising the polynucleotide of Em 24, wherein the polynucleotide is under the control of a constitutive promoter or an inducible promoter. Em26. A host cell comprising the polynucleotide of Em24, or the expression vector of Em25.

[1420] Em27. A process for the preparation of a polypeptide according to any one of Ems 1 to 23, comprising expressing the polypeptide from a polynucleotide according to Em24, and recovering the polypeptide.

[1421] Em28. A composition comprising the polypeptide of any one of Ems 1 to 19.

[1422] Em29. A composition comprising a polynucleotide according to Em 24, an expression vector according Em 25 or a host cell according to Em 26.

[1423] Em30. A composition according to Em 28 or 29, which is a dried composition.

[1424] Em31. A composition comprising a polypeptide according to any of Ems 1 to 12 and a polypeptide according to any of Ems 13 to 19.

[1425] Em32. A composition comprising one or more polypeptides according to Ems 1 to 19, or a composition according to Em 31, which is immobilized on a solid support.

[1426] Em33. A process for hydrolysing a polyamide polymer or polyamide oligomer comprising contacting the polyamide polymer or polyamide oligomer with a polypeptide according to any of Ems 1 to 19, a composition according to Em 28, a composition comprising a host cell according to Em 26 or a composition according to Em 3 lor 32; optionally wherein the polyamide polymer or polyamide oligomer is a polyamide 6,6 polymer or a polyamide 6,6 oligomer.

[1427] Em34. The process according to Em33, wherein the polyamide polymer is a polyamide 6,6 polymer and wherein the polyamide 6,6 polymer is partially chemically hydrolysed to provide polyamide 6,6 oligomers which are contacted with a polypeptide according to any of Ems 1 to 19, a composition according to Em 28, a composition comprising a host cell according to Em 26 or a composition according to Em 3 lor 32.

[1428] Em35. The process according to Ems 33 or 34, wherein the partial chemical hydrolysis provides a composition comprising water soluble polyamide 6,6 oligomers; optionally wherein the chemical hydrolysis provides a mixture of oligomers comprising at least dimers and trimers; optionally comprising at least dimers, trimers, tetramers, pentamers and hexamers.

[1429] Em36. The process according to any of Em 33 to 35, wherein the partial chemical hydrolysis provides a composition additionally comprising insoluble oligomers.

[1430] Em37. The process according to any of Ems 33 to 36, wherein the partial chemical hydrolysis is an acid catalysed partial hydrolysis.

[1431] Em38. The process according to Ems 33 to 37, which is carried out in an aqueous medium, wherein the optical density of the aqueous medium is reduced following contacting the polyamide polymer or polyamide oligomer with the polypeptide. Em39. The process according to any of Ems 33 to 38, wherein the polymer or oligomer is a polyamide 6,6 polymer or oligomer and the products of the hydrolysis comprises adipic acid and / or hexamethylene diamine; optionally wherein the adipic acid and / or the hexamethylene diamine are recovered.

[1432] Em40. A process for the preparation of polyamide 6,6, comprising carrying out a process according to any of Ems 33 to 39, wherein the polyamide is polyamide 6,6 polymer or polyamide 6,6 oligomer, recovering adipic acid and / or hexamethylene diamine monomers resulting from the process and synthesising a polyamide 6,6 polymer from the recovered adipic acid and / or the hexamethylene diamine monomers.

[1433] Em41. A method for increasing the polyamide 6,6 hydrolase activity of an amidase enzyme comprising:

[1434] (a) identifying the amino acid sequence of a polypeptide having either polyamide 6,6 hydrolase activity or 6-aminohexanoate dimer hydrolase activity, wherein the amidase lacks

[1435] (i) a glycine residue at position 181 or a histidine residue at position 266, or lacks both a glycine residue at position 181 and a histidine residue at position 266 with reference to SEQ ID NO:1;

[1436] (ii) an arginine residue at position 181 or a histidine residue at position 266, or lacks both an arginine residue at position 181 and a histidine residue at position 266 with reference to SEQ ID NO:l;or

[1437] (iii) an alanine residue at position 181 or a histidine residue at position 266, or lacks both an alanine residue at position 181 and a histidine residue at position 266 with reference to SEQ ID NO:1

[1438] (b) substituting the amino acid at position 181 with a glycine residue and / or substituting the amino acid at position 266 with a histidine residue; or substituting the amino acid at position 181 with an arginine residue and / or substituting the amino acid at position 266 with a histidine residue; or substituting the amino acid at position 181 with an alanine residue and / or substituting the amino acid at position 266 with a histidine residue; wherein positions are relative to the amino acid sequence of SEQ ID NO: 1 ; and optionally if the amidase sequence has other than an alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine or valine at position 187 in relation to SEQ ID NO:1; then

[1439] (c) replacing the amino acid at position 187 with respect to SEQ ID NO:1 with an amino acid selected from the group consisting of alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine.

[1440] Em42. A method for increasing the polyamide 6,6 hydrolase activity of an amidase enzyme comprising: (a) identifying the amino acid sequence of a polypeptide having either polyamide 6,6 hydrolase activity, or 6-aminohexanoate dimer hydrolase activity, wherein the amidase sequence has an amino acid other than an alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine at position 187 in relation to SEQ ID NO:1; and

[1441] (b) replacing the amino acid at position 187 with respect to SEQ ID NO:1 with an amino acid selected from the group consisting of alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine; and optionally (i) if the sequence does not comprise a glycine residue at position 181 with respect to SEQ ID NO:1, then replacing the amino acid with a glycine; (ii) if the sequence does not comprise an alanine residue at position 181 with respect to SEQ ID NO:1, then replacing the amino acid with an alanine; or(iii) if the sequence does not comprise an arginine residue at position 181 with respect to SEQ ID NO:1, then replacing the amino acid with an arginine; and(iv) if the sequence does not comprise a histidine residue at position 266 with respect to SEQ ID NO:1, replacing the amino acid at position 266 with a histidine residue.

[1442] Em43. The method according to Em41 or 42, wherein the identified sequence of the polypeptide having either polyamide 6,6 hydrolase activity, or 6-aminohexanoate dimer hydrolase activity has at least one of the following additional features:(i) an amino acid sequence of at least 50% identity to SEQ ID NO: 1 ;(ii) a catalytic triad comprising a serine residue at position 112, a lysine residue at a position 115 and a tyrosine or phenylalanine residue at a position 215 (iii) a nitrogen inverting residue at position 170; optionally wherein the nitrogen inverting residue is a tyrosine residue

[1443] Em44. The method according to any of Ems 41 to 43, wherein polyamide 6,6 hydrolase activity increased is one or more of polyamide 66 hexamer hydrolase, polyamide 6,6 N-pentamer hydrolase, polyamide 6,6 C-pentamer hydrolase, polyamide 6,6 tetramer hydrolase, polyamide 6,6 N-trimer hydrolase, polyamide 66 C-trimer hydrolase, or polyamide 66 dimer hydrolase activity.

[1444] Em45. The method according to any of Ems 4 Ito 44 wherein the polypeptide having either polyamide 66 hydrolase activity, or 6-aminohexanoate dimer hydrolase activity identified in step (a) has at least 60% sequence identity to a sequence selected from SEQ ID NO:1, SEQ ID NO:14 to SEQ ID NO:21, SEQ ID NO:33.

[1445] Em46. The method according to any of Ems 41 to 45, wherein the polypeptide has increased hydrolytic activity towards polyamide 6,6 oligomers having terminal amino groups and / or increased activity towards polyamide 6,6 oligomers having terminal carboxylate groups.

[1446] Em47. A polypeptide prepared according to any one of Ems 41 to 46

[1447] Em48. An assay for indicating a hydrolytic activity of an amidase enzyme towards a polyamide oligomer, wherein the hydrolytic activity releases at least one diamine monomer; the process comprising: contacting the amidase enzyme with the polyamide oligomer; to release a diamine monomer, with a putrescine oxidase in the presence of oxygen, to produce an oxidised diamine monomer and a peroxide; contacting the peroxide with a peroxidase in the presence of peroxidase substrate; and detecting the activity of the peroxidase on the peroxidase substrate to indicate the activity of the amidase enzyme towards a polyamide oligomer.

[1448] Em49. The assay according to Em 47, wherein the putrescine oxidase is Rhodococcus erythropolis putrescine oxidase.

[1449] Em50. The assay according to Ems 48 or 49 wherein the peroxide is H2O2.

[1450] Em51. The assay according to any of Ems 48 to 50 where in the peroxidase substrate is a colourogenic substrate and the activity of the peroxidase on the peroxidase substrate is detected by detecting the colour of the product; optionally wherein the colourogenic substrate is 2,2'-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid (ABTS).

[1451] Em52. The assay according to any of Ems 48 to 51, wherein the peroxidase is HRP.

[1452] Em53. The assay according to any of Ems 48 to 52, wherein the polyamide oligomer is 6-[(6- aminohexyl) amino] -6-oxo-hexanoic acid.

[1453] Em54. A method for monitoring the progress of a polyamide hydrolysis to produce at least one diamine product, the method comprising detecting the presence or the quantity of the diamine product using an assay according to any of Ems 48 to 53.

[1454] REFERENCE

[1455] Johnson P.D. (1995) Nylon 6,6 oligomers : Synthesis, Characterization & Relevance To The Polymer. A Doctoral thesis submitted to Sheffield Hallam University 1995. ProQuest Number: 10697184. Published by ProQuest LLC(2017).

[1456] SEQUENCE TABLE

Claims

CLAIMS1. A polypeptide capable of hydrolysing an amide bond in a linear amide polymer wherein the polypeptide comprises a glycine, alanine or arginine residue at position 181 and a histidine residue at position 266 with respect to SEQ ID NO:1; optionally with the exclusion of SEQ ID NOs:4 to 13.

2. The polypeptide according to claim 1, comprising an amino acid selected from the group consisting of alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine at position 187 with respect to SEQ ID NO:1.

3. The polypeptide according to claim 1 or claim 2, comprising one or more of the following features:(i) at least 50% amino acid sequence identity to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:381 or SEQ ID NO:117;(ii) a catalytic triad comprising a serine residue at position 112, a lysine residue at a position 115 and a tyrosine or phenylalanine, optionally a tyrosine residue, at a position 215 with respect to SEQ ID NO:1;(iii) a nitrogen inverting residue at position 170 with respect to SEQ ID NO:1; optionally wherein the nitrogen inverting residue is a tyrosine residue;(iv) the polypeptide has 6-aminohexanoate dimer hydrolase activity and / or polyamide 6,6 dimer hydrolase activity or polyamide 6,6 dimer hydrolase activity.

4. The polypeptide according to any of claims 1 to 3, comprising either or both of:(i) a catalytic triad comprising a serine residue at position 112, a lysine residue at position 115 and a tyrosine or phenylalanine residue, optionally a tyrosine residue, at position 215, with respect to SEQ ID NO:1;(ii) a nitrogen inverting residue at position 170 with respect to SEQ ID NO:1; optionally wherein the nitrogen inverting residue is a tyrosine residue.

5. The polypeptide according to any of claims 1 to 4, which has at least 70% sequence identity to SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:381 or SEQ ID NO:117.

6. The polypeptide according to any of claims 1 to 5, comprising the amino acid sequence of amino acids 2 to 419 of SEQ ID NO:30, wherein:X at position 5 is T or A;X at position 9 is H, D or YX at position 12 is Y or F;X at position 16 is present or absent;X at position 31 is N or S;X at position 34 is T or A;X at position 36 is A or S;X at position 37 is H or N;X at position 39 is G or A;X at position 41 is I, V or L;X at position 42 is V, L or M;X at position 46 is V, A or T;X at position 47 is V or I;X at position 63 is D, N or G;X at position 65 is V or L;X at position 66 is present or absent;X at position 67 is present or absent;X at position 69 is L or M;X at position 71 is G or D;X at position 77 is E or D;X at position 80 is Y, F or H;X at position 81 is T or A;X at position 86 is V or I;X at position 87 is V or L;X at position 90 is N, D or G;X at position 91 is Q, D, E or K;X at position 93 is V or I;X at position 94 is G or A;X at position 96 is Y or H;X at position 102 is A or G;X at position 105 is D or Q;X at position 109 V or L;X at position 112 is V or I;X at position 116 is M or L;X at position 120 is L or V;X at position 121 is V, M or I;X at position 123 is A or S;X at position 124 is L or V;X at position 125 is I or V;X at position 126 is D or E;X at position 128 is G or A;X at position 130 is I or V;X at position 136 is I or V;X at position 140 is I, V or L;X at position 147 is A or V;X at position 148 is Y or F;X at position 153 is I or V;X at position 155 is H, N, D or Q;X at position 156 is V or L;X at position 161 is I or V;X at position 163 is I, V or L;X at position 168 is D, N or E;X at position 174 is S or A;X at position 180 is G, R, or AX at position 182 is S or A;X at position 183 is A, S or G;X at position 186 is R or is A, C D, E, F, G, H, 1, 1, L, Q, T or V.X at position 195 is D or G;X at position 201 is T or A;X at position 202 is T or S ;X at positions 208 is N or absent;X at positions 209 is present or absent;X at positions 210 is present or absent;X at positions 211 is present or absent;X at position 215 is E, N, Q or K;X at position 222 is N or H;X at position 225 is V or I;X at position 227 is A or G;X at position 229 is I or V;X at position 234 is T or S ;X at position 241 is A or V;X at position 248 is S or A;X at position 249 is K or R;X at position 250 is L or I;X at position 251 is D or G;X at position 255 is D or E; X at position 257 is T or A; X at position 258 is I or V; X at position 263 is A, T or S; X at position 270 is G or A; X at position 272 is V or I; X at position 284 is Q or R; X at position 285 is L, I or M; X at position 286 is M, L or V; X at position 288 is D, N or E; X at position 292 is G or A; X at position 296 is R or K; X at position 307 is L or F; X at position 311 is D or S ; X at position 313 is E, D or N; X at position 319 is S or G; X at position 322 is A or S ; X at position 324 is H or F; X at position 338 is N or H; X at position 343 is V or I; X at position 344 is T or S ; X at position 345 is G or A; X at position 349 is Y or H; X at position 355 is I, L or V; X at position 359 is T, S or A; X at position 360 is D, Q or N; X at position 361 is T or S ; X at position 362 is V or A; X at position 366 is M, L or F; X at position 368 is T, A or S; X at position 371 is E or D; X at position 373 is E or D; X at position 376 is H, E or Q; X at position 378 is H or N; X at position 381 is Q or D; X at position 382 is N, T or S; X at position 384 is L, I or V;X at position 386 is L or I;X at position 391 is S, A or T; and / orX at position 392 is L or M; wherein X at other positions is any amino acid.

7. The polypeptide according to claim 5, having the amino acid sequence of amino acids 2 to 412 of SEQ ID NO: 31, wherein:X at position 2 is T or N;X at position 10 is D or S;X at position 45 is V or T;X at position 47 is A or S;X at position 50 is any amino acid, particularly R or L;X at position 51 is any amino acid particularly R or P;X at position 53 is any amino acid particularly N or A;X at position 54 is any amino acid particularly R, S or T;X at position 55 is V or A;X at position 59 is D or G;X at position 62 is D or N;X at position 68 is D or G;X at position 70 is any amino acid particularly K or L;X at position 71 is R or H;X at position 84 is L or V;X at position 91 is G or A;X at position 130 is S or T;X at position 132 is any amino acid, particularly R or P;X at position 137 is V or I;X at position 152 is H or N;X at position 161 N or D;X at position 177 is G, A or R.X at position 183 is R or is A, C D, E, F, G, H, 1, 1, L, Q, T or V.X at position 188 is any amino acid, particularly E or G;X at position 206 is V or A;X at position 222 is I or V;X at position 244 is D or G;X at position 284 is K or E;X at position 285 is G or A;X at position 286 is any amino acid, particularly P or H;X at position 298 is S or N;X at position 309 is A or V;X at position 319 is any amino acid, particularly Q or G;X at position 332 is any amino acid, particularly G or E;X at position 336 is V or I;X at position 351 is any amino acid, particularly A or P;X at position 353 is D or Q;X at position 356 is I or M;X at position 359 is M or L;X at position 367 is S or N;X at position 376 is E or Q;X at position 383 is H or R;X at position 389 is any amino acid, particularly I or T;X at position 392 is E or D;X at position 396 is any amino acid, particularly T or D;X at position 397 is present or absent if present it is any amino acid, particularly a D;X at position 398 is present or absent if present it is any amino acid, particularly an E;X at position 399 is present or absent if present it is any amino acid, particularly an A;X at position 400 is present or absent if present it is any amino acid, particularly an A;X at position 401 is present or absent if present it is any amino acid, particularly an A;X at position 403 is T or A;X at position 404 is T or A;X at position 406 is E or K; and / orX at position 410 is N or K; optionally X at positions 397, 398, 399, 400 and 401 are present; optionally they are D, E, A, A and A respectively.

8. The polypeptide according to any of claims 1 to 4, comprising the amino acid sequence of SEQ ID NO:22 to SEQ ID NO:29; or an amino acid having 70% sequence identity to any of SEQ ID NO:22 to SEQ ID NO:29; and optionally wherein the polypeptide comprises an amino acid selected from the group consisting of alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine at position 187 with respect to SEQ ID NO:1.

9. The polypeptide according to any of claims 1 to 4, comprising the amino acid sequence of SEQ ID NO:373, wherein:X at position 2 is S, T or absentX at position 3 is A, D, V or absentX at position 4 is A or absentX at position 5 is L, D, T or absentX at position 6 is G, A, or absentX at position 7 is S, A or absentX at position 8 is G, D or absentX at position 9 is D, H or absentX at position 10 is N, L, P or absentX at position 11 is T, P, D or absentX at position 12 is S, E, A R or absentX at position 13 is H, E, P, A, G or absentX at position 14 is D, R, V, N, E, P or absentX at position 15 is T, R, P, G, I or absentX at position 16 is D, A, K, S or absentX at position 17 is S, R, I, A, P or absentX at position 18 is R, P, Q, H, or absentX at position 19 is R, H, N, A, T, D or absentX at position 20 is R, G, A, T, D, S, P or absentX at position 21 is R, S, P, G, A, T or absentX at position 22 is T, P, S, A, L, G, V, D or absentX at position 23 is T or absentX at position 24 is G or absentX at position 25 is P, Q, S, A, T or absentX at position 26 is H or absentX at position 27 is P or absentX at position 28 is A, G, D, P, S or absentX at position 29 is R or absentX at position 30 is Y, F or absentX at position 31 is P, A or absentX at position 32 is G, D or absentX at position 33 is G, A or absentX at position 34 is A, E, G, P or absentX at position 35 is P, A, R or absentX at position 36 is G, S, E, N or absentX at position 37 is E, Q, R, V, A, H or absentX at position 39 is R, N, D or SX at position 41 is E or DX at position 42 is S, G or TX at position 45 is E, D, Q or TX at position 46 is A, P or GX at position 48 is Q, H, D, Y or GX at position 52 is A or TX at position 54 is S, A or GX at position 55 is H, R, G or NX at position 56 is L or MX at position 57 is G, N or AX at position 59 is I, L or MX at position 60 is L, V, M or TX at position 61 is P or HX at position 62 is T, S, V, MX at position 64 is V, T, A or SX at position 65 is I or VX at position 66 is P, S or AX at position 68 is R, H, N or TX at position 69 is S, D, P, R, V, A, D or absentX at position 70 is P, V or EX at position 71 is A, N, E, H, S, T or GX at position 72 is A, T, V, S, D or absentX at position 73 is P, R or SX at position 74 is S, V, A, G, L, Y, P, T or absentX at position 75 is G, A or absentX at position 76 is A or absentX at position 77 is A or absentX at position 78 is E or absentX at position 79 is E, H, A, S, G, D, E or TX at position 80 is A, P, GX at position 81 is T, V, L, A, FX at position 82 is V, A, S, DX at position 83 is R, H, NX at position 85 is D, G, EX at position 86 is S, A, V, P, L or absentX at position 87 is L, I, V, F or absentX at position 88 is T, A, E, L, G or absentX at position 89 is P, S, T, A or absentX at position 90 is R, Q, A, H or GX at position 91 is L, M or VX at position 92 is P or DX at position 93 is D, G, E, A or TX at position 95 is Q, E, V, L, R or GX at position 96 is Q, R, A or DX at position 97 is R or QX at position 99 is E or DX at position 100 is Q, E, R or AX at position 101 is S or TX at position 102 is Y, F or CX at position 104 is D or NX at position 106 is F or LX at position 110 is R or HX at position 111 is G or RX at position 112 is G, D, T, S, A, D, E or NX at position 113 E, R, D, H or QX at position 114 is V or LX at position 115 is L, V or IX at position 117 is E or DX at position 118 is Y, F or HX at position 119 is Y, S or AX at position 120 is R, A, G or KX at position 121 is A, S, P or DX at position 124 is A or GX at position 125 is P or AX at position 126 is D or HX at position 127 is D, Q, A or GX at position 128 is R, L or PX at position 131 is L, V or IX at position 133 is S or AX at position 134 is V or IX at position 138 is L, M or IX at position 140 is G or AX at position 141 is T, I or LX at position 142 is V, T or absentX at position 143 is V, F, A or MX at position 145 is A, V or SX at position 146 is L, F or VX at position 147 is V, I or AX at position 148 is D, E or GX at position 149 is E, G, D, Q, A, R or SX at position 150 is G, A, S or DX at position 151 is R, L, A, S or EX at position 152 is I, L or VX at position 153 is D or IX at position 154 is P or AX at position 155 is A, S, E, R or DX at position 156 is R, Q, G or AX at position 157 is P, R or AX at position 158 V or IX at position 159 is T or VX at position 160 E, A, H, T, R or DX at position 161 is Y or HX at position 162 is V or IX at position 163 is P or RX at position 166 is E, A or GX at position 167 is G or DX at position 169 is V or AX at position 174 is S, A, Q or TX at position 175 is D or G or absentX at position 176 is D or absentX at position 177 is A, P or absentX at position 178 is A, N or absentX at position 179 is A or absentX at position 180 is P or absentX at position 181 is R or absentX at position 182 is V or absentX at position 183 is T or absentX at position 184 is I, L or VX at position 185 is Q, L or RX at position 186 is Q, H or RX at position 187 is V, L or AX at position 191 is E, Q, V, A or RX at position 192 is V or IX at position 193 is A, S, G or HX at position 194 is I, V or LX at position 195 is N or DX at position 197 is S, N, D or RX at position 199 is D, N or EX at position 201 is V, I, L or TX at position 202 is D or NX at position 204 is A, G, D or TX at position 205 is S or AX at position 209 is T, A or RX at position 211 is G, A or R; particularly GX at position 213 is S or AX at position 217 is R, A, S, C, D, E, F, G, H, 1, 1, L, Q, T or VX at position 218 is S, T, P, A, D, V, R or EX at position 219 is R or AX at position 220 is R, A, L, C or GX at position 221 is D, E, H, T, D, A, P or EX at position 222 is G, D or SX at position 224 is P or AX at position 225 is A, E, P, V, D, E or IX at position 226 is D, N or SX at position 227 is T, D or NX at position 228 is Y or AX at position 229 is E, A or SX at position 232 is T, A or VX at position 233 is T, S or GX at position 235 is R or TX at position 236 is G or TX at position 237 is R or absentX at position 238 is P or absentX at position 239 is D or absentX at position 240 is A or absentX at position 241 is D or absentX at position 242 is A or absentX at position 243 is S, D, N or PX at position 245 is A, S, G, R or TX at position 246 is T, V, L or GX at position 248 is E or AX at position 255 is N, D or HX at position 258 is V or IX at position 260 is A or GX at position 262 is I or VX at position 263 is I or VX at position 267 is T or SX at position 269 is L, M or SX at position 270 is R or PX at position 272 is A, S, G, V, P or HX at position 273 is E, D or AX at position 274 is A, L, V or IX at position 275 is L or VX at position 277 is I, V, T, R, A or EX at position 278 is H, Y, F or RX at position 279 is L or VX at position 281 is A or SX at position 282 is K or RX at position 283 is L or IX at position 284 is D or GX at position 286 is D or GX at position 287 is R, L, H or QX at position 290 is T, I or AX at position 291 is I or VX at position 295 is T, Q, S, R or AX at position 296 is T, A or SX at position 300 is F, C or YX at position 301 is A or PX at position 303 is G or AX at position 304 is G or AX at position 305 is V or IX at position 306 is S or CX at position 307 is C, S, A or TX at position 317 is R or QX at position 318 is M, V, L or AX at position 319 is M, I, V or LX at position 322 is G, S or RX at position 324 is H, A, V, I, S, L, T or KX at position 325 is A or GX at position 326 is P or AX at position 327 is G or RX at position 332 is S or PX at position 333 is E, Q, G or AX at position 334 is T, A, D, G or EX at position 336 is V, T or AX at position 337 is R, S, D or HX at position 338 is S, R, G or AX at position 339 is I, V or TX at position 340 is M or LX at position 341 is E, A, S or DX at position 344 is S or DX at position 345 is R, H, Q, T or PX at position 346 is E, A, T or QX at position 349 is T, P, D or MX at position 350 is Y, D, P or absentX at position 351 is E, K, P, A, S or DX at position 352 is G, D, H or SX at position 354 is T or PX at position 355 is G, N, S, D, E, T or AX at position 356 is T, A, E, V, D, I or RX at position 357 is F or HX at position 359 is D, N, E, G or SX at position 364 is R or SX at position 369 is T or MX at position 371 is N or DX at position 372 is E or DX at position 374 is G, A or SX at position 377 is S or TX at position 378 is G or AX at position 379 is I or VX at position 385 is N, S or YX at position 388 is L or IX at position 389 is D or EX at position 391 is P, L, R, Q, M or AX at position 392 is T or SX at position 394 is S, A or TX at position 396 is I or VX at position 399 is L or FX at position 401 is S, T, A or VX at position 404 is E, D or GX at position 406 is D, Y or EX at position 407 is T, G or SX at position 408 is D, E, R, A or PX at position 409 is H, D, E, F, Y, Q or SX at position 410 is W, G, L or RX at position 411 is H, N or DX at position 412 is G, D, R, A or EX at position 413 is V, L, R, K or AX at position 415 is N, S, Q, T or GX at position 416 is D, G, A, E, S, R, Q or TX at position 417 is V, I or LX at position 419 is L or VX at position 420 is D or EX at position 421 is V, I or LX at position 422 is S or CX at position 423 is R, H, E or TX at position 424 is A or SX at position 427 is A, G, T, R, S, P, Q or absentX at position 428 is M, A, V, L, F, I, P, V or absentX at position 429 is A, P, R, G, S or absentX at position 430 is Y, A or absentX at position 431 is A or absentX at position 432 is A or absentX at position 433 is E or absentX at position 434 is T or absentX at position 444 is A or absentX at position 445 is P or absentX at position 446 is D or absentX at position 447 is S or absentX at position 448 is K or absentX at position 449 is E or absentX at position 450 is N or absentX at position 451 is A or absentX at position 452 is A or absent.

10. The polypeptide according to any of claims 1 to 4, selected from: SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:23, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 122, SEQ ID NO: 128, SEQ ID NO: 131, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 157, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 184 and SEQ ID NO: 192 or a sequence having at least 80% sequence identity to any of these; and wherein the said sequence is optionally substituted at position 187 with an amino acid selected from the group consisting of alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine.

11. The polypeptide according to any of claims 1 to 4, comprising the amino acid sequence of amino acids 2 to 392 of SEQ ID NO:2; amino acids 2 to 342 of SEQ ID NO: 117 or amino acids 2 to 392 of SEQ ID NO: 367 (NylBGLH) or an amino acid sequence having at least 96% sequence identity to any of these.

12. The polypeptide according to any of claims 1 to 4, comprising the amino acid sequence of any of SEQ ID NOs: 117 to 119 or a polypeptide having at least 70% sequence identity thereto; and wherein the arginine at position 187 with reference to SEQ ID NO:1 is substituted with an amino acid selected from the group consisting of alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine.

13. The polypeptide having polyamide hydrolase activity comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:33 or a polypeptide having at least 50% sequence identity to either SEQ ID NO:1 or SEQ ID NO:33 wherein the amino acid at position 187 with reference to SEQ ID NO:1 is substituted by an amino acid selected from the group consisting of alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine; optionally with the proviso that if the amino acid at position 187 with respect to SEQ ID NO:1 is A, K, E or L, then the amino acid at position 181 is A, R, G, or N; and / or the amino acid at position 266 is H, G or M.

14. The polypeptide according to claim 13, comprising:(i) a catalytic triad comprising a serine residue at position 112, a lysine residue at a position 115 and a tyrosine or phenylalanine, optionally a tyrosine residue, at a position 215 with respect to SEQ ID NO:1;(ii) a nitrogen inverting residue at position 170 with respect to SEQ ID NO:1; optionally wherein the nitrogen inverting residue is a tyrosine residue.

15. The polypeptide according to claim 13 or 14, comprising the amino acid sequence of SEQ ID NO:1, SEQ ID NO:33, SEQ ID NOs: 14-21; SEQ ID NO:38, SEQ ID NO:44, SEQ ID NOs:48-50, SEQ ID NOs:68-71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:79-81, SEQ ID NOs:84-86, SEQ ID NO:90, SEQ ID NOs:92-94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO: 101, SEQ ID NO: 104, SEQ ID NO: 109, SEQ ID NO: 110 and SEQ ID NO: 115 or a sequence having at least 80% sequence identity to any of these; and wherein in each case the amino acid at position 187 with reference to SEQ ID NO:1 is substituted with an amino acid selected from the group consisting of alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine; provided that if the amino acid at position 196 is A, then the amino acid at position 190 is A, R, G, or N; and / or the amino acid at position 285 is H, G or M16. The polypeptide according to any of claims 13 to 15, comprising the sequence of amino acids 5 to 402 of SEQ ID NO:379, wherein:X at position 5 is S, A, T or LX at position 8 is Q, P or AX at position 11 is A, P or DX at position 13 is Y or FX at position 16 is A or GX at position 17 is A, E or GX at position 18 is A, P or R X at position 20 is E, Q or R X at position 22 is T, N or H X at position 25 is S or G X at position 29 is P or A X at position 31 is H, Q or D X at position 37 is A or S X at position 38 is H or R X at position 40 is G or S X at position 42 is M, I or L X at position 43 is V or LX at position 45 is S or TX at position 47 is A, V or TX at position 48 is V or IX at position 49 is S or PX at position 52 is P, D or SX at position 53 is V or PX at position 54 is N or AX at position 55 is A or TX at position 57 is G, A, S, L or YX at position 58 is H, E, A or SX at position 59 is A or PX at position 60 is L, V or TX at position 61 is A or VX at position 64 is D or GX at position 65 is A, S or VX at position 66 is I or LX at position 67 is A, T or EX at position 68 is A, T or PX at position 69 is Q or RX at position 70 is L or MX at position 72 is D or GX at position 74 is E or QX at position 75 is Q, R or AX at position 79 is Q or EX at position 80 is T or SX at position 81 is Y or CX at position 83 is D or NX at position 85 is F or LX at position 91 is T, S, G or AX at position 94 is V, L or IX at position 99 is R or AX at position 100 is A or PX at position 104 is A or PX at position 112 is A or SX at position 122 is V or FX at position 126 is V or IX at position 127 is D or EX at position 128 is E, D or QX at position 130 is R or LX at position 134 is A or EX at position 135 is Q or RX at position 137 is V or IX at position 139 is E, H, T or RX at position 140 is Y or HX at position 145 is A, G or EX at position 148 is V or AX at position 153 is S, T or QX at position 154 is G or absentX at position 155 is D or absentX at position 156 is P or absentX at position 157 is N or absentX at position 158 is A or absentX at position 159 is P or absentX at position 160 is R or absentX at position 161 is V or absentX at position 162 is T or absentX at position 163 is V or LX at position 164 is L or QX at position 166 is V or LX at position 170 is Q or EX at position 171 is I or VX at position 172 is S or AX at position 173 is I or VX at position 176 is N or SX at position 183 is A or GX at position 190 is D, A, R, G or NX at position 196 is A, C, D, E, F, G, H, I, L, Q, T or VX at position 197 is T or SX at position 200 is H, D or AX at position 204 is A or EX at position 205 is D or NX at position 206 is T or DX at position 208 is S, R or AX at position 211 is T or AX at position 212 is T or S X at position 216 is D, S or N X at position 218 is S, G, R or A X at position 235 is I or V X at position 236 is V or I X at position 240 is T or S X at position 242 is L or M X at position 243 is R or P X at position 245 is V or A X at position 250 is T, I or V X at position 251 is Y or H X at position 254 is A or S X at position 260 is R or L X at position 268 is T, Q, S or R X at position 269 is T or A X at position 276 is G or A X at position 280 is C or S X at position 285 is N, H, G or M X at position 297 is V, I or H X at position 306 is E, Q or G X at position 307 is D, G, T or A X at position 310 is R or E X at position 311 is R, S or G X at position 312 is V or I X at position 313 is L or M X at position 314 is A, S or E X at position 318 is H or R X at position 319 is E or A X at position 323 is D or Y X at position 324 is K, E or P X at position 325 is G or D X at position 328 is N, S or G X at position 329 is T or A X at position 332 is D, E, G or N X at position 344 is N or D X at position 362 is D or E X at position 364 is L, R, Q or PX at position 369 is I or VX at position 374 is S or TX at position 377 is D or EX at position 379 is Y or DX at position 381 is E, R, D or AX at position 385 is R or GX at position 386 is L or VX at position 388 is N, S or QX at position 389 is G, A or DX at position 390 is I or VX at position 394 is V or IX at position 396 is R or HX at position 400 is A or TX at position 401 is V or MX at position 402 A or absent optionally with the proviso that if the amino acid at position 196 is A, then the amino acid at position 190 is A, R, G, or N; and / or the amino acid at position 285 is H, G or M.

16. The polypeptide according to any of claims 13 to 15, wherein X at position 190 is G or R; particularly G.

17. The polypeptide according to any of claims 13 to 16, wherein X at position 285 is H, G or M; particularly H.

18. The polypeptide according to any of claims 13 to 17, wherein X at position 196 is, C, D, E, F, I, L, Q, or V; particularly I or L.

19. The polypeptide according to any of claims 13 to 17, wherein X at position 196 is A, C, D, G, H, I, L, M, Q, S, T or V; optionally A, C, D, H, I, L, Q, or T; further optionally C, D, H, I, L or Q.

20. The polypeptide of any preceding claim, which is capable of hydrolysing a polyamide polymer or a polyamide oligomer.

21. The polypeptide according to claim 20, wherein the polyamide is polyamide 6 or polyamide 6,6.

22. The polypeptide according to claims 20 or 21 which is capable of hydrolysing a water soluble polyamide 6 oligomer or polyamide 6,6 oligomer.

23. The polypeptide according to any of claims 20 to 22, which hydrolyses a polyamide 6,6 dimer, N- trimer, C-trimer, tetramer, N-pentamer, C-pentamer and / or hexamer or mixtures comprising any two or more of the foregoing.

24. A polynucleotide comprising a polynucleotide sequence encoding the polypeptide of any of claims 1 to 23.

25. An expression vector comprising the polynucleotide of claim 24; optionally wherein the polynucleotide is under the control of a constitutive promoter or an inducible promoter.

26. A host cell comprising the polynucleotide of claim 24, or the expression vector of claim 25.

27. A process for the preparation of a polypeptide according to any one of claims 1 to 23, comprising expressing the polypeptide from a polynucleotide according to claim 24, and recovering the polypeptide.

28. A composition comprising the polypeptide of any one of claims 1 to 19,29. A composition comprising a polynucleotide according to claim 24, an expression vector according to claim 25 or a host cell according to claim 26.

30. A composition according to claim 28 or 29, which is a dried composition.

31. A composition comprising a polypeptide according to any of claims 1 to 12 and a polypeptide according to any of claims 13 to 19.

32. A composition comprising one or more polypeptides according to claims 1 to 19, or a composition according to claim 31 , which is immobilized on a solid support.

33. A process for hydrolysing a polyamide polymer or polyamide oligomer comprising contacting the polyamide polymer or polyamide oligomer with a polypeptide according to any of claims 1 to 19, a composition according to claim 28, a composition comprising a host cell according to claim 26 or a composition according to claim 3 lor 32; optionally wherein the polyamide polymer or polyamide oligomer is a polyamide 6,6 polymer or a polyamide 6,6 oligomer.

34. The process according to claim 33, wherein the polyamide polymer is a polyamide 6,6 polymer and wherein the polyamide 6,6 polymer is partially chemically hydrolysed to provide polyamide 6,6 oligomers which are contacted with a polypeptide according to any of claims 1 to 19, a compositionaccording to claim 28, a composition comprising a host cell according to claim 26 or a composition according to claim 3 lor 32.

35. The process according to claims 33 or 34, wherein the partial chemical hydrolysis provides a composition comprising water soluble polyamide 6,6 oligomers; optionally wherein the chemical hydrolysis provides a mixture of oligomers comprising at least dimers and trimers; optionally comprising at least dimers, trimers, tetramers, pentamers and hexamers.

36. The process according to any of claims 33 to 35, wherein the partial chemical hydrolysis provides a composition additionally comprising insoluble oligomers.

37. The process according to any of claims 33 to 36, wherein the partial chemical hydrolysis is an acid catalysed partial hydrolysis.

38. The process according to claims 33 to 37, which is carried out in an aqueous medium; optionally wherein the optical density of the aqueous medium is reduced following contacting the polyamide polymer or polyamide oligomer with the polypeptide.

39. The process according to any of claims 33 to 38, wherein the polymer or oligomer is a polyamide 6,6 polymer or oligomer and the products of the hydrolysis comprise adipic acid and / or hexamethylene diamine; optionally wherein the adipic acid and / or the hexamethylene diamine are recovered.

40. A process for the preparation of polyamide 6,6, comprising carrying out a process according to any of claims 33 to 39, wherein the polyamide is polyamide 6,6 polymer or polyamide 6,6 oligomer, recovering adipic acid and / or hexamethylene diamine monomers resulting from the process and synthesising a polyamide 6,6 polymer from the recovered adipic acid and / or the hexamethylene diamine monomers.

41. A method for increasing the polyamide 6,6 hydrolase activity of an amidase enzyme comprising:(a) identifying the amino acid sequence of a polypeptide having either polyamide 6,6 hydrolase activity or 6-aminohexanoate dimer hydrolase activity, wherein the amidase lacks:(i) a glycine residue at position 181 or a histidine residue at position 266, or lacks both a glycine residue at position 181 and a histidine residue at position 266 with reference to SEQ ID NO:1;(ii) an arginine residue at position 181 or a histidine residue at position 266, or lacks both an arginine residue at position 181 and a histidine residue at position 266 with reference to SEQ ID NO:1;or(iii) an alanine residue at position 181 or a histidine residue at position 266, or lacks both an alanine residue at position 181 and a histidine residue at position 266 with reference to SEQ ID NO:1(b) substituting the amino acid at position 181 with a glycine residue and / or substituting the amino acid at position 266 with a histidine residue; or substituting the amino acid at position 181 with an arginine residue and / or substituting the amino acid at position 266 with a histidine residue; or substituting the amino acid at position 181 with an alanine residue and / or substituting the amino acid at position 266 with a histidine residue; wherein positions are relative to the amino acid sequence of SEQ ID NO:1; and optionally if the amidase sequence has other than an alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine or valine at position 187 in relation to SEQ ID NO:1; then(c) replacing the amino acid at position 187 with respect to SEQ ID NO:1 with an amino acid selected from the group consisting of alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine.

42. A method for increasing the polyamide 6,6 hydrolase activity of an amidase enzyme comprising:(a) identifying the amino acid sequence of a polypeptide having either polyamide 6,6 hydrolase activity or 6-aminohexanoate dimer hydrolase activity, wherein the amidase sequence has an amino acid other than an alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine at position 187 in relation to SEQ ID NO:1; and(b) replacing the amino acid at position 187 with respect to SEQ ID NO:1 with an amino acid selected from the group consisting of alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, glutamine, threonine and valine; and optionally(i) if the sequence does not comprise a glycine residue at position 181 with respect to SEQ ID NO:1, then replacing the amino acid with a glycine;(ii) if the sequence does not comprise an alanine residue at position 181 with respect to SEQ ID NO:1, then replacing the amino acid with an alanine; or(iii) if the sequence does not comprise an arginine residue at position 181 with respect to SEQ ID NO:1, then replacing the amino acid with an arginine; and(iv) if the sequence does not comprise a histidine residue at position 266 with respect to SEQ ID NO:1, replacing the amino acid at position 266 with a histidine residue.

43. The method according to any of claims 41 or 42, wherein the identified sequence of the polypeptide having either polyamide 6,6 hydrolase activity or 6-aminohexanoate dimer hydrolase activity has at least one of the following additional features:(i) an amino acid sequence of at least 50% identity to SEQ ID NO:1;(ii) a catalytic triad comprising a serine residue at position 112, a lysine residue at a position 115 and a tyrosine or phenylalanine residue at a position 215;(iii) a nitrogen inverting residue at position 170; optionally wherein the nitrogen inverting residue is a tyrosine residue.

44. The method according to any of claims 41 to 43, wherein polyamide 6,6 hydrolase activity increased is one or more of polyamide 6,6 hexamer hydrolase, polyamide 6,6 N-pentamer hydrolase, polyamide 6,6 C-pentamer hydrolase, polyamide 6,6 tetramer hydrolase, polyamide 6,6 N-trimer hydrolase, polyamide 6,6 C-trimer hydrolase or polyamide 6,6 dimer hydrolase activity.

45. The method according to any of claims 41 to 44, wherein the polypeptide having either polyamide 6,6 hydrolase activity or 6-aminohexanoate dimer hydrolase activity identified in step (a) has at least 60% sequence identity to a sequence selected from: SEQ ID NO:1, SEQ ID NO:14 to SEQ ID NO:21 and SEQ ID NO:33.

46. The method according to any of claims 41 to 43, wherein the polypeptide has increased hydrolytic activity towards polyamide 6,6 oligomers having terminal amino groups and / or increased activity towards polyamide 6,6 oligomers having terminal carboxylate groups.

47. A polypeptide prepared according to any one of claims 41 to 44.

48. An assay for indicating a hydrolytic activity of an amidase enzyme towards a polyamide oligomer, wherein the hydrolytic activity releases at least one diamine monomer; the process comprising:contacting the amidase enzyme with the polyamide oligomer; to release a diamine monomer, with a putrescine oxidase in the presence of oxygen, to produce an oxidised diamine monomer and a peroxide; contacting the peroxide with a peroxidase in the presence of peroxidase substrate; and detecting the activity of the peroxidase on the peroxidase substrate to indicate the activity of the amidase enzyme towards a polyamide oligomer.

49. The assay according to claim 48, wherein the putrescine oxidase is Rhodococcus erythropolis putrescine oxidase.

50. The assay according to claims 48 or 49, wherein the peroxide is H2O2.

51. The assay according to any of claims 48 to 50, wherein the peroxidase substrate is a colourogenic substrate and the activity of the peroxidase on the peroxidase substrate is detected by detecting the colour of the product; optionally wherein the colourogenic substrate is 2,2'-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid (ABTS).

52. The assay according to any of claims 48 to 51, wherein the peroxidase is HRP.

53. The assay according to any of claims 48 to 52, wherein the polyamide oligomer is 6-[(6- aminohexyl) amino] -6-oxo-hexanoic acid.

54. A method for monitoring the progress of a polyamide hydrolysis to produce at least one diamine product, the method comprising detecting the presence or the quantity of the diamine product using an assay according to any of claims 48 to 53.

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