Polyamide-degrading polypeptides and methods of use

Abstract

Various embodiments relate generally to the technical field of enzyme technology and specifically relates to enzymes having depolymerisation activity and to nucleic acids encoding those as well as method of the manufacture of said enzymes. In particular, the enzymes are nylon hydrolases. Further encompassed are methods and uses of these enzymes.

Description

POLYAMIDE-DEGRADING POLYPEPTIDES AND METHODS OF USECROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority of Singapore Patent Application No. 10202402624V filed 26 August 2024, the content of which being hereby incorporated by reference in its entirety for all purposes.TECHNICAL FIELD

[0002] The present invention lies in the technical field of enzyme technology and specifically relates to polypeptides having depolymerisation activity and to nucleic acids encoding those as well as methods of the manufacture of said enzymes. Further encompassed are methods and uses of these enzymes.BACKGROUND

[0003] Unsustainable growth in plastic waste has fueled urgency to develop solutions for a more sustainable future. The exponential increase in plastic waste, particularly from synthetic polymers such as polyamides, has emerged as a critical environmental challenge. The persistence and non- biodegradable nature of plastics like polyamide 6 (PA 6) and polyamide 6,6 (PA 6,6) contribute significantly to environmental pollution and microplastic accumulation. Consequently, there is an urgent need to develop innovative, sustainable, and scalable solutions to address plastic waste. Among these, bio-based approaches, such as enzymatic degradation, are particularly attractive due to their lower energy requirements, use of non-toxic reaction conditions, and potential for high specificity and environmental compatibility. Enzyme-based depolymerization can be integrated into circular economy models for plastic recycling, enabling recovery and reuse of monomers under mild and environmentally friendly conditions.

[0004] To date, only two enzymes have been reported to significantly degrade PA 6 and PA 6,6 polymers: manganese peroxidase (MnP), produced by certain white rot fungi, and the N-terminal threonine hydrolase NylC. While MnP operates via oxidative mechanisms with limited substrate specificity and high dependence on redox conditions, NylC is a well-characterized hydrolase that catalyses the hydrolysis of short oligomers as well as polymeric forms of PA 6 and PA 6,6. Furthermore, NylC has been engineered to significantly enhance its thermal stability. Furthermore, engineering efforts have led to variants of NylC with improved thermostability, thereby enhancing its industrial applicability. However, despite these advancements, the enzymatic activity of NylC remains insufficient for efficient plastic recycling at scale.

[0005] Therefore, there is still a need in the art for providing novel and alternative enzymes capable of efficiently depolymerizing polyamide materials to address the drawbacks of existing enzymes. In particular, there is a need in the art to provide novel and alternative hydrolases with enhanced enzymatic activity and methods of their use to enable more effective degradation and recycling ofpolyamide waste, thereby offering a viable and sustainable solution to the global plastic pollution crisis.SUMMARY

[0006] In one aspect, there is provided a polypeptide having depolymerisation activity, comprising or consisting of: (i) an amino acid sequence as set forth in SEQ ID NO:15 (NylCA); (ii) an amino acid sequence that shares at least 50% sequence identity with the amino acid sequence set forth in SEQ ID NO:15 over its entire length; or (iii) a functional fragment of any one of (i)-(ii) having depolymerisation activity, wherein said polypeptide comprises: an amino acid residue Y at the position corresponding to position 146 of SEQ ID NO:15; an amino acid residue K at the position corresponding to position 189 of SEQ ID NO:15; an amino acid residue N at the position corresponding to position 219 of SEQ ID NO: 15; amino acid residues GNT at the positions corresponding to positions 265-267 of SEQ ID NO:15; amino acid residues DGD at the positions corresponding to positions 306-308 of SEQ ID NO:15; and at least one amino acid substitution at a position within ±5 amino acid residues of positions 146, 189, 219, 265-267 and / or 306-308, wherein position numbering is relative to the amino acid sequence set forth in SEQ ID NO:15.

[0007] In various embodiments, the at least one amino acid substitution is at one or more positions selected from: (i) positions 141 -145 and / or 147-151 ; (ii) positions 184-188 and / or 190-194; (iii) positions 214-218 and / or 220-224; (iv) positions 260-264 and / or 268-272; and (v) positions 301 -305 and / or 309-313, wherein all positions are relative to SEQ ID NO:15.

[0008] In various embodiments, the polypeptide comprises at least one amino acid substitution at one or more positions selected from 142, 144, 145, 148-150, 184, 186, 187, 190, 191 , 192, 193, 214, 215, 216, 217, 218, 220, 221 , 223, 224, 260, 261 , 262, 263, 264, 269, 270, 271 , 272, 303, 304, 305, 309, 312, and 313 of SEQ ID NO:15.

[0009] In various embodiments, the polypeptide comprises: (i) an amino acid sequence set forth in any one of SEQ ID NO:24-36 at positions corresponding to positions 141 -151 of SEQ ID NO:15; (ii) an amino acid sequence set forth in any one of SEQ ID NO:38-50 at positions corresponding to positions 184-194 of SEQ ID NO:15, (iii) an amino acid sequence set forth in any one of SEQ ID NO:51-63 at positions corresponding to positions 214-224 of SEQ ID NO:15, (iv) an amino acid sequence set forth in any one of SEQ ID NO:66-78 at positions corresponding to positions 260-272 of SEQ ID NO:15, and (v) an amino acid sequence set forth in any one of SEQ ID NO:79-90 at positions corresponding to positions 301-313 of SEQ ID NO:15.

[0010] In various embodiments, the polypeptide comprises or consists of an amino acid sequence set forth in any one of SEQ ID NO:1-13, or functional variants or fragments thereof.

[0011] In various embodiments, the polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 5, 12 or 13, or functional variants or fragments thereof.

[0012] In another aspect there is provided a nucleic acid molecule encoding the polypeptide disclosed herein, preferably the nucleic acid molecule is comprised in a vector.

[0013] In another aspect there is provided a host cell comprising the nucleic acid molecule disclosed herein.

[0014] In another aspect there is provided a method for producing the polypeptide disclosed herein, comprising culturing a host cell disclosed herein under conditions that allow expression of the polypeptide, and isolating said polypeptide from the host cell or culture medium.

[0015] In another aspect there is provided a composition comprising the polypeptide disclosed herein, wherein the composition is formulated for use in depolymerising a polyamide material.

[0016] In another aspect there is provided a solid support material comprising the polypeptide disclosed herein immobilized thereon.

[0017] In various embodiments, the solid support material comprises a particulate polymer resin, optionally selected from agarose, polystyrene, polymethacrylate, or a magnetic resin, and is suitable for use in a chromatography column, bioreactor or magnetic separation system.

[0018] In another aspect there is provided a bioreactor comprising the solid support material disclosed herein, configured for continuous or batch enzymatic depolymerisation of polyamide material.

[0019] In another aspect there is provided a method of degrading polyamide material, the method comprising contacting the polyamide material with the polypeptide disclosed herein, the nucleic acid disclosed herein, the host cell disclosed herein, the composition disclosed herein, orthe solid support material disclosed herein under conditions suitable for depolymerisation and degradation of the polyamide material.

[0020] In various embodiments, the polyamide material is selected from the group consisting of polyamide-6 (PA6), polyamide-6,6 (PA66), aramids, caprolactam-containing polymers, and oligomers thereof.

[0021] In another aspect there is provided the use of the polypeptide disclosed herein, the nucleic acid disclosed herein, the host cell disclosed herein, the composition disclosed herein, or the solidsupport material disclosed herein for depolymerisation of a polyamide material, or in a process for degrading polyamide material, or recycling or upcycling polyamide material.

[0022] In another aspect there is provided a method of producing a degradation product from a polyamide material, the method comprising contacting the polyamide material with the polypeptide disclosed herein, the nucleic acid disclosed herein, the host cell disclosed herein, the composition disclosed herein, or the solid support material disclosed herein under suitable conditions for depolymerisation; and recovering the degradation product comprising one or more monomers or oligomers.

[0023] In another aspect there is provided a method of recycling polyamide material comprising passing the polyamide material through the bioreactor disclosed herein under conditions suitable for enzymatic degradation.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Various embodiments will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings.

[0025] FIG. 1 shows a phylogenetic tree of hydrolases (square boxes and round dots represents new hydrolases from this work).

[0026] FIG. 2A illustrates a fluorogenic assay for detecting acti ity. The peptidases / protease / peptide assays are used to screen as a proxy the breakage of amide bonds like that found in nylon or related polyamides. Here, a peptide (polyamide) substrate is labelled with a fluorophore on one end and a dark quencher on the other. In the intact state, fluorescence is quenched due to proximity between the fluorophore and quencher. Upon cleavage of the peptide (polyamide) by a hydrolase, the fluorophore is separated from the quencher, resulting in a detectable fluorescent signal upon excitation. This provides a readout of enzymatic activity based on amide bond hydrolysis; and FIG. 2B shows a bar graph comparing normalized activity to known NylC references (NylC and NylCa) for a peptide assay and caprolactam-MAFC assay. Each ID is a tag-gene combination - Table 3.

[0027] FIG. 3 shows the 24 hour protease activity of Nyl_FT5 and Nyl_FH5 compared to NylC and NylCa, at 4 different temperatures. For each temperature, the individual column from left to right represents the reading for Nyl_FT5 (ID 2413), Nyl_FH5 (ID 3612), NylC, NylCa and Blank, respectively. At all temperatures, Nyl_FH5 showed the highest reading.

[0028] FIG. 4 shows the sequence identity of residues surrounding the active-site in nylon hydrolases. The active site residues in NylCA are indicated with stars, with numbering corresponding to their positions in the NylCA sequence. For each active-site residue, the neighbouring 5 residuepositions (i-5 to i+5) were analysed. Positions conserved among the reference nylon hydrolases and the newly discovered Nyl_FH5, Nyl_FT5, and Nyl_FT6 are highlighted with a shaded background.DETAILED DESCRIPTION

[0029] The following detailed description refers to, by way of illustration, specific details and embodiments in which the invention may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention. Other embodiments may be utilized and structural and logical changes may be made without departing from the scope of the invention. Embodiments described below in context of the polypeptides are analogously valid for the respective nucleic acid molecules, host cells, methods, uses and vice versa. The various embodiments are not necessarily mutually exclusive, as some embodiments can be combined with one or more other embodiments to form new embodiments.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "comprises" means "includes." In case of conflict, the present specification, including explanations of terms, will prevail. "About”, as used herein in connection with numerical values refers to the referenced numerical value ±10% or ±5%.

[0031] The present invention is based on the inventors’ identification of novel polypeptides having depolymerisation activity for degrading polyamide materials or substrates. Specifically, the present invention addresses the need to provide a new collection of polyamide-degrading enzymes with depolymerisation activity identified through targeted genome mining and solubility-optimized expression. Notably, some novel polypeptides show high catalytic performance despite low sequence identity to previously characterized NylC enzymes, revealing unexpected structure-function diversity. Detailed analysis confirmed the conservation of catalytic residues while uncovering novel sequence variation in residues surrounding the active site (FIG. 4). These enzymes represent a significant advancement in the field of bio-based plastic recycling, offering promising candidates for further development into efficient industrial biocatalysts for polyamide depolymerisation.

[0032] Based on the above findings, the invention, in a first aspect, covers polypeptides having depolymerisation activity comprising, or consisting of an amino acid sequence as set forth in SEQ ID NO:15 (NylCA), comprising at least one amino acid substitution located at amino acid residues neighbouring conserved active site residues, or functional variants or fragments thereof.

[0033] In various embodiments, the polypeptide may be termed as a polyamide-degrading enzyme, comprising at least one amino acid substitution located at amino acid residues neighbouring active site residues of NylCA, with the proviso that said polypeptide is not NylC, NylCA, NylCa-TS, NylCp2,NylCp2-TS, NylCK, NylCk-TS, Tt-NylC or M-NylC having the amino acid sequence set forth in SEQ ID NO:14-22, respectively.

[0034] Herein, the terms “peptide”, “polypeptide", “protein”, and “enzyme” refer to a chain of amino acids linked by peptide bonds, regardless of the number of amino acids forming said chain.

[0035] As used herein, the term “polyamide-degrading enzyme” refers to any polypeptide capable of catalysing the cleavage of amide bonds within polyamide materials, including synthetic polymers such as nylon. This includes enzymes that exhibit depolymerisation activity. Polyamide-degrading enzymes encompass NylC hydrolases, which are bacterial enzymes originally characterized for their ability to hydrolyse nylon-6 oligomers, as well as functional variants and engineered derivatives such as thermostable forms (e.g., NylCa) and NylC-like enzymes bearing substitutions near conserved catalytic residues. The term further includes other hydrolases, from natural or synthetic sources, that exhibit similar depolymerisation activity toward polyamides, as well as any polypeptide, regardless of sequence origin or classification, that demonstrates the ability to degrade polyamide substrates.

[0036] The term “amino acid”, as used herein refers to natural and / or unnatural or synthetic amino acids, including both the D and L optical isomers, amino acid analogues (for example norleucine is an analogue of leucine) and derivatives known in the art. The term "natural amino acid”, as used herein, relates to the 20 naturally occurring L-amino acids, namely Gly (G), Ala (A), Vai (V), Leu (L), lie (I), Phe (F), Cys (C), Met (M), Pro (P), Thr (T), Ser (S), Glu (E), Gin (Q), Asp (D), Asn (N), His (H), Lys (K), Arg (R), Tyr (Y), and Trp (W). Generally, in the context of the present application, the polypeptides are shown in the N- to C-terminal orientation. All amino acid residues are generally referred to herein by reference to their one letter code and, in some instances, their three letter code. This nomenclature is well known to those skilled in the art and used herein as understood in the field. As a person skilled in the art would appreciate, amino acids can be categorized in different classes depending upon the chemical and physical properties of the amino acid residue. Amino acids may be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala, Vai, Leu, lie, Pro, Phe, Trp, Met; (2) uncharged polar: Gly, Ser, Thr, Cys, Tyr, Asn, Gin; (3) acidic: Asp, Glu; and (4) basic: Lys, Arg, His. Alternatively, naturally occurring residues may be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, lie; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. As one of skill in the art would appreciate, amino acids can be categorized in different classes depending upon the chemical and physical properties of the amino acid residue. Typically, hydrophobic amino acids can be further classified as having an aliphatic side chain or an aromatic side chain. Aliphatic amino acids and aromatic amino acids are known one of skill in the art. Typically, aliphatic amino acids have a side chain containing hydrogen and carbon atoms. Examples of aliphatic amino acids include alanine, isoleucine, proline, and valine. Typically, aromatic amino acids contain a side chain containing anaromatic ring. Examples of aromatic amino acids include phenylalanine, tyrosine, histidine and tryptophan.

[0037] The term “amino acid substitution", as used herein, refers to any substitution of an amino acid residue at a position corresponding to the reference NylCA sequence, for example, the amino acid sequence set forth in SEQ ID NO:15. The substitution may be a conservative and / or non-conservative substitution. The term "conservative amino acid substitution" means the exchange (substitution) of one amino acid residue for another amino acid residue, where such exchange does not lead to a considerable change in the polarity or charge or size at the position of the exchanged amino acid, e.g. the exchange of a nonpolar amino acid residue for another nonpolar amino acid residue. Conservative amino acid substitutions in the context of the invention encompass, for example, G=A=S, l=V=L=M, D=E, N=Q, N=Q=S=T, K=R, K=R=H, Y=F=W, S=T, S=T=C, G=A=I=V=L=M=Y=F=W=P=S=T.

[0038] As used herein, the term "depolymerisation activity" refers to the enzymatic activity of a NylC hydrolase that catalyses the cleavage of amide bonds within polymeric or oligomeric substrates, particularly synthetic polyamides. This activity involves the hydrolytic breakdown of high molecular weight polymer chains into smaller oligomers or monomeric units through scission of non-peptidic amide linkages. In the context of the present disclosure, the term encompasses depolymerisation resulting in monomers as well as low-molecular-weight oligomers, including dimers, trimers, tetramers, pentamers, and hexamers, derived from the polymeric or oligomeric substrate. The depolymerisation activity of NylC hydrolase enables the degradation of substrates such as 6-aminohexanoic acid oligomers or caprolactam-based polymers, mimicking peptide bonds yet comprising non-natural synthetic structures. The depolymerisation activity may occur in an endo-type or exo-type manner, and does not require the presence ofterminal amino or carboxyl groups on the substrate. Such activity is of particular relevance for the biodegradation or bioconversion of synthetic plastic waste materials.

[0039] The polypeptides of the present invention may have depolymerisation activity at 60 % or more, preferably 70 % or more, more preferably 80% or more, relative to a reference enzyme, or parental polypeptide sequence. In various embodiments, the polypeptides of the present invention exhibit enhanced depolymerisation activity. This means that the polypeptides are capable of performing depolymerisation at 1 10% or more, preferably 125% or more, and more preferably 150% or more, relative to the depolymerisation activity of a reference enzyme, such as a wild-type NylC or parental polypeptide comprising the amino acid sequence of SEQ ID NO:14 or 15.

[0040] In various embodiments, the polypeptide disclosed herein may exhibit one or more improved enzyme activity, such as depolymerisation and protease activities. The term “improved enzyme property” refers to any enzyme property made better or more desirable for a particular purpose as compared to that property found in a reference enzyme. For the polypeptides disclosed herein, the comparison is generally made to a reference hydrolase which does not contain the particular substitutions which improve enzyme activity.

[0041] In various embodiments, the polypeptides disclosed herein may exhibit improved or increased depolymerisation activity relative to NylC (SEQ ID NO:14) or NylCA (SEQ ID NO:15).

[0042] In various embodiments, the polypeptides disclosed herein may exhibit amide or polyamide hydrolytic activity. As used herein, the term "amide or polyamide hydrolytic activity" may refer to the enzymatic ability to hydrolyse amide bonds, including peptide bonds in natural polypeptides or proteins, and non-peptidic amide linkages in synthetic polyamides or oligomers thereof. In various embodiments, the presence and extent of such activity may be conveniently assessed using a protease-based assay, for example by monitoring the hydrolysis of a proteinaceous or peptide-based substrate such as fluorescein isothiocyanate (FITC)-labelled casein. Hydrolysis of the test substrate, evidenced by the generation of smaller peptide fragments or free amino acids, serves as a proxy measure for the enzyme’s capability to cleave non-peptidic amide bonds in synthetic polyamide materials (e.g., nylon). Accordingly, such assay results may be indicative of the enzyme’s depolymerisation potential, and the presence of amide or polyamide hydrolytic activity in a NylC hydrolase may reflect its utility in diverse biotechnological applications, including polymerdegradation, peptide processing, and protein modification.

[0043] In various embodiments, the polypeptides disclosed herein having depolymerisation activity are derived from one or more bacterial species selected from the group consisting of Agromyces spp. (e.g., Agromyces mediolanus), Sediminivirga spp. (e.g., Sediminivirga luteola), Nonomuraea spp. (e.g., Nonomuraea turkmeniaca), Microbacterium spp. (e.g., Microbacterium sp. JB110 or Microbacterium testaceum), Brevibacterium spp. (e.g., Brevibacterium aurantiacum), Cryptosporangium spp. (e.g., Cryptosporangium aurantiacum), Leucobacter spp., Prauserella spp. (e.g., Prauserella flava), and Rhodococcus spp. (e.g., Rhodococcus qingshengii). The amino acid sequences of the hydrolases identified from these species are disclosed herein, each exhibiting distinct catalytic motifs and active site residues. These sequence-identified enzymes constitute novel hydrolases distinct from previously reported nylon oligomer hydrolases and provide an expanded repertoire of biocatalysts for enzymatic polyamide degradation and recycling applications.

[0044] In various embodiments, the polypeptide may be in isolated form and more specifically, may be an isolated polypeptide. “Isolated”, as used herein, relates to the polypeptide in a form where it has been at least partially separated from other cellular components it may naturally occur or associate with. The polypeptide may be a recombinant polypeptide, i.e. polypeptide produced in a genetically engineered organism that does not naturally produce said polypeptide. Both native and recombinant polypeptides may be post-translationally modified by N-linked glycosylation.

[0045] In various embodiments, the polypeptide disclosed herein further comprises one or more peptide or protein tags. The tag may be located at the N-terminus, C-terminus, or at an internal site, and is expressed as part of a continuous single polypeptide chain. Suitable tags include, but are notlimited to, polyhistidine (His) tags for affinity purification, hemagglutinin (HA) tags for immunological detection, maltose-binding protein (MBP) tags for enhancing solubility and stability, glutathione S- transferase (GST) tags for purification and immobilization, and thioredoxin (TRX) tags optionally linked to a Tobacco Etch Virus (TEV) protease cleavage site to allow post-purification removal. The incorporation of such tags may facilitate recombinant production, simplify downstream purification, improve folding and thermostability, and allow immobilization of the polypeptide on solid supports for use in bioreactors or recycling applications.

[0046] In various embodiments, the polypeptide disclosed herein comprises at least one amino acid substitution located within ±5 amino acid residues of one or more active sites relative to NylCA (SEQ ID NO:15). In particular, the at least one amino acid substitution is positioned at or adjacent to amino acid residues that flank (immediately flank) conserved residues of active site residues. For illustrative purposes, if active site residues are denoted as position i, then the substitution may occur at any position from i-5 to i+5. The numbering of amino acid residues is made with reference to the amino acid sequence set forth in SEQ ID NO:15.

[0047] The active site of the polypeptide refers to the region in which substrate binding and catalysis occur, encompassing both the catalytic site (the functional core responsible for the chemical reaction underlying depolymerisation activity) and additional residues that facilitate substrate recognition, positioning, and stabilization. The catalytic site is a subset of the active site comprising key catalytic residues that may directly participate in the hydrolysis of bonds within the polymeric material. These catalytic residues contribute to the enzymatic activity. In various embodiments, the polypeptide comprises the active site residues indicated in FIG. 4 with stars.

[0048] In various embodiments, the polypeptide comprises the catalytic amino acid residue Y at the position corresponding to position 146; the catalytic amino acid residue K at the position corresponding to position 189; the catalytic amino acid residue N at the position corresponding to position 219. In various embodiments, the polypeptide comprises the catalytic amino acid residues G at the position corresponding to position 265; the catalytic amino acid residues N at the position corresponding to position 266; and the catalytic amino acid residues T at the position corresponding to position 267, thus forming the catalytic residues / motif GNT at the positions corresponding to positions 265-267. In various embodiments, the polypeptide comprises the catalytic amino acid residues D at the position corresponding to position 306; the catalytic amino acid residues G at the position corresponding to position 307; and the catalytic amino acid residues D at the position corresponding to position 308, thus forming the catalytic residues / motif DGD at the positions corresponding to positions 306-308. The position numbering of the amino acid residues and substitutions thereof are in accordance with the amino acid residue numbering of SEQ ID NO:15. It has been found that these amino acid residues and motifs are conserved and important for the depolymerisation activity of the polypeptide. In preferred embodiments, the polypeptides thus comprise all of the above indicated residues at the given or corresponding positions.

[0049] All amino acid residues are generally referred to herein by reference to their one letter code and, in some instances, theirthree letter code. This nomenclature is well known to those skilled in the art and used herein as understood in the field.

[0050] In various embodiments, the polypeptide comprises at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50 amino acid substitutions at positions within ±5 amino acid residues of the catalytic residues and motifs. In various embodiments, the at least one amino acid substitution within ±5 amino acid residues of the catalytic residues and motifs may be at one or more positions selected from: (I) 141-145 and / or 147-151 ; (ii) 184-188 and / or 190-194; (iii) 214-218 and / or 220-224; (iv) 260-264 and / or 268-272; and (v) 301 -305 and / or 309-313, wherein position numbering is relative to the amino acid sequence set forth in SEQ ID NO:15.

[0051] In various embodiments, the polypeptide comprises an amino acid residue Y at the position corresponding to position 146, and at least one amino acid substitution at any position of 141 -145 and / or 147-151 of SEQ ID NO:15. In various embodiments, the polypeptide comprises at least one amino acid substitution at one or more positions selected from 142, 144, 145 and 148-150. In various embodiments, the polypeptide comprises an amino acid residue S at the position corresponding to position 141 ; an amino acid residue A at the position corresponding to position 143; an amino acid residue D at the position corresponding to position 147; and an amino acid residue R at the position corresponding to position 151 of SEQ ID NO:15. In various embodiments, the polypeptide comprises an amino acid sequence SXAXXYDXXXR at positions corresponding to positions 141-151 of SEQ ID NO:15, where X is any amino acid residue. In various embodiments, the polypeptide comprises an amino acid sequence of any one of SEQ ID NO: 24-36 at positions corresponding to positions 141- 151 of SEQ ID NO:15.

[0052] In various embodiments, the polypeptide comprises an amino acid residue K at the position corresponding to position 189, and at least one amino acid substitution at any position of 184-188 and / or 190-194 of SEQ ID NO:15. In various embodiments, the polypeptide comprises at least one amino acid substitution at one or more positions selected from 184, 186, 187, 190, 191 , 192 and 193. In various embodiments, the polypeptide comprises an amino acid residue A at the position corresponding to position 185; an amino acid residue G at the position corresponding to position 188; and an amino acid residue R at the position corresponding to position 194. In various embodiments, the polypeptide comprises an amino acid sequence XAXXGKXXXXR at positions corresponding to positions 184-194 of SEQ ID NO:15, where X is any amino acid residue. In various embodiments, the polypeptide comprises an amino acid sequence of any one of SEQ ID NO: 38-50 at positions corresponding to positions 184-194 of SEQ ID NO:15.

[0053] In various embodiments, the polypeptide comprises an amino acid residue N at the position corresponding to position 219, and at least one amino acid substitution at any position of 214-218 and / or 220-224 of SEQ ID NO:15. In various embodiments, the polypeptide comprises at least one amino acid substitution at one or more positions selected from 214, 215, 216, 217, 218, 220, 221 , 223, and 224. In various embodiments, the polypeptide comprises an amino acid residue G at the position corresponding to position 222. In various embodiments, the polypeptide comprises an amino acid sequence XXXXXNXXGXX at position corresponding to position 214-224 of SEQ ID NO:15, where X is any amino acid residue. In various embodiments, the polypeptide comprises an amino acid sequence of any one of SEQ ID NO: 51-63 at positions corresponding to positions 214-224 of SEQ ID NO:15.

[0054] In various embodiments, the polypeptide comprises amino acid residues GNT at the positions corresponding to positions 265-267, and at least one amino acid substitution at any position of 260- 264 and / or 268-272 of SEQ ID NO:15. In various embodiments, the polypeptide comprises at least one amino acid substitution at one or more positions selected from 260, 261 , 262, 263, 264, 269, 270, 271 and 272. In various embodiments, the polypeptide comprises an amino acid residue T at the position corresponding to position 268. In various embodiments, the polypeptide comprises an amino acid sequence XXXXXGNTTXXXX at position corresponding to position 260-272 of SEQ ID NO:15, where X is any amino acid residue. In various embodiments, the polypeptide comprises an amino acid sequence of any one of SEQ ID NO: 66-78 at positions corresponding to positions 260-272 of SEQ ID NO:15.

[0055] In various embodiments, the polypeptide comprises amino acid residues DGD at the positions corresponding to positions 306-308, and at least one amino acid substitution at any position of 301-305 and / or 309-313 of SEQ ID NO:15. In various embodiments, the polypeptide comprises at least one amino acid substitution at one or more positions selected from 303, 304, 305, 309, 312, and 313. In various embodiments, the polypeptide comprises an amino acid residue F at the position corresponding to position 301 ; an amino acid residue H at the position corresponding to position 302; an amino acid residue L at the position corresponding to position 310; and an amino acid residue F at the position corresponding to position 311. In various embodiments, the polypeptide comprises an amino acid sequence FHXXXDGDXLFXX at positions corresponding to positions 301-313 of SEQ ID NO:15, where X is any amino acid residue. In various embodiments, the polypeptide comprises an amino acid sequence of any one of SEQ ID NO: 79-90 at positions corresponding to positions 301- 313 of SEQ ID NO:15.

[0056] In various embodiments, the at least one amino acid substitution is at one or more positions selected from: (i) positions 141 -145 and 147-151 ; (ii) positions 184-188 and 190-194; (iii) positions 214-218 and 220-224; (iv) positions 260-264 and 268-272; and (v) positions 301 -305 and 309-313, wherein all positions are relative to SEQ ID NO: 15.

[0057] In various embodiments, the polypeptide comprises at least one amino acid substitution at one or more positions selected from 142, 144, 145, 148-150, 184, 186, 187, 190, 191 , 192, 193, 214, 215, 216, 217, 218, 220, 221 , 223, 224, 260, 261 , 262, 263, 264, 269, 270, 271 , 272, 303, 304, 305, 309, 312, and 313 of SEQ ID NO:15.

[0058] In various embodiments, the polypeptide comprises an amino acid sequence: (i) SXAXXYDXXXR at position corresponding to position 141 -151 , (ii) XAXXGKXXXXR at position corresponding to position 184-194, (iii) XXXXXNXXGXX at position corresponding to position 214- 224, (iv) XXXXXGNTTXXXX at position corresponding to position 260-272, and (v) FHXXXDGDXLFXX at position corresponding to position 301-313, where X is any amino acid residue, and wherein all positions are relative to SEQ ID NO:15.

[0059] In various embodiments, the polypeptide comprises: (i) an amino acid sequence set forth in any one of SEQ ID NO:24-36 at positions corresponding to positions 141-151 ; (ii) an amino acid sequence set forth in any one of SEQ ID NO:38-50 at position corresponding to position 184-194; (iii) an amino acid sequence set forth in any one of SEQ ID NO:51-63 at position corresponding to position 214-224; (iv) an amino acid sequence set forth in any one of SEQ ID NO:66-78 at position corresponding to position 260-272; and (v) an amino acid sequence set forth in any one of SEQ ID NO:79-90 at position corresponding to position 301-313, wherein all positions are relative to SEQ ID NO:15.

[0060] In various embodiments, the at least one amino acid substitution is selected from those listed in the below Table 1 , with amino acid (AA) position numbering relative to WT-NylCA (SEQ ID NO:15).

[0061] Table 1 : Amino Acid Substitutions

[0062] In various embodiments, the polypeptide disclosed herein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 1 (Nyl_FH1) or a functional variant or fragment thereof. In various embodiments, the polypeptide disclosed herein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 1 or a functional variant or fragment thereof and comprises the amino acid sequence set forth in SEQ ID NO: 24 at positions corresponding to positions 141-151 , the amino acid sequence set forth in SEQ ID NO: 38 at positions corresponding to positions 184-194, the amino acid sequence set forth in SEQ ID NO: 52 at positions corresponding to positions 214-224, the amino acid sequence set forth in SEQ ID NO: 66 at positions corresponding to positions 260-272, and the amino acid sequence set forth in SEQ ID NO: 80 at positions corresponding to positions 301- 313, wherein all positions are relative to SEQ ID NO:15.

[0063] In various embodiments, the polypeptide disclosed herein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 2 (Nyl_FH2) or a functional variant or fragment thereof. In various embodiments, the polypeptide disclosed herein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 2 or a functional variant or fragment thereof and comprises the amino acid sequence set forth in SEQ ID NO: 25 at positions corresponding to positions 141-151 , the amino acid sequence set forth in SEQ ID NO: 39 at positions corresponding to positions 184-194, the amino acid sequence set forth in SEQ ID NO: 53 at positions corresponding to positions 214-224, the amino acid sequence set forth in SEQ ID NO: 67 at positions corresponding to positions 260-272, and the amino acid sequence set forth in SEQ ID NO: 81 at positions corresponding to positions 301 - 313, wherein all positions are relative to SEQ ID NO:15.

[0064] In various embodiments, the polypeptide disclosed herein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 3 (Nyl_FH3) or a functional variant or fragment thereof. In various embodiments, the polypeptide disclosed herein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 3 or a functional variant or fragment thereof and comprises the amino acid sequence set forth in SEQ ID NO: 26 at positions corresponding to positions 141-151 , theamino acid sequence set forth in SEQ ID NO: 40 at positions corresponding to positions 184-194, the amino acid sequence set forth in SEQ ID NO: 51 at positions corresponding to positions 214-224, the amino acid sequence set forth in SEQ ID NO: 68 at positions corresponding to positions 260-272, and the amino acid sequence set forth in SEQ ID NO: 82 at positions corresponding to positions 301- 313, wherein all positions are relative to SEQ ID NO:15.

[0065] In various embodiments, the polypeptide disclosed herein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 4 (Nyl_FH4) or a functional variant or fragment thereof. In various embodiments, the polypeptide disclosed herein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 4 or a functional variant or fragment thereof and comprises the amino acid sequence set forth in SEQ ID NO: 27 at positions corresponding to positions 141-151 , the amino acid sequence set forth in SEQ ID NO: 41 at positions corresponding to positions 184-194, the amino acid sequence set forth in SEQ ID NO: 54 at positions corresponding to positions 214-224, the amino acid sequence set forth in SEQ ID NO: 69 at positions corresponding to positions 260-272, and the amino acid sequence set forth in SEQ ID NO: 83 at positions corresponding to positions 301 - 313, wherein all positions are relative to SEQ ID NO:15.

[0066] In various embodiments, the polypeptide disclosed herein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 5 (Nyl_FH5) or a functional variant or fragment thereof. In various embodiments, the polypeptide disclosed herein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 5 or a functional variant or fragment thereof and comprises the amino acid sequence set forth in SEQ ID NO: 28 at positions corresponding to positions 141-151 , the amino acid sequence set forth in SEQ ID NO: 42 at positions corresponding to positions 184-194, the amino acid sequence set forth in SEQ ID NO: 55 at positions corresponding to positions 214-224, the amino acid sequence set forth in SEQ ID NO: 70 at positions corresponding to positions 260-272, and the amino acid sequence set forth in SEQ ID NO: 84 at positions corresponding to positions 301 - 313, wherein all positions are relative to SEQ ID NO:15.

[0067] In various embodiments, the polypeptide disclosed herein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 6 (Nyl_FH6) or a functional variant or fragment thereof. In various embodiments, the polypeptide disclosed herein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 6 or a functional variant or fragment thereof and comprises the amino acid sequence set forth in SEQ ID NO: 29 at positions corresponding to positions 141-151 , the amino acid sequence set forth in SEQ ID NO: 43 at positions corresponding to positions 184-194, the amino acid sequence set forth in SEQ ID NO: 56 at positions corresponding to positions 214-224, the amino acid sequence set forth in SEQ ID NO: 71 at positions corresponding to positions 260-272, and the amino acid sequence set forth in SEQ ID NO: 85 at positions corresponding to positions 301 - 313, wherein all positions are relative to SEQ ID NO:15.

[0068] In various embodiments, the polypeptide disclosed herein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 7 (Nyl_FH7) or a functional variant or fragment thereof. In various embodiments, the polypeptide disclosed herein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 7 or a functional variant or fragment thereof and comprises the amino acid sequence set forth in SEQ ID NO: 30 at positions corresponding to positions 141-151 , the amino acid sequence set forth in SEQ ID NO: 44 at positions corresponding to positions 184-194, the amino acid sequence set forth in SEQ ID NO: 57 at positions corresponding to positions 214-224, the amino acid sequence set forth in SEQ ID NO: 72 at positions corresponding to positions 260-272, and the amino acid sequence set forth in SEQ ID NO: 85 at positions corresponding to positions 301- 313, wherein all positions are relative to SEQ ID NO:15.

[0069] In various embodiments, the polypeptide disclosed herein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 8 (Nyl_FT1) or a functional variant or fragment thereof. In various embodiments, the polypeptide disclosed herein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 8 or a functional variant or fragment thereof and comprises the amino acid sequence set forth in SEQ ID NO: 31 at positions corresponding to positions 141-151 , the amino acid sequence set forth in SEQ ID NO: 45 at positions corresponding to positions 184-194, the amino acid sequence set forth in SEQ ID NO: 58 at positions corresponding to positions 214-224, the amino acid sequence set forth in SEQ ID NO: 73 at positions corresponding to positions 260-272, and the amino acid sequence set forth in SEQ ID NO: 79 at positions corresponding to positions 301 - 313, wherein all positions are relative to SEQ ID NO:15.

[0070] In various embodiments, the polypeptide disclosed herein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 9 (Nyl_FT2) or a functional variant or fragment thereof. In various embodiments, the polypeptide disclosed herein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 9 or a functional variant or fragment thereof and comprises the amino acid sequence set forth in SEQ ID NO: 32 at positions corresponding to positions 141-151 , the amino acid sequence set forth in SEQ ID NO: 46 at positions corresponding to positions 184-194, the amino acid sequence set forth in SEQ ID NO: 59 at positions corresponding to positions 214-224, the amino acid sequence set forth in SEQ ID NO: 74 at positions corresponding to positions 260-272, and the amino acid sequence set forth in SEQ ID NO: 87 at positions corresponding to positions 301 - 313, wherein all positions are relative to SEQ ID NO:15.

[0071] In various embodiments, the polypeptide disclosed herein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 10 (Nyl_FT3) or a functional variant or fragment thereof. In various embodiments, the polypeptide disclosed herein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 10 or a functional variant or fragment thereof and comprises the amino acid sequence set forth in SEQ ID NO: 33 at positions corresponding to positions 141-151 , the amino acid sequence set forth in SEQ ID NO: 47 at positions corresponding to positions 184-194, the amino acid sequence set forth in SEQ ID NO: 60 at positions corresponding to positions 214-224, theamino acid sequence set forth in SEQ ID NO: 75 at positions corresponding to positions 260-272, and the amino acid sequence set forth in SEQ ID NO: 88 at positions corresponding to positions 301- 313, wherein all positions are relative to SEQ ID NO:15.

[0072] In various embodiments, the polypeptide disclosed herein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 11 (Nyl_FT4) or a functional variant or fragment thereof. In various embodiments, the polypeptide disclosed herein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 11 or a functional variant or fragment thereof and comprises the amino acid sequence set forth in SEQ ID NO: 34 at positions corresponding to positions 141-151 , the amino acid sequence set forth in SEQ ID NO: 48 at positions corresponding to positions 184-194, the amino acid sequence set forth in SEQ ID NO: 61 at positions corresponding to positions 214-224, the amino acid sequence set forth in SEQ ID NO: 76 at positions corresponding to positions 260-272, and the amino acid sequence set forth in SEQ ID NO: 79 at positions corresponding to positions 301 - 313, wherein all positions are relative to SEQ ID NO:15.

[0073] In various embodiments, the polypeptide disclosed herein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 12 (Nyl_FT5) or a functional variant or fragment thereof. In various embodiments, the polypeptide disclosed herein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 12 or a functional variant or fragment thereof and comprises the amino acid sequence set forth in SEQ ID NO: 35 at positions corresponding to positions 141-151 , the amino acid sequence set forth in SEQ ID NO: 49 at positions corresponding to positions 184-194, the amino acid sequence set forth in SEQ ID NO: 62 at positions corresponding to positions 214-224, the amino acid sequence set forth in SEQ ID NO: 77 at positions corresponding to positions 260-272, and the amino acid sequence set forth in SEQ ID NO: 89 at positions corresponding to positions 301 - 313, wherein all positions are relative to SEQ ID NO:15.

[0074] In various embodiments, the polypeptide disclosed herein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 13 (Nyl_FT6) or a functional variant or fragment thereof. In various embodiments, the polypeptide disclosed herein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 13 or a functional variant or fragment thereof and comprises the amino acid sequence set forth in SEQ ID NO: 36 at positions corresponding to positions 141-151 , the amino acid sequence set forth in SEQ ID NO: 50 at positions corresponding to positions 184-194, the amino acid sequence set forth in SEQ ID NO: 63 at positions corresponding to positions 214-224, the amino acid sequence set forth in SEQ ID NO: 78 at positions corresponding to positions 260-272, and the amino acid sequence set forth in SEQ ID NO: 90 at positions corresponding to positions 301 - 313, wherein all positions are relative to SEQ ID NO:15.

[0075] The term “functional variant’1, as used herein in relation to the polypeptide disclosed herein, relates to polypeptides that comprise or consist of an amino acid sequence that is at least 50%, 55% 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.25%, or 99.5% identical or homologous to the reference amino acid sequence (e.g any one of SEQ ID NO:1-22), over their entire length, but retain the functionality of the reference sequence (i.e. depolymerisation activity). The term “functional variant” also encompasses variants that comprise N- and / or C-terminal extensions of 1 or more amino acids.

[0076] In various embodiments, the functional variant of the polypeptide has a sequence identity of less than 70% to a reference sequence (e.g., SEQ ID NO:14-22), provided that the variant exhibits depolymerisation activity. In various embodiments, the functional variant of the polypeptide has a sequence identity of between 50% and 69.9% to a reference sequence (e.g, SEQ ID NO:14-22) provided that the variant exhibits depolymerisation activity.

[0077] In various embodiments, the polypeptide disclosed herein has an amino acid sequence that shares at least 50, 60, 70, 80, or 90 % sequence identity with the amino acid sequence set forth in SEQ ID NO:15 over its entire length, and comprises the conserved catalytic residues at positions 146, 189, 219, 265-267 and 306-308, and the at least one amino acid substitution at positions within ±5 amino acid residues of positions 146, 189, 219, 265-267 and 306-308, wherein all positions are relative to SEQ ID NO:15.

[0078] In various embodiments, the polypeptide disclosed herein has an amino acid sequence that shares at least 50, 60, 70, 80, or 90 % sequence identity with the amino acid sequence set forth in any one of SEQ ID NO:1 -13 over its entire length, and comprises: (i) an amino acid sequence set forth in any one of SEQ ID NO:24-36 at positions corresponding to positions 141 -151 ; (ii) an amino acid sequence set forth in any one of SEQ ID NO:38-50 at position corresponding to position 184- 194; (iii) an amino acid sequence set forth in any one of SEQ ID NO:51-63 at position corresponding to position 214-224; (iv) an amino acid sequence set forth in any one of SEQ ID NO:66-78 at position corresponding to position 260-272; and (v) an amino acid sequence set forth in any one of SEQ ID NO:79-90 at position corresponding to position 301-313, wherein all positions are relative to SEQ ID NO:15.

[0079] The identity of amino acid sequences (or nucleotide sequences) is generally determined by means of a sequence comparison. This sequence comparison is based on the BLAST algorithm that is established in the existing art and commonly used (cf. e.g. Altschul et al. (1990) “Basic local alignment search tool”, J. Mol. Biol. 215:403-410, and Altschul et al. (1997): “Gapped BLAST and PSI-BLAST : a new generation of protein database search programs”; Nucleic Acids Res., 25, p. 3389- 3402) and is effected in principle by mutually associating similar successions of nucleotides or amino acids in the nucleic acid sequences and amino acid sequences, respectively. A tabular association of the relevant positions is referred to as an "alignment." Sequence comparisons (alignments), in particular multiple sequence comparisons, are commonly prepared using computer programs which are available and known to those skilled in the art. A comparison of this kind also allows a statementas to the similarity to one another of the sequences that are being compared. This is usually indicated as a percentage identity, i.e. the proportion of identical nucleotides or amino acid residues at the same positions or at positions corresponding to one another in an alignment. The more broadly construed term "homology", in the context of amino acid sequences, also incorporates consideration of the conserved amino acid exchanges, i.e. amino acids having a similar chemical activity, since these usually perform similar chemical activities within the protein. The similarity of the compared sequences can therefore also be indicated as a "percentage homology" or "percentage similarity." Indications of identity and / or homology can be encountered over entire polypeptides or genes, or only over individual regions. Homologous and identical regions of various nucleic acid sequences or amino acid sequences are therefore defined by way of matches in the sequences. Such regions often exhibit identical functions. They can be small, and can encompass only a few nucleotides or amino acids. Small regions of this kind often perform functions that are essential to the overall activity of the protein. It may therefore be useful to refer sequence matches only to individual, and optionally small, regions. Unless otherwise indicated, however, indications of identity and homology herein refer to the full length of the respectively indicated amino acid sequence (or nucleic acid sequence).

[0080] The term “functional fragment”, as used herein in relation to the polypeptides disclosed herein, relates to polypeptides that differ from the reference polypeptide by a deletion of one or more amino acids from its C- and / or N-terminus while retaining the depolymerisation activity. Said fragments preferably retain full functionality. In various embodiments, such fragment differs from the reference sequence and they may lack 1 -20 amino acids from their N- and / or C-terminus, for example 1 -15 amino acids or 1 -10 amino acids or 1 -5 amino acids, and encompasses an amino acid sequence that matches the initial molecule over a length of at least 200, 250, 300, 310, 320, 325, 330, 335, 340, 345, 350 continuously connected amino acids. The functional fragment comprises the conserved catalytic residues at positions 146, 189, 219, 265-267 and 306-308, and the at least one amino acid substitution at a position within ±5 amino acid residues of positions 146, 189, 219, 265-267 and 306- 308. In various embodiments, the functional fragments of the polypeptides described herein retain enzymatic activity, that is depolymerisation activity. It is preferred that they have at least 50 %, more preferably at least 70, most preferably at least 90 % of the depolymerisation activity of the initial molecule, preferably of the polypeptide having the amino acid sequence of SEQ ID NO: 14 (NylC) or 15 (NylCA).

[0081] In addition to the above-described substitutions, polypeptides according to the embodiments disclosed herein can comprise additional amino acid modifications other than those described above (i.e. amino acid mutations (substitutions) at positions outside of the positions within ±5 amino acid residues of positions 146, 189, 219, 265-267 and 306-308 of SEQ ID NO:15, and present in any one of the amino acid sequences set forth in SEQ ID NO:1-13. These additional amino acid modifications preferably do not affect, alter or reverse the catalytic residues and motifs. This means that the abovedefined features of these residues / motifs are not changed by these additional mutations beyond that what is defined above. Such polypeptides are, for example, further developed by targeted geneticmodification, i.e. by way of mutagenesis methods, and optimized for specific purposes or with regard to special properties (for example, with regard to their catalytic activity, stability, etc.). If such additional modifications are introduced into the polypeptides of the invention, these preferably do not affect, alter the depolymerisation activity of the reference enzymes. This means that the abovedefined features of these residues are not changed by these additional mutations beyond what is defined above. In addition, nucleic acids contemplated herein can be introduced into recombination formulations and thereby used to generate entirely novel polyamide degrading enzymes.

[0082] Accordingly, in one aspect, the present invention relates to a polypeptide having depolymerization activity towards polyamide substrates, comprising or consisting of: (i) an amino acid sequence as set forth in any one of SEQ ID NO:14-22; (ii) an amino acid sequence that shares at least 50, 60, 70, 80, or 90 % sequence identity with the amino acid sequence set forth in any one of SEQ ID NO:14-22 over its entire length; or (iii) a functional fragment of any one of (i)-(ii) having depolymerisation activity, wherein said polypeptide comprises: an amino acid residue Y at the position corresponding to position 146; an amino acid residue K at the position corresponding to position 189; an amino acid residue N at the position corresponding to position 219; amino acid residues GNT at the positions corresponding to positions 265-267; amino acid residues DGD at the positions corresponding to positions 306-308; and at least one amino acid substitution at a position within ±5 amino acid residues of positions 146, 189, 219, 265-267 and 306-308, wherein position numbering is relative to the amino acid sequence set forth in SEQ ID NO:15.

[0083] In various embodiments, the polypeptide disclosed herein may comprise or consist of: (i) an amino acid sequence as set forth in SEQ ID NO:15 (NylCA); (ii) an amino acid sequence that shares at least 50, 60, 70, 80, or 90 % sequence identity with the amino acid sequence set forth in SEQ ID NO:15 over its entire length; or (iii) a functional fragment of any one of (i)-(ii) having depolymerisation activity, wherein said polypeptide comprises: an amino acid residue Y at the position corresponding to position 146; an amino acid residue K at the position corresponding to position 189; an amino acid residue N at the position corresponding to position 219; amino acid residues GNT at the positions corresponding to positions 265-267; amino acid residues DGD at the positions corresponding to positions 306-308; and at least one amino acid substitution at a position within ±5 amino acid residues of positions 146, 189, 219, 265-267 and 306-308, wherein position numbering is relative to the amino acid sequence set forth in SEQ ID NO:15.

[0084] In various embodiments, the polypeptide disclosed herein may comprise or consist of: (i) an amino acid sequence as set forth in any one of SEQ ID NO:1 -13; (ii) an amino acid sequence that shares at least 50, 60, 70, 80, or 90 % sequence identity with the amino acid sequence set forth in any one of SEQ ID NO:1 -13 over its entire length; or (iii) a functional fragment of any one of (i)-(ii) having depolymerisation activity, wherein said polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NO:24-36 at positions corresponding to positions 141 -151 ; an amino acid sequence set forth in any one of SEQ ID NO:38-50 at position corresponding to position 184-194; anamino acid sequence set forth in any one of SEQ ID NO:51-63 at position corresponding to position 214-224; an amino acid sequence set forth in any one of SEQ ID NO:66-78 at position corresponding to position 260-272; and an amino acid sequence set forth in any one of SEQ ID NO:79-90 at position corresponding to position 301 -313, wherein all positions are relative to SEQ ID NO:15.

[0085] In various embodiments, the polypeptide disclosed herein comprises or consists of an amino acid sequence set forth in any one of SEQ ID NO: 1-13.

[0086] In various embodiments, the polypeptide disclosed herein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 5, 12 or 13.

[0087] In various embodiments, the polypeptide disclosed herein may comprise or consist of: (i) an amino acid sequence as set forth in SEQ ID NO: 5, 12 or 13; (ii) an amino acid sequence that shares at least 50, 60, 70, 80, or 90 % sequence identity with the amino acid sequence set forth in SEQ ID NO:5, 12 or 13 over its entire length; (iii) a functional fragment of any one of (i)-(ii) having depolymerisation activity, wherein said polypeptide comprises an amino acid sequence set forth in SEQ ID NO:28, 35, or 36 at positions corresponding to positions 141-151 ; an amino acid sequence set forth in SEQ ID NO:42, 49, or 50 at position corresponding to position 184-194; an amino acid sequence set forth in SEQ ID NO: 55, 62 or 63 at position corresponding to position 214-224; an amino acid sequence set forth in SEQ ID NO:70, 77, or 78 at position corresponding to position 260- 272; and an amino acid sequence set forth in SEQ ID NO:84, 89 or 90 at position corresponding to position 301-313, wherein all positions are relative to SEQ ID NO:15.

[0088] In various embodiments, the polypeptide disclosed herein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 5, or 12.

[0089] In various embodiments, the polypeptide disclosed herein may comprise or consist of: (i) an amino acid sequence as set forth in SEQ ID NO: 5 or 12; (ii) an amino acid sequence that shares at least 50, 60, 70, 80, or 90 % sequence identity with the amino acid sequence set forth in SEQ ID NO:5, 12 or 13 over its entire length; (iii) a functional fragment of any one of (i)-(ii) having depolymerisation activity, wherein said polypeptide comprises an amino acid sequence set forth in SEQ ID NO:28 or 35 at positions corresponding to positions 141 -151 ; an amino acid sequence set forth in SEQ ID NO:42, or 49 at position corresponding to position 184-194; an amino acid sequence set forth in SEQ ID NO: 55, or 62 at position corresponding to position 214-224; an amino acid sequence set forth in SEQ ID NO:70, or 77 at position corresponding to position 260-272; and an amino acid sequence set forth in SEQ ID NO:84, or 89 at position corresponding to position 301 -313, wherein all positions are relative to SEQ ID NO:15.

[0090] As used herein, the terms "polyamide material" or "polyamide substrate" refer to a synthetic polymeric material comprising a backbone with recurring amide (CONH) linkages betweenmonomeric units. Such materials encompass homopolymers, copolymers, oligomers, blends, and composites in which amide bonds are a principal structural feature of the polymer chain. Polyamide materials include, but are not limited to, polyamide 6 (PA6), polyamide 6,6 (PA6,6), and other synthetic polyamides such as polyamide 1 1 (PA11), polyamide 12 (PA12), polyamide 4,6 (PA4,6), polyamide 6,10 (PA6,10), polyamide 6,12 (PA6.12), caprolactam-containing polymers or oligomers, as well as copolymers and oligomeric derivatives thereof. These may be homopolymers, copolymers, blends, or composites in which amide bonds form a principal structural component of the polymer chain. Within the scope of the present disclosure, the term "nylon" may be used interchangeably with synthetic polyamides and refers to this subset of materials, including but not limited to polyamide 6 (PA6), polyamide 6,6 (PA66), aramids (e.g., poly(p-phenylene terephthalamide) and poly(m- phenylene isophthalamide)), and other related aliphatic, semi-aromatic, or fully aromatic polyamides. Such materials are typically produced via ring-opening polymerization of lactams (e.g., caprolactam for PA6) or via condensation of aliphatic or aromatic diamines with dicarboxylic acids (e.g., hexamethylene diamine with adipic acid for PA66, or p-phenylenediamine with terephthaloyl chloride for aramids). The polyamide material may be provided in various physical forms including, but not limited to, fibres, films, resins, powders, granules, moulded articles, or textile blends. Unless otherwise specified, naturally occurring polyamides such as proteins (e.g., silk, wool, keratin) are excluded from the scope of the present disclosure.

[0091] In various embodiments, the polyamide material may be polyamide-6 (PA6) and / or polyamide-6,6 (PA66) and / or aramids.

[0092] In various embodiment, the polypeptide disclosed herein may be thermostabilized. Thermostabilisation may be achieved through rational amino acid substitutions at flexible loop regions, introduction of disulfide bonds, consensus sequence design, or directed evolution approaches, thereby enhancing structural rigidity and resistance to thermal denaturation. The resulting thermostabilised hydrolases retain catalytic activity at elevated temperatures, enabling efficient depolymerisation of polyamide substrates. The polypeptide here displays increased thermostability, that is the ability to resist irreversible change in enzyme function or activity (i.e. denature) when exposed to extreme temperatures. In various embodiment, the polypeptide disclosed herein retains its depolymerization activity after exposure to a range of temperatures from about 4°C to about 95°C. The thermostability of a polypeptide may be evaluated by any suitable means known to persons skilled in the art. For example, thermostability can be assessed by measuring the residual enzyme activity of the polypeptide after incubation at different temperatures.

[0093] Nucleic acid molecules encoding the polypeptide disclosed herein are also provided. All embodiments disclosed above in relation to the polypeptide similarly apply to the nucleic acid molecules and vice versa.

[0094] The nucleic acid molecules can be DNA molecules or RNA molecules. They can exist as an individual strand, as an individual strand complementary to said individual strand, or as a double strand. With DNA molecules in particular, the sequences of both complementary strands in all three possible reading frames are to be considered in each case. Also to be considered is the fact that different codons, i.e. base triplets, can code for the same amino acids, so that a specific amino acid sequence can be coded by multiple different nucleic acids. As a result of this degeneracy of the genetic code, all nucleic acid sequences that can encode one of the above-described polypeptides are included in this subject of the invention. The skilled artisan is capable of unequivocally determining these nucleic acid sequences, since despite the degeneracy of the genetic code, defined amino acids are to be associated with individual codons. The skilled artisan can therefore, proceeding from an amino acid sequence, readily ascertain nucleic acids coding forthat amino acid sequence. In addition, in the context of nucleic acids according to the present invention one or more codons can be replaced by synonymous codons. This aspect refers in particular to heterologous expression of the enzymes contemplated herein. For example, every organism, e.g. a host cell of a production strain, possesses a specific codon usage. "Codon usage" is understood as the translation of the genetic code into amino acids by the respective organism. Bottlenecks in protein biosynthesis can occur if the codons located on the nucleic acid are confronted, in the organism, with a comparatively small number of loaded tRNA molecules. Also it codes forthe same amino acid, the result is that a codon becomes translated in the organism less efficiently than a synonymous codon that codes for the same amino acid. Because of the presence of a larger number of tRNA molecules forthe synonymous codon, the latter can be translated more efficiently in the organism. By way of methods commonly known today such as, for example, chemical synthesis or the polymerase chain reaction (PCR) in combination with standard methods of molecular biology or protein chemistry, a skilled artisan has the ability to manufacture, on the basis of known DNA sequences and / or amino acid sequences, the corresponding nucleic acids all the way to complete genes. Such methods are known, for example, from Sambrook, J., Fritsch, E. F., and Maniatis, T, 2001 , Molecular cloning: a laboratory manual, 3rd edition, Cold Spring Laboratory Press.

[0095] The nucleic acid molecules encoding the polypeptides described herein, as well as a circular DNA molecule containing such a nucleic acid, in particular a plasmid, vector, cosmid, bacterial artificial chromosome (BAC), bacteriophage, viral vector or hybrids thereof also form part of the present invention.

[0096] In various embodiments, the nucleic acid molecules may be comprised in a vector or is a vector. "Vectors" are understood for purposes herein as elements - made up of nucleic acids - that contain a nucleic acid contemplated herein as a characterizing nucleic acid region. They enable said nucleic acid to be established as a stable genetic element in a species or a cell line over multiple generations or cell divisions. In particular when used in bacteria, vectors are special plasmids, i.e. circular genetic elements. In the context herein, a nucleic acid as contemplated herein is cloned into a vector. Included among the vectors are, for example, those whose origins are bacterial plasmids,viruses, or bacteriophages, or predominantly synthetic vectors or plasmids having elements of widely differing derivations. Using the further genetic elements present in each case, vectors are capable of establishing themselves as stable units in the relevant host cells over multiple generations. They can be present extrachromosomally as separate units, or can be integrated into a chromosome resp. into chromosomal DNA.

[0097] In various embodiments, the vector may be an expression vector. Expression vectors encompass nucleic acid sequences which are capable of replicating in the host cells, by preference microorganisms, particularly preferably bacteria, that contain them, and expressing therein a contained nucleic acid. In various embodiments, the vectors described herein thus also contain regulatory elements that control expression of the nucleic acids encoding a polypeptide or fusion protein of the invention. Expression is influenced in particular by the promoter or promoters that regulate transcription. Expression can occur in principle by means of the natural promoter originally located in front of the nucleic acid to be expressed, but also by means of a host-cell promoter furnished on the expression vector or also by means of a modified, or entirely different, promoter of another organism or of another host cell. In the present case at least one promoter for expression of a nucleic acid as contemplated herein is made available and used for expression thereof. Expression vectors can furthermore be regulated, for example by way of a change in culture conditions or when the host cells containing them reach a specific cell density, or by the addition of specific substances, in particular activators of gene expression. One example of such a substance is the galactose derivative isopropyl-beta-D-thiogalactopyranoside (IPTG), which is used as an activator of the bacterial lactose operon (lac operon). In various embodiments, the expression vector is derived from a pET-28 series backbone. pET-28 is a well-known Escherichia coli expression vector that utilises a bacteriophage T7 RNA polymerase-dependent promoter for high-level transcription of the heterologous nucleic acid sequence. The vector may comprise an antibiotic resistance gene as a selection marker for selection in E. coli, a multiple cloning site (MCS) permitting in-frame fusion of the target polypeptide with tags, such as an N-terminal and / or C-terminal polyhistidine (His6) tag, and optional protease cleavage sites to enable removal of the tag following purification.

[0098] In various embodiments, the nucleic acid molecule disclosed herein may comprise an "expression construct” which refers to a functional unit built in the vector for the purpose of recombinantly expressing the polypeptide or fusion protein disclosed herein, when introduced into an appropriate host cell. The term "recombinant", as used herein (e g. a recombinant polypeptide, a recombinant fusion protein, a recombinant nucleic acid, or the like), refers to any molecule which is prepared, expressed, created or isolated by recombinant means, and which is not naturally occurring. "Recombinant" can be used synonymously with "engineered" or "non-natural" and can refer to to an organism, microorganism, cell, nucleic acid molecule, or vector that includes at least one genetic alteration or has been modified by introduction of an exogenous nucleic acid molecule, wherein such alterations or modifications are introduced by genetic engineering (i.e., human intervention). Genetic alterations include, for example, modifications introducing expressible nucleic acid moleculesencoding proteins, fusion proteins or enzymes, or other nucleic acid molecule additions, deletions, substitutions or other functional disruption of a cell's genetic material. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a polynucleotide, gene or operon.

[0099] In various embodiments, the nucleic acid molecule may be comprised in a bacterial plasmid or is a bacterial plasmid. The term “bacterial plasmid” as used herein refers to a circular DNA molecule capable of replication in a bacterial host cell. A bacterial plasmid may contain an appropriate origin of replication, which is a sequence of DNA sufficient to enable the replication of the plasmid in a host bacterial cell. A bacterial plasmid may also contain a selectable marker sequence, which encodes a selectable marker conferring cellular resistance to antibiotics such as ampicillin, kanamycin, chloramphenicol, and tetracycline.

[0100] In various embodiments, the nucleic acid molecule, or vector, or plasmid, containing the nucleic acid molecule, further comprises regulatory elements for controlling expression of said nucleic acid molecule.

[0101] The term "operably linked" as used herein refers to the relationship between two or more nucleotide sequences that interact physically or functionally. For example, a promoter or regulatory nucleotide sequence is said to be operably linked to a nucleotide sequence that codes for an RNA or a protein if the two sequences are situated such that the regulatory nucleotide sequence will affect the expression level of the coding or structural nucleotide sequence. “Regulatory nucleotide sequences” or “regulatory elements” as used herein refer to nucleotide sequences that influence the timing and level / amount of transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters; translation leader sequences; introns; enhancers; stem-loop structures; repressor binding sequences; termination sequences; and polyadenylation recognition sequences. Particular regulatory sequences may be located upstream and / or downstream of a coding sequence operably linked thereto.

[0102] In various embodiments, the nucleic acid molecule further comprises one or more nucleotide sequences encoding a peptide or protein tag. The tag may be positioned at the N-terminus, C- terminus, or internally, and is typically encoded by a contiguous nucleotide sequence within the same open reading frame as the polypeptide disclosed herein. In various embodiments, the tag may include, but is not limited to, a polyhistidine (His) tag, HA (hemagglutinin) tag, MBP (maltose-binding protein), and TRX TEV (thioredoxin fusion with a Tobacco Etch Virus protease cleavage site). These tags may serve various purposes, including facilitating purification, enhancing solubility, improving stability, enabling detection, or allowing multimeric assembly or immobilization. In various embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide operably linked to a nucleotide sequence encoding a tag, such that the tag and polypeptide are expressed as a single continuous polypeptide chain.

[0103] In various embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a polyhistidine (His) tag. The His tag facilitates purification of the recombinant protein using immobilized metal affinity chromatography (IMAC), for example via nickel- or cobalt-charged resins. The His tag may be positioned at the N-terminus or C-terminus and may optionally include a protease cleavage site for removal following purification. In various embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a hemagglutinin (HA) tag. The HA tag may allow for immunological detection using anti-HA antibodies, for example in Western blotting, ELISA, or immunoprecipitation, and may be useful in expression screening or cellular localization studies. In various embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a maltose-binding protein (MBP) tag. The MBP tag is widely used to improve solubility and stability of recombinant proteins expressed in Escherichia coli, and may additionally facilitate purification via amylose resin chromatography. In various embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a thioredoxin (TRX) tag linked to a Tobacco Etch Virus (TEV) protease cleavage site. The TRX portion enhances solubility and correct folding, while the TEV site allows for precise removal of the tag after purification using a site-specific protease. In various embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding an NT11 tag.

[0104] In various embodiments, the nucleic acid molecule comprises a translation initiation region (TIR) operably linked to the coding sequence of the polypeptide and tag, for example His-tag. The TIR, may include modifications to the ribosome binding site, intergenic spacing, and start codon context, to achieve soluble expression of the encoded polypeptide in a microbial host. In various embodiments, TIR optimization is specifically applied to His-tagged constructs, and may not confer similar solubility benefits when alternative tags are used. Accordingly, the expression constructs disclosed herein may include a His-tag, an TIR, and a nucleotide sequence encoding the polypeptide. In various embodiments, the polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 5 or 12 (i.e. FH5 or FT5).

[0105] Another aspect of the invention relates to a host cell comprising the polypeptide or nucleic acid molecule disclosed herein. All embodiments disclosed above in relation to the polypeptide, and nucleic acid molecule disclosed herein, similarly apply to the host cell, and vice versa.

[0106] All cells are in principle suitable as host cells, i.e. prokaryotic or eukaryotic cells. Those host cells can be manipulated in genetically advantageous fashion, “he term “host cell" as used herein refers to a living cell into which the polypeptide, or nucleic acid molecule is to be or has been introduced The living cell includes both a cultured cell and a cell within a living organism. In various embodiments, host cells can be engineered to incorporate a desired gene or expression construct on its chromosome or in its genome. The host cell may be any cell that is commonly used for expression, i.e. transcription and translation of the nucleic acid molecule for the production of the polypeptide or fusion protein disclosed herein. In particular, the term “host cell” relates to prokaryotes, eukaryotes,plants, insect cells or mammalian cells, cell lines and cell culture systems. Host cells include, without limitation, bacterial, microbial, plant or animal cells. In various embodiments, the host cell is represented by those host cells whose activity can be regulated on the basis of genetic regulation elements that are made available, for example, on the vector, but can also be present a priori in those cells. They can be stimulated to expression, for example, by controlled addition of chemical compounds that serve as activators, by modifying the culture conditions, or when a specific cell density is reached. This makes possible economical production of the polypeptides contemplated herein.

[0107] A nucleic acid molecule disclosed herein or a vector / plasmid containing said nucleic acid molecule may be transfected, transduced or transformed into the host cell, which all generally refers to the incorporation of the nucleic acid molecule into the host cell. Methods for the transformation, transduction or transfection of cells are established in the existing art and are sufficiently known to the skilled artisan. The host cells contemplated herein are cultured and fermented in a usual manner, for example in discontinuous or continuous systems. In the former case a suitable nutrient medium is inoculated with the host cells, and the product is harvested from the medium after a period of time to be ascertained experimentally. Continuous fermentations are notable for the achievement of a flow equilibrium in which, over a comparatively long period of time, cells die off in part but are also in part renewed, and the protein formed can simultaneously be removed from the medium.

[0108] Preferred host cells are prokaryotic or bacterial cells, such as E. coli cells. Bacteria are notable for short generation times and few demands in terms of culturing conditions. As a result, economical culturing methods for producing the polypeptides can be established. In addition, the skilled artisan has ample experience in the context of bacteria in fermentation technology. Gramnegative or Gram-positive bacteria may be suitable for a specific production instance, for a wide variety of reasons to be ascertained experimentally in the individual case, such as nutrient sources, product formation rate, time requirement, etc. In various embodiments, the host cells may be E.coli cells.

[0109] Host cells contemplated herein can be modified in terms of their requirements for culture conditions, can comprise other or additional selection markers, or can also express other or additional proteins. They can, in particular, be those host cells that transgenically express multiple proteins or enzymes.

[0110] The host cell can, however, also be a eukaryotic cell, which is characterized in that it possesses a cell nucleus. A further embodiment is therefore represented by a host cell which is characterized in that it possesses a cell nucleus. In contrast to prokaryotic cells, eukaryotic cells are capable of post-translationally modifying the protein that is formed. Examples thereof are fungi such as Actinomycetes, or yeasts such as Saccharomyces or Kluyveromyces or insect cells, such as Sf9 cells. This may be particularly advantageous, for example, when the proteins, in connection with theirsynthesis, are intended to experience specific modifications made possible by such systems. Among the modifications that eukaryotic systems carry out in particular in conjunction with protein synthesis are, for example, the bonding of low-molecular-weight compounds such as membrane anchors or oligosaccharides. In various embodiments, the host cells are thus eukaryotic cells, such as insect cells, for example Sf9 cells.

[0111] Host cells contemplated herein may be used to manufacture the polypeptides described herein.

[0112] Accordingly, a further aspect of the invention is therefore a method of producing / manufacturing a polypeptide disclosed herein, comprising culturing a host cell contemplated herein under conditions that allow expression of the polypeptide; and isolating the polypeptide from the culture medium orfrom the host cell. Culture conditions and mediums can be selected bythose skilled in the art based on the host organism used by resorting to general knowledge and techniques known in the art.

[0113] In this regard, the host cell may be readily manipulated in microbiological and biotechnological terms. This refers, for example, to easy culturability, high growth rates, low demands in terms of fermentation media, and good production and secretion rates for the polypeptides. The polypeptides can furthermore be modified, after their manufacture, by the cells producing them, for example by the addition of sugar molecules, formylation, amination, etc. Post-translation modifications of this kind can functionally influence the polypeptide.

[0114] In various embodiments, the method may further comprise purifying the isolated polypeptide disclosed herein. “Purification” or “purifying” herein means the process of removing components from a host cell or culture, the presence of which is not desired. Purification is a relative term and does not require that all traces of the undesirable component be removed.

[0115] In yet another aspect of the invention, there is provided a cell-free method for producing a polypeptide disclosed herein, comprising subjecting the nucleic acid molecule disclosed herein to reaction conditions that allow the transcription and translation of the polypeptide, and isolating said a polypeptide disclosed herein.

[0116] As used herein, the term "cell-free method" refers to an in vitro biochemical system that enables the synthesis of polypeptides in the absence of living cells, by providing the necessary molecular machinery for transcription and / or translation in a controlled, cell-free environment. Such systems may typically comprise cell extracts or reconstituted enzyme mixtures containing ribosomes, tRNAs, amino acids, nucleotides, energy sources (e.g., ATP, GTP), cofactors, and other components required for gene expression. In various embodiments, the cell-free method may be derived from prokaryotic sources (e g., E. coli lysates), eukaryotic sources (e g., wheat germ, rabbit reticulocyte,or insect cell extracts), or synthetic transcription-translation (TX-TL) systems assembled from purified components. The nucleic acid molecule encoding the polypeptide or fusion protein may be introduced into the system to initiate expression, and the resulting polypeptide may be subsequently isolated and purified from the reaction mixture using standard biochemical techniques. Cell-free methods are advantageous for rapid, scalable, and high-throughput protein production, especially for toxic, unstable, or non-naturally occurring proteins that are difficult to express in living cells.

[0117] There is also provided a composition comprising the polypeptide, nucleic acid molecule encoding the polypeptide, or the host cell comprising the nucleic acid molecule disclosed herein.

[0118] In various embodiments, the composition may comprise one or more additional components or agents that enhance the function, delivery, or stability, of the polypeptide, nucleic acid molecule, or host cell for one or more of the uses described below. Accordingly, the compositions disclosed herein may be formulated and adapted for use in a wide range of applications.

[0119] In various embodiments, the composition may comprise the polypeptide, nucleic acid molecule encoding the polypeptide, or the host cell present in a liquid medium. The liquid medium may be an aqueous liquid medium, such as water, a buffered solution, or the like. In certain embodiments, the liquid medium is a cell culture medium. The cell culture medium may be bacterial or yeast cell culture mediums.

[0120] In various embodiments, the composition may comprise a buffer solution, wherein the composition is formulated for use in depolymerising a polyamide material.

[0121] In various embodiments, the composition may be frozen or lyophilized. In various embodiments, the polypeptide, nucleic acid molecule encoding the polypeptide, or the host cell may be formulated into suspensions, sprayable solutions, hydrogels, or otherwise easily dispersible formulations.

[0122] In various embodiments, the composition may be liquid or dry, for instance in the form of a powder. In various embodiments, the composition is a lyophilizate. The composition may further comprise excipients and / or reagents. Appropriate excipients encompass buffers commonly used in biochemistry, and / or agents for adjusting pH.

[0123] In various embodiments, the composition is solubilized in an aqueous medium together with one or several excipients, especially excipients which are able to stabilize or protect the polypeptide, nucleic acid molecule encoding the polypeptide, or the host cell from degradation. For instance, the polypeptide, nucleic acid molecule encoding the polypeptide, or the host cell may be solubilized in water, eventually with additional components. The resulting mixture may then be dried so as to obtain a powder. Methods for drying such mixture are well known to the one skilled in the art and include,without limitation, lyophilization, freeze-drying, spray-drying, supercritical drying, down-draught evaporation, thin-layer evaporation, centrifugal evaporation, conveyer drying, fluidized bed drying, drum drying or any combination thereof.

[0124] In various embodiments, the composition may further comprise additional polypeptide(s) exhibiting enzymatic activity, not limited to nylonases.

[0125] There is also provided a kit comprising the polypeptide, nucleic acid molecule encoding the polypeptide, the host cell, or the composition disclosed herein.

[0126] Immobilizing enzymes on solid supports has a long history with a primary goal of lowering enzyme consumption by repetitively using the same batch of enzymes. In addition, site-separation of solid-phase immobilization reduces aggregation, leading to increased stability and activity of biocatalysts, and simplifies the purification by avoiding contamination of products by enzymes. Consequently, immobilized biocatalysts have been developed for industrial uses. Compared with conventional industrial processes using chemical catalysts, immobilized enzymes are economically attractive and environmentally friendly.

[0127] There are three main-stream immobilization technologies, including attachment to carriers either covalent or non-covalently, physical entrapment, and self-crosslinking. For biocatalysts such as hydrolases with an exposed substrate-binding surface for biomolecule-based substrates, strategies based on attachment to hydrophilic porous resins by either covalent-binding and affinitybinding methods are direct, convenient, and feasible to facilitate their performance in aqueous conditions.

[0128] Thus, the immobilized polypeptides having depolymerization activity are stable, reusable and highly efficient in mediating depolymerization reactions to degrade polyamide materials or substrates.

[0129] Accordingly, in one aspect ofthe invention, the polypeptides having depolymerization activity may be immobilized on a suitable support material. Suitable support materials include various resins and polymers that are used in chromatography columns and the like. The support may have the form of beads, including magnetic beads, or may be the surface of larger structure, such as a microtiter plate. Immobilization allows for a very easy and simple contacting with the substrate, as well as easy separation of enzyme and substrate after the synthesis. If the polypeptide with the enzymatic function is immobilized on a solid column material or magnetic resin, the depolymerization may be a continuous process and / or the substrate / product solution may be cycled over the column or contacted with the magnetic particles for repeated reaction cycles.

[0130] Accordingly, the present invention, in one aspect, also covers a solid support material comprising the polypeptide according to the invention immobilized thereon. The solid support materialmay comprise a polymer resin, preferably in particulate form, such as those mentioned above. The polypeptide can be immobilized on the solid support material by covalent or non-covalent interactions. The solid support may be, for example, an agarose bead.

[0131] In various embodiments, the polypeptides may be glycosylated and may be immobilized by means of concanavalin A (Con A), a lectin (carbohydrate-binding protein) that is isolated from Canavalia ensiformis (jack bean). It binds specifically to a-D-mannose and a-D-glucose containing biomolecules, including glycoproteins and glycolipids. Said ConA protein is used in immobilized form on affinity columns to immobilize glycoproteins and glycolipids. Accordingly, in various embodiments, the polypeptide disclosed herein is glycosylated and non-covalently bound to a carbohydrate-binding moiety, preferably concanavalin A, coupled to the solid support material surface. Embodiments of glycosylated polypeptides of the invention have been described above.

[0132] The solid support materials described above can be used for the on-column depolymerization of at least one polyamide material or substrate in a method for degrading a polyamide material or substrate, comprising contacting a solution comprising the polyamide material or substrate with the solid support material described above under conditions that allow depolymerization of the polyamide material or substrate.

[0133] In various embodiments, the polypeptide disclosed herein is biotinylated and the immobilization is facilitated by interaction with a biotin-binding moiety, preferably a streptavidin, avidin or neutravidin moiety or variant thereof, covalently linked to the solid support. Functionalization of the polypeptide with the biotin may be achieved using methods known in the art, such as functionalization with a biotin esterwith N-Hydroxysuccinimide (NHS), such as succinimidyl-6-(biotinamido)hexanoate. The solid support may be an agarose bead and the biotin-binding moiety may be an avidin variant, such as neutravidin (deglycosylated avidin).

[0134] In various embodiments, the polypeptide disclosed herein is immobilized on the solid support by reaction of free amino groups in the polypeptide, for example from lysine side chains, with an N- hydroxysuccinimide functional group on the surface of the solid support.

[0135] In various embodiments, the solid support material comprises a particulate polymer resin, optionally selected from agarose, polystyrene, polymethacrylate, or a magnetic resin (e g., a polymer resin incorporating magnetisable particles such as ferrite or iron oxide) and is suitable for use in a chromatography column, bioreactor, or magnetic separation system.

[0136] Accordingly, in one aspect, there is provided a bioreactor comprising the solid support material disclosed herein. The bioreactor may be configured for continuous or batch enzymatic depolymerisation of polyamide material or substrate.

[0137] In various embodiments, the present invention provides a bioreactor system comprising a solid support material having immobilized thereon a polypeptide with depolymerisation activity toward a polyamide material. The bioreactor may be configured for continuous or batch processing and may be used for the enzymatic degradation of synthetic polyamide materials, including but not limited to polyamide 6 (PA6), polyamide 6,6 (PA6,6), PA4,6, PA6,10, PA6.12, PA11 , PA12, and copolymers or oligomeric derivatives thereof.

[0138] In various embodiments, the bioreactor may comprise a packed-bed column in which a solution or suspension containing the polyamide material or substrate is flowed through the immobilized enzyme bed under conditions suitable for enzymatic depolymerisation. Alternatively, the bioreactor may operate in batch mode, where the polyamide material or substrate is incubated with the immobilized enzyme in a stirred tank or reaction vessel.

[0139] Operating conditions for the bioreactor may be optimized for the activity of the immobilized polypeptide, including temperatures between 30°C and 60°C and pH values between 7.5 and 9.0. The enzymatic depolymerization reaction results in the cleavage of amide bonds, yielding degradation products such as e-caprolactam, diamines, dicarboxylic acids, or oligomeric intermediates, depending on the type and composition of the polyamide material or substrate. In various embodiments, the bioreactor is integrated into a closed-loop process for recycling or upcycling synthetic nylon waste.

[0140] In various embodiments, the bioreactor may further comprise one or more inlets and outlets for introducing the polyamide material or substrate and removing degradation products. It may also be equipped with sensors and control elements for temperature, pH, and flow rate. In various embodiments, the bioreactor may be coupled to downstream separation units such as filters or chromatographic columns. The modular format of the immobilized enzyme support may allow for facile regeneration or replacement of the catalytic matrix when enzymatic activity declines due to operational fatigue.❖ Methods of Use

[0141] All embodiments disclosed herein in relation to the polypeptides, nucleic acids, compositions, support material, and bioreactor are similarly applicable to the uses and methods described herein and vice versa.

[0142] The polypeptides described herein exhibit depolymerization activity toward polyamide materials, including synthetic polymers such as nylon. Owing to this activity, the polypeptides can be employed in a variety of applications aimed at the enzymatic degradation, recycling, or upcycling of polyamide waste. These include methods for breaking down polyamide materials into monomeric or oligomeric products, processes for recovering valuable degradation intermediates, and use in biocatalytic systems such as immobilized enzyme bioreactors. The polypeptides may also beincorporated into industrial or environmental platforms for managing polymer waste, including the treatment of post-consumer or industrial nylon waste in the form of fibers, films, molded articles, or resins.

[0143] Accordingly, in one aspect, there is provided a method for degrading a polyamide material, the method comprising contacting the polyamide material with a polypeptide or compositions or support material as described herein under conditions suitable for enzymatic depolymerisation.

[0144] In various embodiments, the polyamide material to be degraded is provided in the form of industrial or post-consumer nylon waste, including but not limited to synthetic fibers (e.g., textiles, carpets, or ropes), films (e.g., packaging materials), molded articles (e.g., automotive parts, electronics casings), resins, pellets, or granules. The method is applicable to polyamide materials in virgin, recycled, or blended forms, and may be used to address environmental and economic concerns associated with synthetic polymer accumulation. In various embodiments, the polyamide material to be degraded is nylon.

[0145] The enzymatic depolymerisation reaction is preferably carried out under conditions optimized for depolymerization activity and substrate accessibility. The conditions suitable for degradation of the polyamide material may include a temperature between 0°C and 90°C, preferably between 20°C and 75°C, or more preferably between 30°C and 68°C. In various embodiments, the degrading process is implemented at 37°C. In various embodiments, the degrading process is implemented at 30°C. In various embodiments, the degrading process is implemented at 60°C. In various embodiments, the reaction is performed at a temperature ranging from 30°C to 60°C, and at a pH between 7.5 and 9.0, which are conditions compatible with the activity of many nylon hydrolases. Buffer systems such as Tris-HCI, phosphate, or carbonate may be used to maintain the desired pH.

[0146] Degradation of the polyamide material can be assessed by monitoring the formation of monomeric or oligomeric degradation products, including s-caprolactam, hexamethylenediamine, adipic acid, or other chain fragments. These degradation products may be detected using chromatographic techniques such as HPLC or GC-MS, or colorimetric assays based on amide bond hydrolysis. In various embodiments, recovery of the degradation products enables subsequent chemical repolymerisation, purification, or upcycling into value-added products.

[0147] In still another aspect, the invention relates to the use of the polypeptides or compositions or support material described herein for the enzymatic depolymerisation of polyamide materials.

[0148] In still another aspect, the invention relates to the use of the polypeptides or compositions or support material described herein in a recycling or upcycling method, wherein polyamide materials are enzymatically degraded and the resulting degradation products are reused in manufacturing orrepurposed into novel materials. The method may be integrated into circular economy frameworks or green chemistry approaches for sustainable polymer life cycle management.

[0149] In various embodiments, there is also provided a method for producing a degradation product from a polyamide material. The method comprises contacting the polyamide material with a polypeptide or composition or support material as described herein under conditions suitable for enzymatic depolymerisation, and subsequently recovering one or more degradation products, such as monomers or short-chain oligomers. The recovered products may be used in synthesis, analysis, or further conversion processes.

[0150] In various embodiments, the degradation product comprises 6-aminohexanoic acid. The method comprises contacting a polyamide-6 (nylon-6) material with the polypeptide or composition or support material as described herein, under conditions suitable for hydrolysis of amide bonds. The enzymatic reaction results in cleavage of the polyamide backbone and release of 6-aminohexanoic acid as a principal degradation product. The 6-aminohexanoic acid may be recovered from the reaction mixture by conventional separation or purification techniques, and may optionally be used as a chemical feedstock, a monomer for polymer re-synthesis, or as an intermediate in further synthetic or bioconversion processes.

[0151] In still another aspect, the invention relates to a method of recycling polyamide material comprising passing the polyamide material through a bioreactor as described herein, under conditions suitable for enzymatic degradation. The bioreactor may be operated in continuous or batch mode. This configuration enables scalable and efficient depolymerisation of polyamide material, with reduced enzyme consumption and simplified product recovery.

[0152] The invention is further illustrated by the following non-limiting examples and the appended claims.EXAMPLEMaterials and Methods

[0153] FITC-casein assay: 1 pg of nylon hydrolase and 5 pg of FITC-labelled casein were diluted in 100 pL of 50 mM Tris, 100 mM sodium chloride pH 8.5 buffer in 1.5 mL Eppendorf tubes, and incubated at 45 °C. After 24 hours, 20 pL of the reaction was transferred to a black 384-well plate and the increase in fluorescence was measured on a plate reader at an excitation / emission wavelength of 485 / 520 nm.

[0154] Caprolactam Assay: 100 pL of 10 mM caprolactam solution was added into a glass vial. 50 pL of protein eluate was added, and topped up to a total volume of 200 pL with phosphate buffer before incubation at 45 °C and 500 rpm for 1 week in an Eppendorf ThermoMixer. After thecaprolactam reaction was completed, 200 pL of 10 mM Meldrum's acid furfural conjugate (MAFC) solution was added into each vial. The mixture was incubated at 37 °C, 750 rpm for 30 min. After the reaction, 200 pL of the solution is transferred into a clear 96-well plate and absorbance was measured at 494 nm.Results and Discussion

[0155] Through a series of genome mining and solubility optimisation that led to the discovery of new hydrolases with depolymerisation activity, including variants with better activity than the current state of the art, a thermostable NylC. Table 2 below provides the amino acid sequence of the 13 newly discovered hydrolases (FIG. 1).

[0156] Table 2: Newly identified polyamide hydrolases, where catalytic site residues are in bolded and bordered with 5 neighbouring residues underlined. Also included are reference known hydrolases for comparison. The initial methionine (M) is omitted from some sequences, and may be included to represent the amino acid at position 1 .

[0157] The genes of the candidate hydrolases were optimized using a solubility tag platform to create a variant library of unique solubility tag-gene sequences.

[0158] Table 3 below lists recombinant proteins generated for the discovered new hydrolases to further test for depolymerisation activity. These constructs are designated as Nyl FT 1-6 and Nyl_FH1-7. Each construct was assigned a unique identifier (e g., ID NO:) and was cloned into an appropriate expression vector for heterologous expression in Escherichia coli.

[0159] The genes of the newly identified hydrolases (i.e. Nyl_FH1 to Nyl_FH7 and Nyl_FT1 to Nyl_FT6) were inserted into a plasmid backbones such as PET28. A range of affinity and solubility tags were employed. These included polyhistidine (His), NT11 (a short amino acid tag, more particularly an 11 aa tag in length), HA, MBP (maltose-binding protein), and TRX TEV (thioredoxin with a TEV cleavage site) tags.

[0160] The resulting constructs were used in downstream assays, including caprolactam-based and protease-based activity screens, to test the enzymatic properties of the identified hydrolases for polyamide degradation.

[0161] Expressed constructs that were soluble were tested in two different assays at temperatures of 45°C and pH 8.5. These assays include a caprolactam-based assay (FIG. 2B, PA66 substratelike) and a protease assay (based on fluorescein isothiocyanate (FITC)-labelled casein, FIG. 2A, and FIG. 2B, peptide based amide bond). Most of the genes were found to be active, where an activity similar or better to engineered and native controls, NylCa and NylC (See Table 4) were observed.

[0162] Table 4. ID with its sequence identity to the closest homolog.*M-NylC has low activity, otherwise closest enzyme is <60% sequence identity.

[0163] Discovery of new hydrolases less than 70% is not straightforward, and the discovery of new hydrolases over 70% when the closest neighbor is inactive is also not straightforward. The more active variants include Nyl_FH1 , FH5 (<70% sequence identity to M-NylC, Microbacterium sp. derived) and Nyl_FT5, FT6 (>70% sequence identity to M-NylC). This was a surprising result as M- NylC performed poorly in the general screen by Bell, Elizabeth L., et al. "Natural diversity screening, assay development, and characterization of nylon-6 enzymatic depolymerization." Nature Communications 15.1 (2024): 1217.

[0164] Further analysis of Nyl_FH5 (2413) and FT5 (3612) at various temperatures also showed better activity compared to control engineered enzyme, NylC (FIG. 3).

[0165] The active site and neighbouring residues of the reference nylon hydrolases were compared with those of the newly discovered Nyl_FH5, Nyl_FT5, and Nyl_FT6. The reference enzymes demonstrate a high degree of conservation, with 58 out of 59 residues conserved. However, while the catalytic site residues remain unchanged in the newly identified enzymes, their neighbouring residues exhibit significant variability (FIG. 4 and Table 5 and 6).

[0166] Table 5 below is a representation of information presented in FIG. 4, in addition with other identified hydrolases with amino acid residues that differ from those in NylCA being underlined and bolded in neighbouring residues of the catalytic residues and active sites.

[0167] Table 6: Pairwise amino acid sequence identities of residues surrounding the active site in nylon hydrolases (FIG. 4). The newly identified Nyl_FH5, Nyl_FT5, and Nyl_FT6 proteins were compared with the reference nylon hydrolases.p-values that are significant at the 99.9% confidence level using the Mann-Whitney U test.

[0168] The invention has been described broadly and generically herein. Each of the narrowerspecies and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0169] One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. Further, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The polypeptides, nucleic acid molecules, compositions, kits, methods and uses described herein are presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention are defined by the scope of the claims. The listing or discussion of a previously published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0170] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, it should be understood that although the present invention has been specifically disclosed by exemplary embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0171] The content of all documents and patent documents cited herein is incorporated by reference in their entirety.

Claims

CLAIMS1. A polypeptide having depolymerisation activity, comprising or consisting of:(i) an amino acid sequence as set forth in SEQ ID NO:15 (NylCA);(ii) an amino acid sequence that shares at least 50% sequence identity with the amino acid sequence set forth in SEQ ID NO:15 over its entire length; or(iii) a functional fragment of any one of (i)-(ii) having depolymerisation activity, wherein said polypeptide comprises: an amino acid residue Y at the position corresponding to position 146 of SEQ ID NO:15; an amino acid residue K at the position corresponding to position 189 of SEQ ID NO:15; an amino acid residue N at the position corresponding to position 219 of SEQ ID NO:15; amino acid residues GNT at the positions corresponding to positions 265-267 of SEQ ID NO:15; amino acid residues DGD at the positions corresponding to positions 306-308 of SEQ ID NO:15; and at least one amino acid substitution at a position within ±5 amino acid residues of positions 146, 189, 219, 265-267 and / or 306-308, wherein position numbering is relative to the amino acid sequence set forth in SEQ ID NO:15.

2. The polypeptide of claim 1 , wherein the at least one amino acid substitution is at one or more positions selected from:(i) positions 141-145 and / or 147-151 ;(ii) positions 184-188 and / or 190-194;(iii) positions 214-218 and / or 220-224;(iv) positions 260-264 and / or 268-272; and(v) positions 301-305 and / or 309-313, wherein all positions are relative to SEQ ID NO:15.

3. The polypeptide of claim 1 , wherein the polypeptide comprises at least one amino acid substitution at one or more positions selected from 142, 144, 145, 148-150, 184, 186, 187, 190, 191 , 192, 193, 214, 215, 216, 217, 218, 220, 221 , 223, 224, 260, 261 , 262, 263, 264, 269, 270, 271 , 272, 303, 304, 305, 309, 312, and 313 of SEQ ID NO:15.

4. The polypeptide of claim 1 , wherein the polypeptide comprises:(i) an amino acid sequence set forth in any one of SEQ ID NO:24-36 at positions corresponding to positions 141-151 of SEQ ID NO:15;(ii) an amino acid sequence set forth in any one of SEQ ID NO:38-50 at positions corresponding to positions 184-194 of SEQ ID NO:15,(iii) an amino acid sequence set forth in any one of SEQ ID NO:51-63 at positions corresponding to positions 214-224 of SEQ ID NO:15,(iv) an amino acid sequence set forth in any one of SEQ ID NO:66-78 at positions corresponding to positions 260-272 of SEQ ID NO:15, and(v) an amino acid sequence set forth in any one of SEQ ID NO:79-90 at positions corresponding to positions 301-313 of SEQ ID NO:15.

5. The polypeptide of claim 1 , wherein the polypeptide comprises or consists of an amino acid sequence set forth in any one of SEQ ID NO: 1-13, or functional variants or fragments thereof.

6. The polypeptide of claim 1 , wherein the polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 5, 12 or 13, or functional variants or fragments thereof.

7. A nucleic acid molecule encoding the polypeptide according to any one of claims 1 to 6, preferably the nucleic acid molecule is comprised in a vector.

8. A host cell comprising the nucleic acid molecule of claim 7.

9. A method for producing the polypeptide of any one of claims 1 to 6, comprising culturing a host cell according to claim 8 under conditions that allow expression of the polypeptide, and isolating said polypeptide from the host cell or culture medium.

10. A composition comprising the polypeptide of any one of claims 1 to 6, wherein the composition is formulated for use in depolymerising a polyamide material.

11. A solid support material comprising the polypeptide according to any one of claims 1 to 6 immobilized thereon.

12. The solid support material of claim 11 , wherein the solid support material comprises a particulate polymer resin, optionally selected from agarose, polystyrene, polymethacrylate, or a magnetic resin, and is suitable for use in a chromatography column, bio reactor or magnetic separation system.

13. A bioreactor comprising the solid support material of claim 12, configured for continuous or batch enzymatic depolymerisation of polyamide material.

14. A method of degrading polyamide material, the method comprising contacting the polyamide material with the polypeptide of claim 1 , the nucleic acid of claim 7, the host cell of claim 8, the composition of claim 10, or the solid support material of claim 11 under conditions suitable for depolymerisation and degradation of the polyamide material.

15. The method of claim 14, wherein the polyamide material is selected from the group consisting of polyamide-6 (PA6), polyamide-6,6 (PA66), aramids, caprolactam-containing polymers, and oligomers thereof.

16. Use of the polypeptide of claim 1 , the nucleic acid of claim 7, the host cell of claim 8, the composition of claim 10, or the solid support material of claim 11 for depolymerisation of a polyamide material, or in a process for degrading polyamide material, or recycling or upcycling polyamide material.

17. A method of producing a degradation product from a polyamide material, the method comprising contacting the polyamide material with the polypeptide of claim 1 , the nucleic acid of claim 7, the host cell of claim 8, the composition of claim 10, or the solid support material of claim 11 under suitable conditions for depolymerisation; and recovering the degradation product comprising one or more monomers or oligomers.

18. A method of recycling polyamide material comprising passing the polyamide material through the bioreactor of claim 13 under conditions suitable for enzymatic degradation.

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