Enzyme variants and uses thereof

Abstract

The present disclosure generally relates to polypeptides capable of hydrolyzing amide bonds in polyamides, and more particularly to polypeptides comprising the amino acid sequence of amino acid residues 2-398 of SEQ ID NO:2, or an amino acid sequence having at least 70% sequence identity thereto, or the amino acid sequence of amino acid residues 2-394 of SEQ ID NO:88, or an amino acid sequence having at least 75% sequence identity thereto. The disclosure also extends to methods of using such polypeptides. In another embodiment, the disclosed polypeptides are capable of hydrolyzing nylon 6,6 oligomers to produce adipic acid and hexamethylenediamine.

Description

[Technical Field]

[0001] The present invention relates to novel synthetic enzymes, particularly enzymes that catalyze the hydrolysis of nylon polymers, and uses thereof. [Background technology]

[0002] All references cited in this specification, including any patents or patent applications, are incorporated herein by reference to enable a complete understanding of the invention, although such references should not be read as constituting an admission that any of these documents form part of the general knowledge in the art in Australia or any other country.

[0003] Global industrialization has had significant environmental impacts, particularly the increased manufacturing and reliance on plastics and plastic products, which include a variety of synthetic or semi-synthetic polymers such as polyethylene terephthalate, PVC, polypropylene, polystyrene, and polyamides.

[0004] Polyamides (also commonly known as nylons) are silk-like thermoplastics that can be melt-processed into fibers, films, or shapes, including those in combination with various additives. Polyamides have many notable commercial applications, for example, in the production of clothing, resins and molded shapes, food packaging, and industrial fibers and ropes.

[0005] Although there have been increasing efforts to find suitable and environmentally sustainable alternatives to plastics, including nylon, including their production and disposal, such products remain a significant problem and contribute to the vast majority of environmental pollutants. The environmental significance of this problem is due, at least in part, to the chemical properties of plastics, as they do not readily decompose in nature. Approaches to addressing the problem of plastic waste have typically included incineration, landfill disposal, and mechanical disintegration. However, these approaches also have significant environmental impacts. For example, incineration of plastics produces potentially harmful by-products that are released into the atmosphere, the rate at which plastics decompose in landfills is typically very slow, posing a risk of toxic materials leaching into groundwater, mechanical disintegration is relatively expensive and inefficient, and the use of the by-products is often limited.

[0006] Nylon can take decades to decompose, and while methods exist to recycle nylon, these methods require energy-intensive grinding and remelting of the polymer. Depending on the nylon and intended use, recycled nylon polymer may need to be blended with virgin nylon, limiting its flexibility and sustainability.

[0007] More recently, biological (enzymatic) degradation of plastics has been explored as an alternative approach to reducing the accumulation of plastic waste. In the case of nylon, a bacterial strain found in the wastewater of a nylon factory was discovered to possess unique enzymes (NylA, NylB, and NylC) capable of digesting the by-products of the production of nylon 6 (a nylon homopolymer of caprolactam). [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] http: / / blast.ncbi.nlm.nih.gov / [Non-patent document 2] http: / / www.ebi.ac.uk / Tools / emboss / [Non-patent document 3] Belousov (1997) Nucleic Acids Res. 25:3440~3444 [Non-patent document 4] Kiumarsi and Parvinzadeh, 2010 J Appl Polymer Sci, 116:3140 [Non-patent document 5] Gashti et al., 2013 Preparative Biochemistry & Biotechnology, 43:798 Summary of the Invention [Problem to be solved by the invention]

[0009] Although the enzymatic degradation of plastics, such as nylon, is an attractive alternative for reducing plastic waste and the environmental impact of its disposal, it has not yet been widely adopted for reasons including its relative inefficiency, slow rates of enzymatic degradation, and low levels of enzyme expression in common industrial host organisms. Thus, there remains an urgent need for improved methods and reagents for the enzymatic degradation of plastics, including nylon. [Means for solving the problem]

[0010] In one aspect disclosed herein, a polypeptide capable of hydrolyzing amide bonds in polyamides is provided, wherein the polypeptide comprises the amino acid sequence of amino acid residues 2-398 of SEQ ID NO:2, or an amino acid sequence having at least 70% sequence identity thereto. In one embodiment, the polypeptide comprises an amino acid sequence having at least 75% sequence identity thereto. In one embodiment, the polypeptide comprises an amino acid sequence having at least 80% sequence identity thereto. In one embodiment, the polypeptide comprises an amino acid sequence having at least 85% sequence identity thereto. In one embodiment, the polypeptide comprises an amino acid sequence having at least 90% sequence identity thereto. In one embodiment, the polypeptide comprises an amino acid sequence having at least 95% sequence identity thereto.

[0011] In one embodiment, the polypeptide comprises an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 125, or an amino acid sequence having at least 80% sequence identity thereto.

[0012] In one embodiment, the polypeptide comprises an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 96, or an amino acid sequence having at least 80% sequence identity thereto.

[0013] In one embodiment, the polypeptide comprises an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 1, or an amino acid sequence having at least 80% sequence identity thereto.

[0014] In one embodiment, the polypeptide comprises an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 125, or an amino acid sequence having at least 80% sequence identity thereto, and comprises the amino acid sequence: i. the amino acid at position 3 is Q or T; ii. the amino acid at position 5 is N or D; iii. the amino acid at position 7 is M or F; iv. the amino acid at position 9 is G or T; v. The amino acid at position 10 is S or F; vi. The amino acid at position 14 is P or A; vii. The amino acid at position 16 is Q or G; viii. The amino acid at position 17 is Q or E; ix. the amino acid at position 25 is Q or T; x. The amino acid at position 26 is A or P; xi. The amino acid at position 29 is N or S; xii. The amino acid at position 32 is A or S; xiii. The amino acid at position 39 is I or L; xiv. The amino acid at position 40 is I or V; xv. The amino acid at position 44 is N or Q; xvi. The amino acid at position 48 is G or A; xvii. The amino acid at position 51 is A or P; xviii. The amino acid at position 52 is A or V; xix. The amino acid at position 53 is S or W; xx. The amino acid at position 57 is A or R; xxi. The amino acid at position 58 is A or S; xxii. The amino acid at position 60 is R or A; xxiii. The amino acid at position 61 is D, A, or G; xxiv. The amino acid at position 63 is D or G; xxv. The amino acid at position 64 is G, E, or R; xxvi. The amino acid at position 67 is F or L; xxvii. The amino acid at position 70 is P or deleted; xxviii. The amino acid at position 72 is G or A; xxix. The amino acid at position 73 is R or K; xxx. The amino acid at position 74 is S or E; xxxi. The amino acid at position 75 is T, M, G, or W; xxxii. The amino acid at position 78 is A or G; xxxiii. The amino acid at position 82 is A or E; xxxiv. The amino acid at position 84 is S or T; xxxv. The amino acid at position 88 is A or G; xxxvi. The amino acid at position 92 is L or M; xxxvii. The amino acid at position 99 is A or S; xxxviii. The amino acid at position 101 is W or H; xxxix. The amino acid at position 110 is S or T; xl. The amino acid at position 114 is L or V; xli. The amino acid at position 123 is G or A; xlii. The amino acid at position 124 is I or T; xliii. The amino acid at position 131 is D or E; xliv. The amino acid at position 132 is R or D; xlv. The amino acid at position 138 is D or A; xlvi. The amino acid at position 143 is H, R, or D; xlvii. The amino acid at position 148 is A or V; xlviii. The amino acid at position 149 is A or K; xlix. The amino acid at position 155 is D or G; l. The amino acid at position 167 is S or G; li. The amino acid at position 171 is E or D; lii. The amino acid at position 173 is D or A; liii. The amino acid at position 178 is D or A; liv. The amino acid at position 179 is G or S; lv. The amino acid at position 180 is D or A; lvi. The amino acid at position 186 is R, E, or Q; lvii. The amino acid at position 188 is T or M; lviii. The amino acid at position 193 is A or P; lix. The amino acid at position 194 is S or P; lx. The amino acid at position 195 is D or P; lxi. The amino acid at position 197 is A or E; lxii. The amino acid at position 203 is R or Y; lxiii. The amino acid at position 206 is L or I; lxiv. The amino acid at position 207 is A or V; lxv. The amino acid at position 210 is R or K; lxvi. The amino acid at position 211 is R, K, or deleted; lxvii. The amino acid at position 212 is S, D, or G; lxviii. The amino acid at position 213 is D or A; lxix. The amino acid at position 215 is P or E; lxx. The amino acid at position 223 is A or V; lxxi. The amino acid at position 234 is L or I; lxxii. The amino acid at position 244 is D or E; lxxiii. The amino acid at position 249 is H or R; lxxiv. The amino acid at position 252 is Q or R; lxxv. The amino acid at position 258 is H or A; lxxvi. The amino acid at position 262 is I or V; lxxvii. The amino acid at position 276 is L or I; lxxviii. The amino acid at position 278 is A or C; lxxix. The amino acid at position 280 is L, A, or P; lxxx. The amino acid at position 288 is Q or E; lxxxi. The amino acid at position 291 is L or R; lxxxii. The amino acid at position 301 is I or V; lxxxiii. The amino acid at position 304 is E, A, or G; lxxxiv. The amino acid at position 305 is A or W; lxxxv. The amino acid at position 308 is A or D; lxxxvi. The amino acid at position 311 is L or R; lxxxvii. The amino acid at position 312 is Q or T; lxxxviii. The amino acid at position 313 is N or G; lxxxix. The amino acid at position 316 is R, K, or P; xc. The amino acid at position 321 is R or Q; xci. The amino acid at position 326 is K or H; xcii. The amino acid at position 327 is F or L; xciii. The amino acid at position 328 is F or L; xciv. The amino acid at position 330 is N or G; xcv. The amino acid at position 335 is S or N; xcvi. The amino acid at position 349 is A or G; xcvii. The amino acid at position 359 is I or V; xcviii. The amino acid at position 368 is A or V; xcix. The amino acid at position 370 is L or F; c. the amino acid at position 375 is L or E; ci. The amino acid at position 379 is D or A; cii. The amino acid at position 384 is L or E; ciii. The amino acid at position 385 is C, M, or N; civ. The amino acid at position 390 is D, E, or R; cv. The amino acid at position 394 is R or A; cvi. the amino acid at position 397 is G or A, and / or cvii. the amino acid at position 398 is G or is deleted; Numbering is relative to amino acid positions in SEQ ID NO:1, SEQ ID NO:96, or SEQ ID NO:125.

[0015] In one embodiment, the polypeptide comprises an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 96, or an amino acid sequence having at least 80% sequence identity thereto, and comprises the amino acid sequence: i. the amino acid at position 3 is Q or T; ii. the amino acid at position 5 is N or D; iii. the amino acid at position 10 is S or F; iv. the amino acid at position 17 is Q or E; v. The amino acid at position 25 is Q or T; vi. The amino acid at position 26 is A or P; vii. The amino acid at position 29 is N or S; viii. The amino acid at position 32 is A or S; ix. the amino acid at position 39 is I or L; x. The amino acid at position 40 is I or V; xi. The amino acid at position 44 is N or Q; xii. The amino acid at position 51 is A or P; xiii. The amino acid at position 52 is A or V; xiv. The amino acid at position 53 is S or W; xv. The amino acid at position 57 is A or R; xvi. The amino acid at position 58 is A or S; xvii. The amino acid at position 63 is D or G; xviii. The amino acid at position 64 is G, E, or R; xix. The amino acid at position 67 is F or L; xx. The amino acid at position 70 is P or deleted; xxi. The amino acid at position 73 is R or K; xxii. The amino acid at position 74 is S or E; xxiii. The amino acid at position 75 is T, M, G, or W; xxiv. The amino acid at position 78 is A or G; xxv. The amino acid at position 82 is A or E; xxvi. The amino acid at position 84 is S or T; xxvii. The amino acid at position 88 is A or G; xxviii. The amino acid at position 92 is L or M; xxix. The amino acid at position 99 is A or S; xxx. The amino acid at position 101 is W or H; xxxi. The amino acid at position 110 is S or T; xxxii. The amino acid at position 114 is L or V; xxxiii. The amino acid at position 124 is I or T; xxxiv. The amino acid at position 131 is D or E; xxxv. The amino acid at position 143 is H, R, or D; xxxvi. The amino acid at position 148 is A or V; xxxvii. The amino acid at position 149 is A or K; xxxviii. The amino acid at position 167 is S or G; xxxix. The amino acid at position 171 is E or D; xl. The amino acid at position 186 is R, E, or Q; xli. The amino acid at position 188 is T or M; xlii. The amino acid at position 193 is A or P; xliii. The amino acid at position 194 is S or P; xliv. The amino acid at position 197 is A or E; xlv. The amino acid at position 207 is A or V; xlvi. The amino acid at position 210 is R or K; xlvii. The amino acid at position 211 is R, K, or deleted; xlviii. the amino acid at position 212 is S, D, or G; xlix. The amino acid at position 215 is P or E; l. The amino acid at position 223 is A or V; li. The amino acid at position 234 is L or I; lii. the amino acid at position 244 is D or E; liii. The amino acid at position 249 is H or R; liv. The amino acid at position 252 is Q or R; lv. The amino acid at position 258 is H or A; lvi. The amino acid at position 276 is L or I; lvii. The amino acid at position 280 is L or A; lviii. The amino acid at position 288 is Q or E; lix. The amino acid at position 291 is L or R; lx. The amino acid at position 301 is I or V; lxi. The amino acid at position 304 is E, A, or G; lxii. The amino acid at position 305 is A or W; lxiii. The amino acid at position 311 is L or R; lxiv. The amino acid at position 312 is Q or T; lxv. the amino acid at position 313 is N or G; lxvi. The amino acid at position 316 is R, K, or P; lxvii. The amino acid at position 326 is K or H; lxviii. The amino acid at position 327 is F or L; lxix. The amino acid at position 328 is F or L; lxx. The amino acid at position 330 is N or G; lxxi. The amino acid at position 349 is A or G; lxxii. The amino acid at position 359 is I or V; lxxiii. The amino acid at position 368 is A or V; lxxiv. The amino acid at position 370 is L or F; lxxv. The amino acid at position 375 is L or E; lxxvi. The amino acid at position 385 is C, M, or N; lxxvii. The amino acid at position 390 is D, E, or R; lxxviii. the amino acid at position 397 is G or A, and / or lxxix. the amino acid at position 398 is G or is deleted; Numbering is relative to amino acid positions in SEQ ID NO:1 or SEQ ID NO:96.

[0016] In one embodiment, the polypeptide comprises an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 96; amino acid residues 2 to 398 of SEQ ID NO: 1, or an amino acid sequence having at least 80% sequence identity thereto; i. the amino acid at position 3 is Q or T; ii. the amino acid at position 5 is N or D; iii. the amino acid at position 25 is Q or T; iv. the amino acid at position 26 is A or P; v. The amino acid at position 32 is A or S; vi. The amino acid at position 40 is I or V; vii. The amino acid at position 51 is A or P; viii. The amino acid at position 52 is A or V; ix. The amino acid at position 53 is S or W; x. The amino acid at position 57 is A or R; xi. The amino acid at position 58 is A or S; xii. The amino acid at position 63 is D or G; xiii. The amino acid at position 64 is G, E, or R; xiv. The amino acid at position 70 is P or deleted; xv. The amino acid at position 73 is R or K; xvi. The amino acid at position 74 is S or E; xvii. The amino acid at position 75 is T, M, or W; xviii. The amino acid at position 78 is A or G; xix. The amino acid at position 82 is A or E; xx. The amino acid at position 84 is S or T; xxi. The amino acid at position 88 is A or G; xxii. The amino acid at position 92 is L or M; xxiii. The amino acid at position 99 is A or S; xxiv. The amino acid at position 101 is W or H; xxv. The amino acid at position 110 is S or T; xxvi. The amino acid at position 114 is L or V; xxvii. The amino acid at position 124 is I or T; xxviii. The amino acid at position 131 is D or E; xxix. The amino acid at position 143 is H, R, or D; xxx. The amino acid at position 148 is A or V; xxxi. The amino acid at position 149 is A or K; xxxii. The amino acid at position 167 is S or G; xxxiii. The amino acid at position 171 is E or D; xxxiv. The amino acid at position 186 is R, E, or Q; xxxv. The amino acid at position 193 is A or P; xxxvi. The amino acid at position 194 is S or P; xxxvii. The amino acid at position 197 is A or E; xxxviii. The amino acid at position 207 is A or V; xxxix. The amino acid at position 210 is R or K; xl. the amino acid at position 211 is R, K, or deleted; xli. The amino acid at position 212 is S, D, or G; xlii. The amino acid at position 215 is P or E; xliii. The amino acid at position 223 is A or V; xliv. The amino acid at position 234 is L or I; xlv. The amino acid at position 244 is D or E; xlvi. The amino acid at position 276 is L or I; xlvii. The amino acid at position 280 is L or A; xlviii. The amino acid at position 288 is Q or E; xlix. The amino acid at position 291 is L or R; l. The amino acid at position 301 is I or V; li. the amino acid at position 304 is E, A, or G; lii. The amino acid at position 305 is A or W; liii. The amino acid at position 311 is L or R; liv. The amino acid at position 312 is Q or T; lv. The amino acid at position 313 is N or G; lvi. The amino acid at position 316 is R, K, or P; lvii. The amino acid at position 328 is F or L; lviii. The amino acid at position 330 is N or G; lix. The amino acid at position 349 is A or G; lx. The amino acid at position 368 is A or V; lxi. The amino acid at position 370 is L or F; lxii. The amino acid at position 375 is L or E; lxiii. The amino acid at position 385 is C or N; lxiv. the amino acid at position 390 is D, E, or R; and / or lxv. the amino acid at position 397 is G or A; Numbering is relative to amino acid positions in SEQ ID NO:1 or SEQ ID NO:96.

[0017] In one embodiment, the polypeptide is selected from the group consisting of an amino acid sequence having amino acid residues 2 to 397 of SEQ ID NO: 2, an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 15, an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 5, an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 10, an amino acid sequence having amino acid residues 2 to 397 of SEQ ID NO: 8, an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 4, an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 12, an amino acid sequence having amino acid residues 2 to 397 of SEQ ID NO: 91, an amino acid sequence having amino acid residues 2 to 397 of SEQ ID NO: 92, and an amino acid sequence having amino acid residues 2 to 397 of SEQ ID NO: 93. , an amino acid sequence having amino acid residues 2 to 397 of SEQ ID NO: 94, an amino acid sequence having amino acid residues 2 to 397 of SEQ ID NO: 95, an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 14, an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 89, an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 6, an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 90, an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 9, an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 7, an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 11, or an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 13.

[0018] In another embodiment, the polypeptide consists of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:15, SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:8, SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:14, SEQ ID NO:89, SEQ ID NO:6, SEQ ID NO:90, SEQ ID NO:9, SEQ ID NO:7, SEQ ID NO:11, or SEQ ID NO:13.

[0019] In one aspect disclosed herein, a polypeptide capable of hydrolyzing amide bonds in polyamides is provided, wherein the polypeptide comprises or consists of the amino acid sequence of amino acid residues 2 to 394 of SEQ ID NO: 88, or an amino acid sequence having greater than 75% sequence identity thereto. In one embodiment, the polypeptide comprises at least 80% sequence identity to SEQ ID NO: 88. In one embodiment, the polypeptide comprises at least 85% sequence identity to SEQ ID NO: 88. In one embodiment, the polypeptide comprises at least 90% sequence identity to SEQ ID NO: 88. In one embodiment, the polypeptide comprises at least 95% sequence identity to SEQ ID NO: 88.

[0020] In one embodiment, the polypeptide comprises an amino acid sequence having amino acid residues 2 to 394 of SEQ ID NO: 123. In one embodiment, the polypeptide consists of the amino acid sequence of SEQ ID NO:123.

[0021] In another embodiment, the polypeptide comprises, or consists of, an amino acid sequence having amino acid residues 2 to 392 of SEQ ID NO: 3, an amino acid sequence having amino acid residues 2 to 394 of SEQ ID NO: 63, or an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 48. In one embodiment, the polypeptide comprises the amino acid sequence of amino acid residues 2 to 394 of the amino acid sequence of SEQ ID NO: 63.

[0022] In one embodiment, the polypeptide consists of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 63, or SEQ ID NO: 48. In one embodiment, the polypeptide consists of the amino acid sequence of SEQ ID NO: 63. In one embodiment, the polypeptide consists of the amino acid sequence of SEQ ID NO: 3.

[0023] In one aspect disclosed herein, a polypeptide capable of hydrolyzing amide bonds in polyamides is provided, wherein the polypeptide comprises, or consists of, the amino acid sequence of amino acid residues 2 to 391 of SEQ ID NO: 45, or an amino acid sequence having at least 61% sequence identity thereto. In one embodiment, the polypeptide comprises at least 70% sequence identity to SEQ ID NO: 45. In one embodiment, the polypeptide comprises at least 75% sequence identity to SEQ ID NO: 45. In one embodiment, the polypeptide comprises at least 80% sequence identity to SEQ ID NO: 45. In one embodiment, the polypeptide comprises at least 85% sequence identity to SEQ ID NO: 45. In one embodiment, the polypeptide comprises at least 90% sequence identity to SEQ ID NO: 45. In one embodiment, the polypeptide comprises at least 95% sequence identity to SEQ ID NO: 45.

[0024] In one embodiment, the polypeptide comprises or consists of an amino acid sequence having amino acid residues 2 to 391 of SEQ ID NO: 25 or an amino acid sequence having amino acid residues 2 to 391 of SEQ ID NO: 22. In one embodiment, the polypeptide consists of the amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 63, or SEQ ID NO: 22.

[0025] In one embodiment, the polypeptides disclosed herein are capable of hydrolyzing nylon polymers. In another embodiment, the polypeptides disclosed herein have adipic acid mono- and di-N-alkylamide hydrolase activity.

[0026] In another embodiment, the polypeptides disclosed herein are capable of hydrolyzing a nylon polymer, wherein the nylon polymer is a nylon oligomer. In one embodiment, the nylon polymer or nylon oligomer is a nylon 6 polymer, a nylon 6 oligomer, a nylon 6,6 polymer, or a nylon 6,6 oligomer. In another embodiment, the nylon polymer or nylon oligomer is a nylon 6,6 polymer or a nylon 6,6 oligomer. In one embodiment, the nylon oligomer is selected from the group consisting of a nylon 6,6 dimer, trimer, tetramer, pentamer, and hexamer. In one embodiment, the nylon oligomer is a dimer. In one embodiment, the nylon oligomer is a trimer. In one embodiment, the nylon oligomer is a tetramer. In a further embodiment, the nylon 6,6 oligomer is a water-soluble nylon 6,6 oligomer.

[0027] In another embodiment, the polypeptides disclosed herein are capable of hydrolyzing nylon 6,6 oligomers to produce adipic acid and hexamethylenediamine.

[0028] In one embodiment, the polypeptides disclosed herein exhibit increased recombinant expression in a host cell system, increased enzymatic activity, and / or increased thermostability compared to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 73-86.

[0029] The present disclosure also extends to compositions comprising the polypeptides described herein.

[0030] The present disclosure also extends to nucleic acid sequences encoding the polypeptides described herein.

[0031] The present disclosure also extends to expression vectors comprising the nucleic acid sequences described herein.

[0032] The present disclosure also extends to host cells containing the nucleic acid sequences or expression vectors described herein.

[0033] In another aspect, the disclosure provides a method of producing a polypeptide capable of hydrolyzing an amide bond in a polyamide, the method comprising: i) providing a polynucleotide described herein; ii) expressing the polynucleotide in a host cell under conditions sufficient to enable the host cell to produce the polypeptide; and iii) collecting the polypeptide produced by the host cell in ii).

[0034] In another aspect, the disclosure provides a method of hydrolyzing a nylon polymer or nylon oligomer, comprising exposing the nylon polymer or nylon oligomer to a polypeptide, composition, or host cell disclosed herein under conditions sufficient to convert the nylon-6,6 polymer to adipic acid and / or hexamethylenediamine.

[0035] In another aspect, the disclosure provides a method of degrading a nylon-containing product, the method comprising exposing the nylon-containing product to a polypeptide, composition, or host cell disclosed herein.

[0036] In one embodiment, the method disclosed herein comprises: i) chemically processing a nylon polymer, nylon oligomer, or nylon-containing product to produce a nylon oligomer; and ii) exposing the nylon oligomer produced in step (i) to a polypeptide, composition, or host cell disclosed herein under conditions sufficient to produce adipic acid and / or hexamethylenediamine. In one embodiment, the nylon oligomer is a nylon dimer, nylon trimer, nylon tetramer, nylon pentamer, or nylon hexamer. In another embodiment, the nylon oligomer is a water-soluble nylon oligomer.

[0037] In one embodiment, the nylon in the nylon polymer, nylon oligomer, or nylon-containing product is nylon 6 or nylon 6,6. In another embodiment, the nylon is nylon 6,6.

[0038] In another aspect, the disclosure provides a method of hydrolyzing a nylon 6,6 polymer, nylon 6,6 oligomer, or nylon 6,6-containing product, comprising exposing the nylon 6,6 polymer, nylon 6,6 oligomer, or nylon 6,6-containing product to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 73, 74, and 87, or an amino acid sequence having at least 70% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 73, 74, and 87. In one embodiment, the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 73, 74, and 87.

[0039] In another embodiment, the methods described herein further comprise recovering the adipic acid and / or hexamethylenediamine produced in step (ii).

[0040] In another aspect, the present disclosure provides compositions comprising adipic acid and / or hexamethylenediamine recovered by the methods disclosed herein.

[0041] In another aspect, the present disclosure provides a method for producing nylon polymers using the adipic acid and / or hexamethylenediamine compositions recovered by the methods disclosed herein. [Brief explanation of the drawings]

[0042] [Figure 1] 1 shows the enzymatic hydrolysis of nylon 6,6 oligomers (A). The red dashed line in (B) indicates the amide bond that is hydrolyzed by the polypeptides disclosed herein. [Figure 2] FIG. 1 shows the amino acid sequences of nylonase NylB, P07061, and P07062. [Figure 3]Figure 1 shows a whole-cell activity assay demonstrating the activity of variant polypeptides having the amino acid sequences of SEQ ID NOS: 2-15 and 88 compared to various extant / ancestral NylB polypeptides in hydrolyzing amide bonds in polyamides. A colorimetric assay is used to detect free amines in solution (absorbance at 335 nm). An increase in the concentration of free amine groups in the reaction solution corresponds to the hydrolysis of the nylon-6,6 trimer substrate to hexamethylenediamine and adipic acid. [Figure 4] FIG. 1 shows the thermal stability of variants A6 (SEQ ID NO: 4) and C2 (SEQ ID NO: 2) as determined by circular dichroism compared to the existing variants A10 (SEQ ID NO: 75), A11 (SEQ ID NO: 76), B11 (SEQ ID NO: 78), C10 (SEQ ID NO: 79), C11 (SEQ ID NO: 80), H10 (SEQ ID NO: 86) as determined by differential scanning fluorimetry. [Figure 5] FIG. 1 shows an alignment of the polypeptides of SEQ ID NOs: 2, 4 to 15. [Figure 6] Figure 4 shows the activity of immobilized enzyme of SEQ ID NO: 4 on nylon 6,6 dimer, trimer, and tetramer when incubated with immobilized enzyme for 1 hour at 40° C. Substrate conversion was quantified by LC-MS and compared to the control (no enzyme at time 0). [Figure 7] Figure 1 shows the activity of variant polypeptides generated from further engineering (Phase II). High-throughput (crude / whole cell activity) screening of candidate nylon 6,6 enzymes against nylon 6,6 oligomers (compared to Gen1_Top (SEQ ID NO: 4) and a no-enzyme control). Candidates that appeared to have the highest whole cell activity were highlighted (A). Purified candidate enzymes were analyzed for activity against nylon 6,6 dimers, trimers, and tetramers using LC-MS (B). Values ​​on the Y-axis correspond to the % normalized conversion of nylon 6,6 oligomers compared to a no-enzyme control. [Figure 8]Figure 1 shows a refined LC-MS assay comparing the whole cell activity of the top candidates from Phase I (SEQ ID NOS: 4, 48, 88, 63, and 4) and Phase II (SEQ ID NOS: 89, 90, 91, 92, 93, 94) against nylon 6,6 dimer (A), trimer (B), and tetramer (C) compared to existing sequences (SEQ ID NOS: 75, 76, 79, 80, 81, 82, 83, 84, 86). Values ​​on the Y-axis correspond to the peak area of ​​nylon 6,6 oligomers measured using LC-MS. [Figure 9] Figure 1 shows the kinetic stability of the top candidates from Gen2 (SEQ ID NOs: 89, 90, 91, 92, 93, 95) towards nylon 6,6 trimer at 40-80°C. Values ​​on the Y-axis correspond to the % normalized conversion of nylon 6,6 trimer compared to the no enzyme control. [Figure 10] FIG. 1 shows the alignment of the polypeptides of SEQ ID NOs: 2, 4 to 15, 89, and 91 to 95 with consensus II (SEQ ID NO: 96). [Figure 11] 1 shows the activity of SEQ ID NOs: 100-115 and 121-123 against nylon 6,6 trimer compared to the actual sequence of SEQ ID NO: 75. A no enzyme control is included. [Figure 12] FIG. 1 shows the alignment of SEQ ID NOs: 2, 4 to 15, 89, 91 to 95, and 97 to 122 in the production of the consensus sequence of consensus IV (SEQ ID NO: 125). DETAILED DESCRIPTION OF THE INVENTION

[0043] 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 to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For purposes of the present invention, the following terms are defined below.

[0044] Unless expressly stated otherwise, the articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0045] As used herein, the term "about" refers to a number, level, value, dimension, size, or amount that varies by up to 10% (e.g., 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%) relative to a reference number, level, value, dimension, size, or amount.

[0046] Unless the context requires otherwise, throughout this specification the words "has," "have," "having," "comprise," "comprises," and "comprising" are to be interpreted as implying the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0047] The present disclosure generally relates to the design and generation of engineered polypeptides, including those with improved hydrolytic enzyme activity, e.g., higher activity or broader activity, against nylon polymers and nylon oligomers, particularly nylon dimers, trimers, tetramers, and pentamers. This disclosure is premised, at least in part, on the inventors' surprising discovery, from ancestral sequence reconstructions of extant and ancestral variants of the NylB family, that the engineered polypeptides have one or more increased or enhanced properties compared to one or more of the extant enzymes. For example, in certain embodiments, the engineered polypeptides disclosed herein have increased activity in hydrolyzing amide bonds in polyamides, or, as shown in one example provided herein, improved ability to hydrolyze amide bonds in nylon polyamides. In certain embodiments, the engineered polypeptides disclosed herein have increased thermal stability. This is a highly surprising discovery for reasons including that the temperature conditions to which a hypothetical ancestral enzyme might have been exposed would not be significantly different from the temperatures or conditions to which one or more corresponding extant enzymes would be exposed. In certain embodiments, the engineered polypeptides disclosed herein relate to increased recombinant expression in host cell systems, where such host cells have been modified by the insertion of a polynucleotide sequence encoding the enzyme. This is particularly advantageous, since currently known nylonase enzymes exhibit only low levels of enzyme expression in common industrial host organisms.

[0048] In one aspect disclosed herein, there is provided a polypeptide capable of hydrolyzing amide bonds in polyamides, wherein the polypeptide comprises an amino acid sequence of amino acid residues 2 to 398 of SEQ ID NO: 2, or an amino acid sequence having at least 70% sequence identity thereto.

[0049] In one embodiment, the polypeptide has a sequence identical to that at amino acid positions 3, 5, 7, 9, 10, 14, 16, 17, 25, 26, 29, 32, 39, 40, 44, 48, 51, 52, 53, 57, 58, 60, 61, 63, 64, 67, 70, 72, 73, 74, 75, 78, 82, 84, 88, 92, 99, 101, 110, 114, 123, 124, 131, 132, 138, 143, 148, 149, 155, 167, 171, 173, 178, 179, 180, 186, 188, 193, 194, 195, 197, 203, 206, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 239, 242, 243, 244, 245, 246, 247, 248, 249, 250, , 207, 210, 211, 212, 213, 215, 223, 234, 244, 249, 252, 258, 262, 276, 278, 280, 288, 291, 301, 304, 305, 308, 311, 312, 313, 316, 321, 326, 327, 328, 330, 335, 349, 359, 368, 370, 375, 379, 384, 385, 390, 394, 397, and 398, wherein the numbering is relative to SEQ ID NO: 125.

[0050] In one embodiment, the polypeptide has a sequence identical to that at amino acid positions 3, 5, 10, 17, 25, 26, 29, 32, 39, 40, 44, 51, 52, 53, 57, 58, 63, 64, 67, 70, 73, 74, 75, 78, 82, 84, 88, 92, 99, 101, 110, 114, 124, 131, 143, 148, 149, 167, 171, 186, 188, 193, 194, 197, 207, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, and at least one amino acid modification at a position selected from the group consisting of: 2, 215, 223, 234, 244, 249, 252, 258, 276, 280, 288, 291, 301, 304, 305, 311, 312, 313, 316, 326, 327, 328, 330, 349, 359, 368, 370, 375, 385, 390, 397, and 398, wherein the numbering is relative to SEQ ID NO:96.

[0051] In another embodiment, the at least one amino acid modification is Q3 or T3, N5 or D5, M7 or F7, G9 or T9, S10 or F10, P14 or A14, Q16 or G16, Q17 or E17, Q25 or T25, A26 or P26, N29 or S29, A32 or S32, I39 or L39, I40 or V40, N44 or Q44, G48 or A48, A51 or P51, A52 or V52, S53 or W53, A57 or R57, A58 or S58, R60 or A60, D61, A61, or G61, D63 or G63, G64, E64, or R6 4, F67 or L67, P70 or 70del, G72 or A72, R73 or K73, S74 or E74, T75, M75, G75 or W75, A78 or G78, A82 or E82, T84 or S84, A88 or G88, L92 or M92, A99 or S99, W101 or H101, S110 or T110, L114 or V114, G123 or A123, I124 or T124, E131 or D131, R132 or D132, D138 or A138, H143, D143 or R143, A148 or V148, A149 or K149, D155 or G155, S167 or G167, E171 or D171, D173 or A173, D178 or A178, G179 or S179, D180 or A180, R186, Q186 or E186, T188 or M188, A193 or P193, S194 or P194, D195 or P195, A197 or E197, R203 or Y203, L206 or I206, A207 or V207, R210 or K210, R211, K211, or 211del, S212, G212 or D212, D213 or A213, P215 or E215, A22 3 or V223, L234 or I234, D244 or E244, H249 or R249, Q252 or R252, H258 or A258, I262 or V262, I276 or L276, A278 or C278, L280 or A280, E288 or Q288, R291 or L291, I301 or V301, G304, E304 or A304, A305 or W305, D308 or A308, L311 or R311, Q312 or T312, N313 or G313, R316, P316 or K316, R321 or Q321, K326 or H326,and G398 or 398del, wherein the numbering is relative to SEQ ID NO: 96 or SEQ ID NO: 125.

[0052] In one embodiment, the polypeptide has a sequence identical to that at amino acid positions 3, 5, 25, 26, 32, 40, 51, 52, 53, 57, 58, 63, 64, 70, 73, 74, 75, 78, 82, 84, 88, 92, 99, 101, 110, 114, 124, 131, 143, 148, 149, 167, 171, 186, 193, 194, 197, 207, 210, 211, 212, 214, 216, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, and at least one amino acid modification at a position selected from the group consisting of: 2, 215, 223, 234, 244, 276, 280, 288, 291, 301, 304, 305, 311, 312, 313, 316, 328, 330, 349, 368, 370, 375, 385, 390, and 397, where the numbering is relative to the amino acid positions in SEQ ID NO:1.

[0053] In another embodiment, the at least one amino acid modification is Q3 or T3, N5 or D5, Q25 or T25, A26 or P26, A32 or S32, I40 or V40, A51 or P51, A52 or V52, S53 or W53, A57 or R57, A58 or S58, D63 or G63, G64, E64, or R64, P70 or 70del, R73 or K73, S74 or E74, T75, M75, or W75, A78 or G78, A82 or E82, T84 of S84, A88 or G88, L92 or M92, A99 or S99, W101 or H101, S110 or T110, L114 or V114, I124 or T124, E131 of D131, H143, D143, or R143, A148 or V148, A149 or K149, S167 or G167, E171 or D171, R186, Q186, or E186, A193 or P193, S194 or P19 4, A197 or E197, A207 or V207, R210 or K210, R211, K211, or 211del, S212, G212, or D212, P215 or E215, A223 or V223, L234 or I234, D244 or E244, I276 or L276, L280 or A280, E288 or Q288, R291 or L291, I301 or V301, G304, E304, or A304, A305 or W30 5, L311 or R311, Q312 or T312, N313 or G313, R316, P316, or K316, F328 or L328, N330 or G330, A349 or G349, V368 or A368, L370 or F370, L375 or E375, C385 or N385, D390, E390, or R390, and G397 or A397, wherein the numbering is relative to the amino acid positions in SEQ ID NO: 1.

[0054] Polypeptides capable of hydrolyzing amide bonds in polyamides are provided, having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 2. Also provided are polypeptides capable of hydrolyzing amide bonds in polyamides, having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 2. Also provided are polypeptides capable of hydrolyzing amide bonds in polyamides, having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2.

[0055] In one embodiment, the polypeptide comprises an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 96, or an amino acid sequence having at least 80% sequence identity thereto.

[0056] In one embodiment, the polypeptide comprises an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 125, i. the amino acid at position 3 is Q or T; ii. the amino acid at position 5 is N or D; iii. the amino acid at position 7 is M or F; iv. the amino acid at position 9 is G or T; v. The amino acid at position 10 is S or F; vi. The amino acid at position 14 is P or A; vii. The amino acid at position 16 is Q or G; viii. The amino acid at position 17 is Q or E; ix. the amino acid at position 25 is Q or T; x. The amino acid at position 26 is A or P; xi. The amino acid at position 29 is N or S; xii. The amino acid at position 32 is A or S; xiii. The amino acid at position 39 is I or L; xiv. The amino acid at position 40 is I or V; xv. The amino acid at position 44 is N or Q; xvi. The amino acid at position 48 is G or A; xvii. The amino acid at position 51 is A or P; xviii. The amino acid at position 52 is A or V; xix. The amino acid at position 53 is S or W; xx. The amino acid at position 57 is A or R; xxi. The amino acid at position 58 is A or S; xxii. The amino acid at position 60 is R or A; xxiii. The amino acid at position 61 is D, A, or G; xxiv. The amino acid at position 63 is D or G; xxv. The amino acid at position 64 is G, E, or R; xxvi. The amino acid at position 67 is F or L; xxvii. The amino acid at position 70 is P or deleted; xxviii. The amino acid at position 72 is G or A; xxix. The amino acid at position 73 is R or K; xxx. The amino acid at position 74 is S or E; xxxi. The amino acid at position 75 is T, M, G, or W; xxxii. The amino acid at position 78 is A or G; xxxiii. The amino acid at position 82 is A or E; xxxiv. The amino acid at position 84 is S or T; xxxv. The amino acid at position 88 is A or G; xxxvi. The amino acid at position 92 is L or M; xxxvii. The amino acid at position 99 is A or S; xxxviii. The amino acid at position 101 is W or H; xxxix. The amino acid at position 110 is S or T; xl. The amino acid at position 114 is L or V; xli. The amino acid at position 123 is G or A; xlii. The amino acid at position 124 is I or T; xliii. The amino acid at position 131 is D or E; xliv. The amino acid at position 132 is R or D; xlv. The amino acid at position 138 is D or A; xlvi. The amino acid at position 143 is H, R, or D; xlvii. The amino acid at position 148 is A or V; xlviii. The amino acid at position 149 is A or K; xlix. The amino acid at position 155 is D or G; l. The amino acid at position 167 is S or G; li. The amino acid at position 171 is E or D; lii. The amino acid at position 173 is D or A; liii. The amino acid at position 178 is D or A; liv. The amino acid at position 179 is G or S; lv. The amino acid at position 180 is D or A; lvi. The amino acid at position 186 is R, E, or Q; lvii. The amino acid at position 188 is T or M; lviii. The amino acid at position 193 is A or P; lix. The amino acid at position 194 is S or P; lx. The amino acid at position 195 is D or P; lxi. The amino acid at position 197 is A or E; lxii. The amino acid at position 203 is R or Y; lxiii. The amino acid at position 206 is L or I; lxiv. The amino acid at position 207 is A or V; lxv. The amino acid at position 210 is R or K; lxvi. The amino acid at position 211 is R, K, or deleted; lxvii. The amino acid at position 212 is S, D, or G; lxviii. The amino acid at position 213 is D or A; lxix. The amino acid at position 215 is P or E; lxx. The amino acid at position 223 is A or V; lxxi. The amino acid at position 234 is L or I; lxxii. The amino acid at position 244 is D or E; lxxiii. The amino acid at position 249 is H or R; lxxiv. The amino acid at position 252 is Q or R; lxxv. The amino acid at position 258 is H or A; lxxvi. The amino acid at position 262 is I or V; lxxvii. The amino acid at position 276 is L or I; lxxviii. The amino acid at position 278 is A or C; lxxix. The amino acid at position 280 is L, A, or P; lxxx. The amino acid at position 288 is Q or E; lxxxi. The amino acid at position 291 is L or R; lxxxii. The amino acid at position 301 is I or V; lxxxiii. The amino acid at position 304 is E, A, or G; lxxxiv. The amino acid at position 305 is A or W; lxxxv. The amino acid at position 308 is A or D; lxxxvi. The amino acid at position 311 is L or R; lxxxvii. The amino acid at position 312 is Q or T; lxxxviii. The amino acid at position 313 is N or G; lxxxix. The amino acid at position 316 is R, K, or P; xc. The amino acid at position 321 is R or Q; xci. The amino acid at position 326 is K or H; xcii. The amino acid at position 327 is F or L; xciii. The amino acid at position 328 is F or L; xciv. The amino acid at position 330 is N or G; xcv. The amino acid at position 335 is S or N; xcvi. The amino acid at position 349 is A or G; xcvii. The amino acid at position 359 is I or V; xcviii. The amino acid at position 368 is A or V; xcix. The amino acid at position 370 is L or F; c. the amino acid at position 375 is L or E; ci. The amino acid at position 379 is D or A; cii. The amino acid at position 384 is L or E; ciii. The amino acid at position 385 is C, M, or N; civ. The amino acid at position 390 is D, E, or R; cv. The amino acid at position 394 is R or A; cvi. the amino acid at position 397 is G or A, and / or cvii. the amino acid at position 398 is G or is deleted; Numbering is relative to amino acid positions in SEQ ID NO:1, SEQ ID NO:96, or SEQ ID NO:125.

[0057] In one embodiment, the polypeptide comprises an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 96, i. the amino acid at position X1 is Q or T; ii. the amino acid at position X2 is N or D; iii. the amino acid at position X3 is S or F; iv. The amino acid at position X4 is Q or E; v. The amino acid at position X5 is Q or T; vi. The amino acid at position X6 is A or P; vii. The amino acid at position X7 is N or S; viii. The amino acid at position X8 is A or S; ix. The amino acid at position X9 is I or L; x. The amino acid at position X10 is I or V; xi. The amino acid at position X11 is N or Q; xii. The amino acid at position X12 is A or P; xiii. The amino acid at position X13 is A or V; xiv. The amino acid at position X14 is S or W; xv. The amino acid at position X15 is A or R; xvi. The amino acid at position X16 is A or S; xvii. The amino acid at position X17 is D or G; xviii. The amino acid at position X18 is G, E, or R; xix. The amino acid at position X19 is F or L; xx. The amino acid at position X20 is P or deleted; xxi. The amino acid at position X21 is R or K; xxii. The amino acid at position X22 is S or E; xxiii. The amino acid at position X23 is T, M, G, or W; xxiv. The amino acid at position X24 is A or G; xxv. The amino acid at position X25 is A or E; xxvi. The amino acid at position X26 is S or T; xxvii. The amino acid at position X27 is A or G; xxviii. The amino acid at position X28 is L or M; xxix. The amino acid at position X29 is A or S; xxx. The amino acid at position X30 is W or H; xxxi. The amino acid at position X31 is S or T; xxxii. The amino acid at position X32 is L or V; xxxiii. The amino acid at position X33 is I or T; xxxiv. The amino acid at position X34 is D or E; xxxv. The amino acid at position X35 is H, R, or D; xxxvi. The amino acid at position X36 is A or V; xxxvii. The amino acid at position X37 is A or K; xxxviii. The amino acid at position X38 is S or G; xxxix. The amino acid at position X39 is E or D; xl. The amino acid at position X40 is R, E, or Q; xli. The amino acid at position X41 is T or M; xlii. The amino acid at position X42 is A or P; xliii. The amino acid at position X43 is S or P; xliv. The amino acid at position X44 is A or E; xlv. The amino acid at position X45 is A or V; xlvi. The amino acid at position X46 is R or K; xlvii. The amino acid at position X47 is R, K, or deleted; xlviii. The amino acid at position X48 is S, D, or G; xlix. The amino acid at position X49 is P or E; l. The amino acid at position X50 is A or V; li. The amino acid at position X51 is L or I; lii. The amino acid at position X52 is D or E; liii. The amino acid at position X53 is H or R; liv. The amino acid at position X54 is Q or R; lv. the amino acid at position X55 is H or A; lvi. The amino acid at position X56 is L or I; lvii. The amino acid at position X57 is L or A; lviii. The amino acid at position X58 is Q or E; lix. The amino acid at position X59 is L or R; lx. The amino acid at position X60 is I or V; lxi. The amino acid at position X61 is E, A, or G; lxii. The amino acid at position X62 is A or W; lxiii. The amino acid at position X63 is L or R; lxiv. The amino acid at position X64 is Q or T; lxv. The amino acid at position X65 is N or G; lxvi. The amino acid at position X66 is R, K, or P; lxvii. The amino acid at position X67 is K or H; lxviii. The amino acid at position X68 is F or L; lxix. The amino acid at position X69 is F or L; lxx. The amino acid at position X70 is N or G; lxxi. The amino acid at position X71 is A or G; lxxii. The amino acid at position X72 is I or V; lxxiii. The amino acid at position X73 is A or V; lxxiv. The amino acid at position X74 is L or F; lxxv. The amino acid at position X75 is L or E; lxxvi. The amino acid at position X76 is C, M, or N; lxxvii. The amino acid at position X77 is D, E, or R; lxxviii. the amino acid at position X78 is G or A, and / or lxxix. the amino acid at position X79 is G or is deleted; Numbering is relative to amino acid positions in SEQ ID NO:1 or SEQ ID NO:96.

[0058] In one embodiment, the polypeptide comprises the following amino acid sequence: i. the amino acid at position 3 is Q or T; ii. the amino acid at position 5 is N or D; iii. the amino acid at position 10 is S or F; iv. the amino acid at position 17 is Q or E; v. The amino acid at position 25 is Q or T; vi. The amino acid at position 26 is A or P; vii. The amino acid at position 29 is N or S; viii. The amino acid at position 32 is A or S; ix. the amino acid at position 39 is I or L; x. The amino acid at position 40 is I or V; xi. The amino acid at position 44 is N or Q; xii. The amino acid at position 51 is A or P; xiii. The amino acid at position 52 is A or V; xiv. The amino acid at position 53 is S or W; xv. The amino acid at position 57 is A or R; xvi. The amino acid at position 58 is A or S; xvii. The amino acid at position 63 is D or G; xviii. The amino acid at position 64 is G, E, or R; xix. The amino acid at position 67 is F or L; xx. The amino acid at position 70 is P or deleted; xxi. The amino acid at position 73 is R or K; xxii. The amino acid at position 74 is S or E; xxiii. The amino acid at position 75 is T, M, G, or W; xxiv. The amino acid at position 78 is A or G; xxv. The amino acid at position 82 is A or E; xxvi. The amino acid at position 84 is S or T; xxvii. The amino acid at position 88 is A or G; xxviii. The amino acid at position 92 is L or M; xxix. The amino acid at position 99 is A or S; xxx. The amino acid at position 101 is W or H; xxxi. The amino acid at position 110 is S or T; xxxii. The amino acid at position 114 is L or V; xxxiii. The amino acid at position 124 is I or T; xxxiv. The amino acid at position 131 is D or E; xxxv. The amino acid at position 143 is H, R, or D; xxxvi. The amino acid at position 148 is A or V; xxxvii. The amino acid at position 149 is A or K; xxxviii. The amino acid at position 167 is S or G; xxxix. The amino acid at position 171 is E or D; xl. The amino acid at position 186 is R, E, or Q; xli. The amino acid at position 188 is T or M; xlii. The amino acid at position 193 is A or P; xliii. The amino acid at position 194 is S or P; xliv. The amino acid at position 197 is A or E; xlv. The amino acid at position 207 is A or V; xlvi. The amino acid at position 210 is R or K; xlvii. The amino acid at position 211 is R, K, or deleted; xlviii. the amino acid at position 212 is S, D, or G; xlix. The amino acid at position 215 is P or E; l. The amino acid at position 223 is A or V; li. The amino acid at position 234 is L or I; lii. the amino acid at position 244 is D or E; liii. The amino acid at position 249 is H or R; liv. The amino acid at position 252 is Q or R; lv. The amino acid at position 258 is H or A; lvi. The amino acid at position 276 is L or I; lvii. The amino acid at position 280 is L or A; lviii. The amino acid at position 288 is Q or E; lix. The amino acid at position 291 is L or R; lx. The amino acid at position 301 is I or V; lxi. The amino acid at position 304 is E, A, or G; lxii. The amino acid at position 305 is A or W; lxiii. The amino acid at position 311 is L or R; lxiv. The amino acid at position 312 is Q or T; lxv. the amino acid at position 313 is N or G; lxvi. The amino acid at position 316 is R, K, or P; lxvii. The amino acid at position 326 is K or H; lxviii. The amino acid at position 327 is F or L; lxix. The amino acid at position 328 is F or L; lxx. The amino acid at position 330 is N or G; lxxi. The amino acid at position 349 is A or G; lxxii. The amino acid at position 359 is I or V; lxxiii. The amino acid at position 368 is A or V; lxxiv. The amino acid at position 370 is L or F; lxxv. The amino acid at position 375 is L or E; lxxvi. The amino acid at position 385 is C, M, or N; lxxvii. The amino acid at position 390 is D, E, or R; lxxviii. the amino acid at position 397 is G or A, and / or lxxix. the amino acid at position 398 is G or is deleted; Numbering is relative to amino acid positions in SEQ ID NO:1 or SEQ ID NO:96.

[0059] In one embodiment, the polypeptide comprises the following amino acid sequence: i. the amino acid at position 3 is Q; ii. the amino acid at position 5 is N; iii. the amino acid at position 10 is F; iv. the amino acid at position 17 is Q; v. the amino acid at position 25 is Q; vi. the amino acid at position 26 is A; vii. the amino acid at position 29 is N; viii. the amino acid at position 32 is S; ix. the amino acid at position 39 is I; x. the amino acid at position 40 is V; xi. the amino acid at position 44 is N; xii. the amino acid at position 51 is P; xiii. the amino acid at position 52 is V; xiv. the amino acid at position 53 is S; xv. the amino acid at position 57 is R; xvi. The amino acid at position 58 is A; xvii. the amino acid at position 63 is G; xviii. the amino acid at position 64 is E; xix. The amino acid at position 67 is F; xx. The amino acid at position 70 is P; xxi. The amino acid at position 73 is K; xxii. the amino acid at position 74 is E; xxiii. the amino acid at position 75 is T; xxiv. The amino acid at position 78 is G; xxv. The amino acid at position 82 is E; xxvi. The amino acid at position 84 is T; xxvii. The amino acid at position 88 is G; xxviii. The amino acid at position 92 is L; xxix. the amino acid at position 99 is A; xxx. The amino acid at position 101 is H; xxxi. The amino acid at position 110 is S; xxxii. The amino acid at position 114 is V; xxxiii. the amino acid at position 124 is T; xxxiv. The amino acid at position 131 is E; xxxv. The amino acid at position 143 is H; xxxvi. The amino acid at position 148 is A; xxxvii. The amino acid at position 149 is A; xxxviii. The amino acid at position 167 is S; xxxix. The amino acid at position 171 is E; xl. the amino acid at position 186 is R; xli. the amino acid at position 188 is T; xlii. the amino acid at position 193 is P; xliii. the amino acid at position 194 is S; xliv. the amino acid at position 197 is A; xlv. The amino acid at position 207 is A; xlvi. The amino acid at position 210 is K; xlvii. the amino acid at position 211 is K; xlviii. the amino acid at position 212 is G; xlix. the amino acid at position 215 is P; l. the amino acid at position 223 is V; li. the amino acid at position 234 is L; lii. the amino acid at position 244 is D; liii. the amino acid at position 249 is H; liv. the amino acid at position 252 is Q; lv. The amino acid at position 258 is H; lvi. The amino acid at position 276 is I; lvii. the amino acid at position 280 is L; lviii. the amino acid at position 288 is E; lix. The amino acid at position 291 is R; lx. The amino acid at position 301 is I; lxi. the amino acid at position 304 is E; lxii. the amino acid at position 305 is A; lxiii. the amino acid at position 311 is L; lxiv. The amino acid at position 312 is T; lxv. the amino acid at position 313 is N; lxvi. the amino acid at position 316 is K; lxvii. the amino acid at position 326 is K; lxviii. the amino acid at position 327 is F; lxix. The amino acid at position 328 is L; lxx. the amino acid at position 330 is N; lxxi. The amino acid at position 349 is A; lxxii. the amino acid at position 359 is I; lxxiii. the amino acid at position 368 is V; lxxiv. the amino acid at position 370 is L; lxxv. the amino acid at position 375 is L; lxxvi. the amino acid at position 385 is C; lxxvii. the amino acid at position 390 is R; lxxviii. the amino acid at position 397 is G; lxxix. the amino acid at position 398 is G; Numbering is with respect to amino acid positions in SEQ ID NO: 1 or SEQ ID NO: 96. In one embodiment, the polypeptide comprises an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 89. In another embodiment, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 89.

[0060] In one embodiment, the polypeptide comprises the following amino acid sequence: i. the amino acid at position 3 is T; ii. the amino acid at position 5 is N; iii. the amino acid at position 10 is F; iv. the amino acid at position 17 is Q; v. the amino acid at position 25 is Q; vi. The amino acid at position 26 is P; vii. the amino acid at position 29 is N; viii. the amino acid at position 32 is A; ix. the amino acid at position 39 is I; x. the amino acid at position 40 is I; xi. the amino acid at position 44 is N; xii. the amino acid at position 51 is P; xiii. the amino acid at position 52 is V; xiv. the amino acid at position 53 is S; xv. the amino acid at position 57 is R; xvi. The amino acid at position 58 is S; xvii. the amino acid at position 63 is G; xviii. the amino acid at position 64 is E; xix. The amino acid at position 67 is F; xx. The amino acid at position 70 is P; xxi. The amino acid at position 73 is K; xxii. the amino acid at position 74 is E; xxiii. the amino acid at position 75 is W; xxiv. The amino acid at position 78 is G; xxv. The amino acid at position 82 is A; xxvi. The amino acid at position 84 is S; xxvii. The amino acid at position 88 is A; xxviii. The amino acid at position 92 is M; xxix. the amino acid at position 99 is A; xxx. The amino acid at position 101 is W; xxxi. The amino acid at position 110 is S; xxxii. The amino acid at position 114 is V; xxxiii. The amino acid at position 124 is I; xxxiv. The amino acid at position 131 is E; xxxv. the amino acid at position 143 is R; xxxvi. The amino acid at position 148 is A; xxxvii. The amino acid at position 149 is A; xxxviii. The amino acid at position 167 is S; xxxix. The amino acid at position 171 is E; xl. the amino acid at position 186 is R; xli. the amino acid at position 188 is T; xlii. the amino acid at position 193 is P; xliii. the amino acid at position 194 is P; xliv. the amino acid at position 197 is A; xlv. The amino acid at position 207 is V; xlvi. The amino acid at position 210 is K; xlvii. the amino acid at position 211 is K; xlviii. the amino acid at position 212 is G; xlix. the amino acid at position 215 is P; l. the amino acid at position 223 is A; li. the amino acid at position 234 is L; lii. the amino acid at position 244 is D; liii. the amino acid at position 249 is H; liv. the amino acid at position 252 is Q; lv. The amino acid at position 258 is H; lvi. The amino acid at position 276 is I; lvii. the amino acid at position 280 is A; lviii. the amino acid at position 288 is E; lix. The amino acid at position 291 is R; lx. The amino acid at position 301 is I; lxi. the amino acid at position 304 is E; lxii. the amino acid at position 305 is W; lxiii. the amino acid at position 311 is L; lxiv. The amino acid at position 312 is Q; lxv. the amino acid at position 313 is G; lxvi. the amino acid at position 316 is R; lxvii. the amino acid at position 326 is K; lxviii. the amino acid at position 327 is F; lxix. The amino acid at position 328 is F; lxx. the amino acid at position 330 is N; lxxi. The amino acid at position 349 is A; lxxii. the amino acid at position 359 is I; lxxiii. the amino acid at position 368 is V; lxxiv. the amino acid at position 370 is L; lxxv. the amino acid at position 375 is E; lxxvi. the amino acid at position 385 is N; lxxvii. the amino acid at position 390 is E; lxxviii. the amino acid at position 397 is A; lxxix. the amino acid at position 398 is G; Numbering is with respect to amino acid positions in SEQ ID NO: 1 or SEQ ID NO: 96. In one embodiment, the polypeptide comprises an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 90. In another embodiment, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 90.

[0061] In one embodiment, the polypeptide comprises the following amino acid sequence: i. the amino acid at position 3 is Q; ii. the amino acid at position 5 is N; iii. the amino acid at position 10 is S; iv. the amino acid at position 17 is E; v. the amino acid at position 25 is Q; vi. the amino acid at position 26 is A; vii. the amino acid at position 29 is S; viii. the amino acid at position 32 is S; ix. the amino acid at position 39 is L; x. the amino acid at position 40 is V; xi. the amino acid at position 44 is Q; xii. the amino acid at position 51 is A; xiii. the amino acid at position 52 is A; xiv. the amino acid at position 53 is S; xv. the amino acid at position 57 is A; xvi. The amino acid at position 58 is A; xvii. the amino acid at position 63 is G; xviii. the amino acid at position 64 is E; xix. The amino acid at position 67 is L; xx. The amino acid at position 70 is P; xxi. The amino acid at position 73 is K; xxii. the amino acid at position 74 is E; xxiii. the amino acid at position 75 is G; xxiv. The amino acid at position 78 is A; xxv. The amino acid at position 82 is E; xxvi. The amino acid at position 84 is S; xxvii. The amino acid at position 88 is G; xxviii. The amino acid at position 92 is L; xxix. the amino acid at position 99 is A; xxx. The amino acid at position 101 is H; xxxi. The amino acid at position 110 is S; xxxii. The amino acid at position 114 is V; xxxiii. the amino acid at position 124 is T; xxxiv. The amino acid at position 131 is E; xxxv. The amino acid at position 143 is D; xxxvi. The amino acid at position 148 is V; xxxvii. The amino acid at position 149 is A; xxxviii. The amino acid at position 167 is S; xxxix. The amino acid at position 171 is E; xl. the amino acid at position 186 is R; xli. the amino acid at position 188 is M; xlii. the amino acid at position 193 is P; xliii. the amino acid at position 194 is S; xliv. the amino acid at position 197 is E; xlv. The amino acid at position 207 is A; xlvi. The amino acid at position 210 is K; xlvii. the amino acid at position 211 is K; xlviii. the amino acid at position 212 is G; xlix. the amino acid at position 215 is P; l. the amino acid at position 223 is V; li. the amino acid at position 234 is L; lii. the amino acid at position 244 is D; liii. the amino acid at position 249 is R; liv. The amino acid at position 252 is R; lv. The amino acid at position 258 is H; lvi. The amino acid at position 276 is I; lvii. the amino acid at position 280 is L; lviii. the amino acid at position 288 is E; lix. The amino acid at position 291 is R; lx. The amino acid at position 301 is I; lxi. the amino acid at position 304 is E; lxii. the amino acid at position 305 is A; lxiii. the amino acid at position 311 is L; lxiv. The amino acid at position 312 is T; lxv. the amino acid at position 313 is N; lxvi. the amino acid at position 316 is R; lxvii. the amino acid at position 326 is H; lxviii. the amino acid at position 327 is L; lxix. The amino acid at position 328 is L; lxx. the amino acid at position 330 is N; lxxi. The amino acid at position 349 is A; lxxii. the amino acid at position 359 is V; lxxiii. the amino acid at position 368 is V; lxxiv. the amino acid at position 370 is L; lxxv. the amino acid at position 375 is L; lxxvi. the amino acid at position 385 is C; lxxvii. the amino acid at position 390 is R; lxxviii. the amino acid at position 397 is G; lxxix. The amino acid at position 398 is deleted, Numbering is with respect to amino acid positions in SEQ ID NO: 1 or SEQ ID NO: 96. In one embodiment, the polypeptide comprises an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 91. In another embodiment, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 91.

[0062] In one embodiment, the polypeptide comprises the following amino acid sequence: i. the amino acid at position 3 is Q; ii. the amino acid at position 5 is N; iii. the amino acid at position 10 is S; iv. the amino acid at position 17 is E; v. the amino acid at position 25 is Q; vi. the amino acid at position 26 is A; vii. the amino acid at position 29 is S; viii. the amino acid at position 32 is S; ix. the amino acid at position 39 is L; x. the amino acid at position 40 is V; xi. the amino acid at position 44 is Q; xii. the amino acid at position 51 is P; xiii. the amino acid at position 52 is A; xiv. the amino acid at position 53 is S; xv. the amino acid at position 57 is A; xvi. The amino acid at position 58 is A; xvii. the amino acid at position 63 is G; xviii. the amino acid at position 64 is E; xix. The amino acid at position 67 is L; xx. The amino acid at position 70 is P; xxi. The amino acid at position 73 is K; xxii. the amino acid at position 74 is E; xxiii. the amino acid at position 75 is G; xxiv. The amino acid at position 78 is A; xxv. The amino acid at position 82 is E; xxvi. The amino acid at position 84 is S; xxvii. The amino acid at position 88 is G; xxviii. The amino acid at position 92 is L; xxix. the amino acid at position 99 is A; xxx. The amino acid at position 101 is H; xxxi. The amino acid at position 110 is S; xxxii. The amino acid at position 114 is V; xxxiii. the amino acid at position 124 is T; xxxiv. The amino acid at position 131 is E; xxxv. The amino acid at position 143 is D; xxxvi. The amino acid at position 148 is V; xxxvii. The amino acid at position 149 is A; xxxviii. The amino acid at position 167 is S; xxxix. The amino acid at position 171 is E; xl. the amino acid at position 186 is R; xli. the amino acid at position 188 is M; xlii. the amino acid at position 193 is P; xliii. the amino acid at position 194 is S; xliv. the amino acid at position 197 is E; xlv. The amino acid at position 207 is A; xlvi. The amino acid at position 210 is K; xlvii. the amino acid at position 211 is K; xlviii. the amino acid at position 212 is G; xlix. the amino acid at position 215 is P; l. the amino acid at position 223 is V; li. the amino acid at position 234 is L; lii. the amino acid at position 244 is D; liii. the amino acid at position 249 is R; liv. The amino acid at position 252 is R; lv. The amino acid at position 258 is A, lvi. The amino acid at position 276 is I; lvii. the amino acid at position 280 is L; lviii. the amino acid at position 288 is E; lix. The amino acid at position 291 is R; lx. The amino acid at position 301 is I; lxi. the amino acid at position 304 is E; lxii. the amino acid at position 305 is A; lxiii. the amino acid at position 311 is L; lxiv. The amino acid at position 312 is T; lxv. the amino acid at position 313 is N; lxvi. the amino acid at position 316 is R; lxvii. the amino acid at position 326 is H; lxviii. the amino acid at position 327 is L; lxix. The amino acid at position 328 is F; lxx. the amino acid at position 330 is N; lxxi. The amino acid at position 349 is A; lxxii. the amino acid at position 359 is V; lxxiii. the amino acid at position 368 is V; lxxiv. the amino acid at position 370 is L; lxxv. the amino acid at position 375 is L; lxxvi. the amino acid at position 385 is M; lxxvii. the amino acid at position 390 is R; lxxviii. the amino acid at position 397 is G; lxxix. The amino acid at position 398 is deleted, Numbering is with respect to amino acid positions in SEQ ID NO: 1 or SEQ ID NO: 96. In one embodiment, the polypeptide comprises an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 92. In another embodiment, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 92.

[0063] In one embodiment, the polypeptide comprises the following amino acid sequence: i. the amino acid at position 3 is Q; ii. the amino acid at position 5 is N; iii. the amino acid at position 10 is S; iv. the amino acid at position 17 is E; v. the amino acid at position 25 is Q; vi. the amino acid at position 26 is A; vii. the amino acid at position 29 is S; viii. the amino acid at position 32 is S; ix. the amino acid at position 39 is L; x. the amino acid at position 40 is V; xi. the amino acid at position 44 is Q; xii. the amino acid at position 51 is P; xiii. the amino acid at position 52 is A; xiv. the amino acid at position 53 is S; xv. the amino acid at position 57 is A; xvi. The amino acid at position 58 is A; xvii. the amino acid at position 63 is G; xviii. the amino acid at position 64 is E; xix. The amino acid at position 67 is L; xx. The amino acid at position 70 is P; xxi. The amino acid at position 73 is K; xxii. the amino acid at position 74 is E; xxiii. the amino acid at position 75 is G; xxiv. The amino acid at position 78 is A; xxv. The amino acid at position 82 is E; xxvi. The amino acid at position 84 is S; xxvii. The amino acid at position 88 is G; xxviii. The amino acid at position 92 is L; xxix. the amino acid at position 99 is A; xxx. The amino acid at position 101 is H; xxxi. The amino acid at position 110 is S; xxxii. The amino acid at position 114 is V; xxxiii. the amino acid at position 124 is T; xxxiv. The amino acid at position 131 is E; xxxv. The amino acid at position 143 is D; xxxvi. The amino acid at position 148 is V; xxxvii. The amino acid at position 149 is A; xxxviii. The amino acid at position 167 is S; xxxix. The amino acid at position 171 is E; xl. the amino acid at position 186 is R; xli. the amino acid at position 188 is M; xlii. the amino acid at position 193 is P; xliii. the amino acid at position 194 is S; xliv. the amino acid at position 197 is E; xlv. The amino acid at position 207 is A; xlvi. The amino acid at position 210 is K; xlvii. the amino acid at position 211 is K; xlviii. the amino acid at position 212 is G; xlix. the amino acid at position 215 is P; l. the amino acid at position 223 is V; li. the amino acid at position 234 is L; lii. the amino acid at position 244 is D; liii. the amino acid at position 249 is R; liv. The amino acid at position 252 is R; lv. The amino acid at position 258 is A, lvi. The amino acid at position 276 is I; lvii. the amino acid at position 280 is L; lviii. the amino acid at position 288 is E; lix. The amino acid at position 291 is R; lx. The amino acid at position 301 is I; lxi. the amino acid at position 304 is E; lxii. the amino acid at position 305 is A; lxiii. the amino acid at position 311 is L; lxiv. The amino acid at position 312 is T; lxv. the amino acid at position 313 is N; lxvi. the amino acid at position 316 is R; lxvii. the amino acid at position 326 is H; lxviii. the amino acid at position 327 is L; lxix. The amino acid at position 328 is L; lxx. the amino acid at position 330 is N; lxxi. The amino acid at position 349 is A; lxxii. the amino acid at position 359 is V; lxxiii. the amino acid at position 368 is V; lxxiv. the amino acid at position 370 is L; lxxv. the amino acid at position 375 is L; lxxvi. the amino acid at position 385 is M; lxxvii. the amino acid at position 390 is R; lxxviii. the amino acid at position 397 is G; lxxix. The amino acid at position 398 is deleted, Numbering is with respect to amino acid positions in SEQ ID NO: 1 or SEQ ID NO: 96. In one embodiment, the polypeptide comprises an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 93. In another embodiment, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 93.

[0064] In one embodiment, the polypeptide comprises the following amino acid sequence: i. the amino acid at position 3 is Q; ii. the amino acid at position 5 is N; iii. the amino acid at position 10 is S; iv. the amino acid at position 17 is E; v. the amino acid at position 25 is Q; vi. the amino acid at position 26 is A; vii. the amino acid at position 29 is S; viii. the amino acid at position 32 is S; ix. the amino acid at position 39 is L; x. the amino acid at position 40 is V; xi. the amino acid at position 44 is Q; xii. the amino acid at position 51 is A; xiii. the amino acid at position 52 is A; xiv. the amino acid at position 53 is S; xv. the amino acid at position 57 is A; xvi. The amino acid at position 58 is A; xvii. the amino acid at position 63 is G; xviii. the amino acid at position 64 is E; xix. The amino acid at position 67 is L; xx. The amino acid at position 70 is P; xxi. The amino acid at position 73 is K; xxii. the amino acid at position 74 is E; xxiii. the amino acid at position 75 is G; xxiv. The amino acid at position 78 is A; xxv. The amino acid at position 82 is E; xxvi. The amino acid at position 84 is S; xxvii. The amino acid at position 88 is G; xxviii. The amino acid at position 92 is L; xxix. the amino acid at position 99 is A; xxx. The amino acid at position 101 is H; xxxi. The amino acid at position 110 is S; xxxii. The amino acid at position 114 is V; xxxiii. the amino acid at position 124 is T; xxxiv. The amino acid at position 131 is E; xxxv. The amino acid at position 143 is D; xxxvi. The amino acid at position 148 is V; xxxvii. The amino acid at position 149 is A; xxxviii. The amino acid at position 167 is S; xxxix. The amino acid at position 171 is E; xl. the amino acid at position 186 is R; xli. the amino acid at position 188 is M; xlii. the amino acid at position 193 is P; xliii. the amino acid at position 194 is S; xliv. the amino acid at position 197 is E; xlv. The amino acid at position 207 is A; xlvi. The amino acid at position 210 is K; xlvii. the amino acid at position 211 is K; xlviii. the amino acid at position 212 is G; xlix. the amino acid at position 215 is P; l. the amino acid at position 223 is V; li. the amino acid at position 234 is L; lii. the amino acid at position 244 is D; liii. the amino acid at position 249 is R; liv. The amino acid at position 252 is R; lv. The amino acid at position 258 is A, lvi. The amino acid at position 276 is I; lvii. the amino acid at position 280 is L; lviii. the amino acid at position 288 is E; lix. The amino acid at position 291 is R; lx. The amino acid at position 301 is I; lxi. the amino acid at position 304 is E; lxii. the amino acid at position 305 is A; lxiii. the amino acid at position 311 is L; lxiv. The amino acid at position 312 is T; lxv. the amino acid at position 313 is N; lxvi. the amino acid at position 316 is R; lxvii. the amino acid at position 326 is H; lxviii. the amino acid at position 327 is L; lxix. The amino acid at position 328 is L; lxx. the amino acid at position 330 is N; lxxi. The amino acid at position 349 is A; lxxii. the amino acid at position 359 is V; lxxiii. the amino acid at position 368 is V; lxxiv. the amino acid at position 370 is L; lxxv. the amino acid at position 375 is L; lxxvi. the amino acid at position 385 is M; lxxvii. the amino acid at position 390 is R; lxxviii. the amino acid at position 397 is G; lxxix. The amino acid at position 398 is deleted, Numbering is with respect to amino acid positions in SEQ ID NO: 1 or SEQ ID NO: 96. In one embodiment, the polypeptide comprises an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 94. In another embodiment, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 94.

[0065] In one embodiment, the polypeptide comprises the following amino acid sequence: i. the amino acid at position 3 is Q; ii. the amino acid at position 5 is N; iii. the amino acid at position 10 is S; iv. the amino acid at position 17 is E; v. the amino acid at position 25 is Q; vi. the amino acid at position 26 is A; vii. the amino acid at position 29 is S; viii. the amino acid at position 32 is S; ix. the amino acid at position 39 is L; x. the amino acid at position 40 is V; xi. the amino acid at position 44 is Q; xii. the amino acid at position 51 is P; xiii. the amino acid at position 52 is A; xiv. the amino acid at position 53 is S; xv. the amino acid at position 57 is A; xvi. The amino acid at position 58 is A; xvii. the amino acid at position 63 is G; xviii. the amino acid at position 64 is R; xix. The amino acid at position 67 is L; xx. The amino acid at position 70 is P; xxi. The amino acid at position 73 is K; xxii. the amino acid at position 74 is E; xxiii. the amino acid at position 75 is G; xxiv. The amino acid at position 78 is A; xxv. The amino acid at position 82 is A; xxvi. The amino acid at position 84 is S; xxvii. The amino acid at position 88 is G; xxviii. The amino acid at position 92 is L; xxix. the amino acid at position 99 is A; xxx. The amino acid at position 101 is H; xxxi. The amino acid at position 110 is S; xxxii. The amino acid at position 114 is V; xxxiii. the amino acid at position 124 is T; xxxiv. The amino acid at position 131 is E; xxxv. The amino acid at position 143 is D; xxxvi. The amino acid at position 148 is V; xxxvii. The amino acid at position 149 is A; xxxviii. The amino acid at position 167 is S; xxxix. The amino acid at position 171 is E; xl. the amino acid at position 186 is R; xli. the amino acid at position 188 is M; xlii. the amino acid at position 193 is P; xliii. the amino acid at position 194 is S; xliv. the amino acid at position 197 is E; xlv. The amino acid at position 207 is A; xlvi. The amino acid at position 210 is K; xlvii. the amino acid at position 211 is K; xlviii. the amino acid at position 212 is G; xlix. the amino acid at position 215 is P; l. the amino acid at position 223 is V; li. the amino acid at position 234 is L; lii. the amino acid at position 244 is D; liii. the amino acid at position 249 is R; liv. The amino acid at position 252 is R; lv. The amino acid at position 258 is H; lvi. The amino acid at position 276 is I; lvii. the amino acid at position 280 is L; lviii. the amino acid at position 288 is E; lix. The amino acid at position 291 is R; lx. The amino acid at position 301 is I; lxi. the amino acid at position 304 is E; lxii. the amino acid at position 305 is A; lxiii. the amino acid at position 311 is L; lxiv. The amino acid at position 312 is T; lxv. the amino acid at position 313 is N; lxvi. the amino acid at position 316 is R; lxvii. the amino acid at position 326 is H; lxviii. the amino acid at position 327 is L; lxix. The amino acid at position 328 is L; lxx. the amino acid at position 330 is N; lxxi. The amino acid at position 349 is A; lxxii. the amino acid at position 359 is V; lxxiii. the amino acid at position 368 is V; lxxiv. the amino acid at position 370 is L; lxxv. the amino acid at position 375 is L; lxxvi. the amino acid at position 385 is C; lxxvii. the amino acid at position 390 is R; lxxviii. the amino acid at position 397 is G; lxxix. The amino acid at position 398 is deleted.

[0066] Numbering is with respect to amino acid positions in SEQ ID NO: 1 or SEQ ID NO: 96. In one embodiment, the polypeptide comprises an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 95. In another embodiment, the polypeptide comprises, or consists of, the amino acid sequence of SEQ ID NO: 95. In one embodiment, the polypeptide comprises an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 1, or an amino acid sequence having at least 80% sequence identity thereto.

[0067] In another embodiment, the polypeptide comprises an amino acid sequence having amino acid residues 2-398 of SEQ ID NO:1, i. the amino acid at position X1 is Q or T; ii. the amino acid at position X2 is N or D; iii. the amino acid at position X3 is Q or T; iv. The amino acid at position X4 is A or P; v. The amino acid at position X5 is A or S; vi. The amino acid at position X6 is I or V; vii. The amino acid at position X7 is A or P; viii. The amino acid at position X8 is A or V; ix. The amino acid at position X9 is S or W; x. The amino acid at position X10 is A or R; xi. The amino acid at position X11 is A or S; xii. The amino acid at position X12 is D or G; xiii. The amino acid at position X13 is G, E, or R; xiv. The amino acid at position X14 is P or is deleted; xv. The amino acid at position X15 is R or K; xvi. The amino acid at position X16 is S or E; xvii. The amino acid at position X17 is T, M, or W; xviii. The amino acid at position X18 is A or G; xix. The amino acid at position X19 is A or E; xx. The amino acid at position X20 is S or T; xxi. The amino acid at position X21 is A or G; xxii. The amino acid at position X22 is L or M; xxiii. The amino acid at position X23 is A or S; xxiv. The amino acid at position X24 is W or H; xxv. The amino acid at position X25 is S or T; xxvi. The amino acid at position X26 is L or V; xxvii. The amino acid at position X27 is I or T; xxviii. The amino acid at position X28 is D or E; xxix. The amino acid at position X29 is H, R, or D; xxx. The amino acid at position X30 is A or V; xxxi. The amino acid at position X31 is A or K; xxxii. The amino acid at position X32 is S or G; xxxiii. The amino acid at position X33 is E or D; xxxiv. The amino acid at position X34 is R, E, or Q; xxxv. The amino acid at position X35 is A or P; xxxvi. The amino acid at position X36 is S or P; xxxvii. The amino acid at position X37 is A or E; xxxviii. The amino acid at position X38 is A or V; xxxix. The amino acid at position X39 is R or K; xl. The amino acid at position X40 is R, K, or deleted; xli. The amino acid at position X41 is S, D, or G; xlii. The amino acid at position X42 is P or E; xliii. The amino acid at position X43 is A or V; xliv. The amino acid at position X44 is L or I; xlv. The amino acid at position X45 is D or E; xlvi. The amino acid at position X46 is L or I; xlvii. The amino acid at position X47 is L or A; xlviii. The amino acid at position X48 is Q or E; xlix. The amino acid at position X49 is L or R; l. The amino acid at position X50 is I or V; li. The amino acid at position X51 is E, A, or G; lii. The amino acid at position X52 is A or W; liii. The amino acid at position X53 is L or R; liv. The amino acid at position X54 is Q or T; lv. the amino acid at position X55 is N or G; lvi. The amino acid at position X56 is R, K, or P; lvii. The amino acid at position X57 is F or L; lviii. The amino acid at position X58 is N or G; lix. The amino acid at position X59 is A or G; lx. The amino acid at position X60 is A or V; lxi. The amino acid at position X61 is L or F; lxii. The amino acid at position X62 is L or E; lxiii. The amino acid at position X63 is C or N; lxiv. the amino acid at position X64 is D, E, or R; and / or lxv. The amino acid at position X65 is G or A.

[0068] In one embodiment, the polypeptide comprises the following amino acid sequence: i. the amino acid at position 3 is T; ii. the amino acid at position 5 is D; iii. the amino acid at position 25 is Q; iv. the amino acid at position 26 is P; v. The amino acid at position 32 is A; vi. the amino acid at position 40 is I; vii. the amino acid at position 51 is P; viii. the amino acid at position 52 is V; ix. the amino acid at position 53 is S; x. the amino acid at position 57 is R; xi. the amino acid at position 58 is A; xii. the amino acid at position 63 is D; xiii. the amino acid at position 64 is E; xiv. The amino acid at position 70 is P; xv. the amino acid at position 73 is K; xvi. The amino acid at position 74 is E; xvii. the amino acid at position 75 is M; xviii. the amino acid at position 78 is G; xix. The amino acid at position 82 is E; xx. the amino acid at position 84 is T; xxi. the amino acid at position 88 is G; xxii. the amino acid at position 92 is L; xxiii. the amino acid at position 99 is S; xxiv. The amino acid at position 101 is H; xxv. The amino acid at position 110 is T; xxvi. The amino acid at position 114 is L; xxvii. The amino acid at position 124 is T; xxviii. The amino acid at position 131 is E; xxix. the amino acid at position 143 is R; xxx. The amino acid at position 148 is V; xxxi. The amino acid at position 149 is K; xxxii. the amino acid at position 167 is G; xxxiii. The amino acid at position 171 is E; xxxiv. the amino acid at position 186 is Q; xxxv. The amino acid at position 193 is P; xxxvi. The amino acid at position 194 is S; xxxvii. The amino acid at position 197 is E; xxxviii. The amino acid at position 207 is A; xxxix. The amino acid at position 210 is K; xl. The amino acid at position 211 is deleted, xli. the amino acid at position 212 is S; xlii. the amino acid at position 215 is E; xliii. the amino acid at position 223 is V; xliv. the amino acid at position 234 is I; xlv. the amino acid at position 244 is E; xlvi. the amino acid at position 276 is L; xlvii. the amino acid at position 280 is L; xlviii. the amino acid at position 288 is Q; xlix. the amino acid at position 291 is L; l. the amino acid at position 301 is I; li. the amino acid at position 304 is E; lii. the amino acid at position 305 is A; liii. the amino acid at position 311 is R; liv. the amino acid at position 312 is Q; lv. The amino acid at position 313 is N, lvi. The amino acid at position 316 is K; lvii. the amino acid at position 328 is F; lviii. the amino acid at position 330 is N; lix. The amino acid at position 349 is G; lx. The amino acid at position 368 is A; lxi. the amino acid at position 370 is F; lxii. the amino acid at position 375 is L; lxiii. the amino acid at position 385 is C; lxiv. The amino acid at position 390 is E; lxv. the amino acid at position 397 is A; Numbering is with respect to amino acid positions in SEQ ID NO: 1 or SEQ ID NO: 96. In one embodiment, the polypeptide comprises an amino acid sequence having amino acid residues 2 to 397 of SEQ ID NO: 2. In another embodiment, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 2.

[0069] In one embodiment, the polypeptide comprises the following amino acid sequence: i. the amino acid at position 3 is T; ii. the amino acid at position 5 is D; iii. the amino acid at position 25 is T; iv. the amino acid at position 26 is P; v. The amino acid at position 32 is A; vi. the amino acid at position 40 is V; vii. the amino acid at position 51 is A; viii. the amino acid at position 52 is A; ix. the amino acid at position 53 is S; x. the amino acid at position 57 is A; xi. the amino acid at position 58 is S; xii. the amino acid at position 63 is G; xiii. the amino acid at position 64 is E; xiv. The amino acid at position 70 is P; xv. the amino acid at position 73 is R; xvi. The amino acid at position 74 is E; xvii. the amino acid at position 75 is M; xviii. the amino acid at position 78 is G; xix. The amino acid at position 82 is A; xx. the amino acid at position 84 is S; xxi. The amino acid at position 88 is A; xxii. the amino acid at position 92 is M; xxiii. the amino acid at position 99 is A; xxiv. The amino acid at position 101 is W; xxv. The amino acid at position 110 is S; xxvi. The amino acid at position 114 is V; xxvii. The amino acid at position 124 is I; xxviii. The amino acid at position 131 is D; xxix. the amino acid at position 143 is H; xxx. The amino acid at position 148 is A; xxxi. The amino acid at position 149 is A; xxxii. the amino acid at position 167 is S; xxxiii. The amino acid at position 171 is D; xxxiv. The amino acid at position 186 is R; xxxv. the amino acid at position 193 is A; xxxvi. The amino acid at position 194 is P; xxxvii. The amino acid at position 197 is A; xxxviii. The amino acid at position 207 is A; xxxix. The amino acid at position 210 is R; xl. the amino acid at position 211 is R; xli. the amino acid at position 212 is G; xlii. the amino acid at position 215 is P; xliii. the amino acid at position 223 is A; xliv. the amino acid at position 234 is I; xlv. the amino acid at position 244 is D; xlvi. the amino acid at position 276 is I; xlvii. the amino acid at position 280 is A; xlviii. the amino acid at position 288 is E; xlix. the amino acid at position 291 is R; l. the amino acid at position 301 is I; li. the amino acid at position 304 is G; lii. the amino acid at position 305 is W; liii. the amino acid at position 311 is L; liv. the amino acid at position 312 is T; lv. The amino acid at position 313 is G, lvi. The amino acid at position 316 is R; lvii. the amino acid at position 328 is F; lviii. the amino acid at position 330 is G; lix. The amino acid at position 349 is A; lx. the amino acid at position 368 is V; lxi. the amino acid at position 370 is L; lxii. the amino acid at position 375 is E; lxiii. the amino acid at position 385 is N; lxiv. The amino acid at position 390 is D; lxv. the amino acid at position 397 is A; Numbering is with respect to amino acid positions in SEQ ID NO: 1 or SEQ ID NO: 96. In one embodiment, the polypeptide comprises an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 4. In another embodiment, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 4.

[0070] In one embodiment, the polypeptide comprises the following amino acid sequence: i. the amino acid at position 3 is T; ii. the amino acid at position 5 is D; iii. the amino acid at position 25 is T; iv. the amino acid at position 26 is P; v. The amino acid at position 32 is A; vi. the amino acid at position 40 is V; vii. the amino acid at position 51 is A; viii. the amino acid at position 52 is A; ix. the amino acid at position 53 is W; x. the amino acid at position 57 is A; xi. the amino acid at position 58 is A; xii. the amino acid at position 63 is G; xiii. the amino acid at position 64 is R; xiv. The amino acid at position 70 is P; xv. the amino acid at position 73 is R; xvi. The amino acid at position 74 is S; xvii. the amino acid at position 75 is M; xviii. the amino acid at position 78 is G; xix. The amino acid at position 82 is A; xx. the amino acid at position 84 is T; xxi. the amino acid at position 88 is G; xxii. the amino acid at position 92 is L; xxiii. the amino acid at position 99 is A; xxiv. The amino acid at position 101 is H; xxv. The amino acid at position 110 is T; xxvi. The amino acid at position 114 is L; xxvii. The amino acid at position 124 is T; xxviii. The amino acid at position 131 is E; xxix. the amino acid at position 143 is H; xxx. The amino acid at position 148 is V; xxxi. The amino acid at position 149 is A; xxxii. the amino acid at position 167 is G; xxxiii. The amino acid at position 171 is D; xxxiv. The amino acid at position 186 is E; xxxv. The amino acid at position 193 is P; xxxvi. The amino acid at position 194 is S; xxxvii. The amino acid at position 197 is A; xxxviii. The amino acid at position 207 is A; xxxix. The amino acid at position 210 is K; xl. The amino acid at position 211 is deleted, xli. the amino acid at position 212 is S; xlii. the amino acid at position 215 is E; xliii. the amino acid at position 223 is V; xliv. the amino acid at position 234 is L; xlv. the amino acid at position 244 is D; xlvi. the amino acid at position 276 is L; xlvii. the amino acid at position 280 is L; xlviii. the amino acid at position 288 is Q; xlix. the amino acid at position 291 is L; l. the amino acid at position 301 is I; li. the amino acid at position 304 is A; lii. the amino acid at position 305 is A; liii. the amino acid at position 311 is R; liv. the amino acid at position 312 is T; lv. The amino acid at position 313 is N, lvi. The amino acid at position 316 is R; lvii. the amino acid at position 328 is F; lviii. the amino acid at position 330 is G; lix. The amino acid at position 349 is G; lx. The amino acid at position 368 is A; lxi. the amino acid at position 370 is F; lxii. the amino acid at position 375 is L; lxiii. the amino acid at position 385 is C; lxiv. The amino acid at position 390 is D; lxv. the amino acid at position 397 is A; Numbering is with respect to amino acid positions in SEQ ID NO: 1 or SEQ ID NO: 96. In one embodiment, the polypeptide comprises an amino acid sequence having amino acid residues 2 to 397 of SEQ ID NO: 5. In another embodiment, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 5.

[0071] In one embodiment, the polypeptide comprises the following amino acid sequence: i. the amino acid at position 3 is Q; ii. the amino acid at position 5 is N; iii. the amino acid at position 25 is Q; iv. the amino acid at position 26 is A; v. The amino acid at position 32 is S; vi. the amino acid at position 40 is V; vii. the amino acid at position 51 is P; viii. the amino acid at position 52 is V; ix. the amino acid at position 53 is S; x. the amino acid at position 57 is R; xi. the amino acid at position 58 is A; xii. the amino acid at position 63 is G; xiii. the amino acid at position 64 is E; xiv. The amino acid at position 70 is P; xv. the amino acid at position 73 is K; xvi. The amino acid at position 74 is E; xvii. the amino acid at position 75 is T; xviii. the amino acid at position 78 is G; xix. The amino acid at position 82 is E; xx. the amino acid at position 84 is T; xxi. the amino acid at position 88 is G; xxii. the amino acid at position 92 is L; xxiii. the amino acid at position 99 is A; xxiv. The amino acid at position 101 is H; xxv. The amino acid at position 110 is S; xxvi. The amino acid at position 114 is V; xxvii. The amino acid at position 124 is T; xxviii. The amino acid at position 131 is E; xxix. the amino acid at position 143 is H; xxx. The amino acid at position 148 is V; xxxi. The amino acid at position 149 is A; xxxii. the amino acid at position 167 is S; xxxiii. The amino acid at position 171 is E; xxxiv. The amino acid at position 186 is R; xxxv. The amino acid at position 193 is P; xxxvi. The amino acid at position 194 is S; xxxvii. The amino acid at position 197 is A; xxxviii. The amino acid at position 207 is A; xxxix. The amino acid at position 210 is K; xl. the amino acid at position 211 is K; xli. the amino acid at position 212 is G; xlii. the amino acid at position 215 is P; xliii. the amino acid at position 223 is V; xliv. the amino acid at position 234 is L; xlv. the amino acid at position 244 is D; xlvi. the amino acid at position 276 is I; xlvii. the amino acid at position 280 is L; xlviii. the amino acid at position 288 is E; xlix. the amino acid at position 291 is R; l. the amino acid at position 301 is I; li. the amino acid at position 304 is E; lii. the amino acid at position 305 is A; liii. the amino acid at position 311 is L; liv. the amino acid at position 312 is T; lv. The amino acid at position 313 is N, lvi. The amino acid at position 316 is K; lvii. the amino acid at position 328 is L; lviii. the amino acid at position 330 is N; lix. The amino acid at position 349 is A; lx. the amino acid at position 368 is V; lxi. the amino acid at position 370 is L; lxii. the amino acid at position 375 is L; lxiii. the amino acid at position 385 is C; lxiv. The amino acid at position 390 is R; lxv. the amino acid at position 397 is G; Numbering is with respect to amino acid positions in SEQ ID NO: 1 or SEQ ID NO: 96. In one embodiment, the polypeptide comprises an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 6. In another embodiment, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 6.

[0072] In one embodiment, the polypeptide comprises the following amino acid sequence: i. the amino acid at position 3 is T; ii. the amino acid at position 5 is N; iii. the amino acid at position 25 is Q; iv. the amino acid at position 26 is P; v. The amino acid at position 32 is A; vi. the amino acid at position 40 is I; vii. the amino acid at position 51 is P; viii. the amino acid at position 52 is V; ix. the amino acid at position 53 is S; x. the amino acid at position 57 is R; xi. the amino acid at position 58 is S; xii. the amino acid at position 63 is G; xiii. the amino acid at position 64 is E; xiv. The amino acid at position 70 is P; xv. the amino acid at position 73 is K; xvi. The amino acid at position 74 is E; xvii. the amino acid at position 75 is W; xviii. the amino acid at position 78 is G; xix. The amino acid at position 82 is E; xx. the amino acid at position 84 is T; xxi. the amino acid at position 88 is G; xxii. the amino acid at position 92 is L; xxiii. the amino acid at position 99 is A; xxiv. The amino acid at position 101 is W; xxv. The amino acid at position 110 is T; xxvi. The amino acid at position 114 is V; xxvii. The amino acid at position 124 is T; xxviii. The amino acid at position 131 is E; xxix. the amino acid at position 143 is R; xxx. The amino acid at position 148 is A; xxxi. The amino acid at position 149 is A; xxxii. the amino acid at position 167 is S; xxxiii. The amino acid at position 171 is E; xxxiv. The amino acid at position 186 is R; xxxv. The amino acid at position 193 is P; xxxvi. The amino acid at position 194 is P; xxxvii. The amino acid at position 197 is A; xxxviii. The amino acid at position 207 is V; xxxix. The amino acid at position 210 is K; xl. the amino acid at position 211 is K; xli. the amino acid at position 212 is G; xlii. the amino acid at position 215 is P; xliii. the amino acid at position 223 is V; xliv. the amino acid at position 234 is L; xlv. the amino acid at position 244 is D; xlvi. the amino acid at position 276 is I; xlvii. the amino acid at position 280 is L; xlviii. the amino acid at position 288 is E; xlix. the amino acid at position 291 is R; l. the amino acid at position 301 is I; li. the amino acid at position 304 is E; lii. the amino acid at position 305 is W; liii. the amino acid at position 311 is L; liv. the amino acid at position 312 is Q; lv. The amino acid at position 313 is N, lvi. The amino acid at position 316 is R; lvii. the amino acid at position 328 is F; lviii. the amino acid at position 330 is N; lix. The amino acid at position 349 is A; lx. the amino acid at position 368 is V; lxi. the amino acid at position 370 is L; lxii. the amino acid at position 375 is L; lxiii. the amino acid at position 385 is C; lxiv. The amino acid at position 390 is D; lxv. the amino acid at position 397 is A; Numbering is with respect to amino acid positions in SEQ ID NO: 1 or SEQ ID NO: 96. In one embodiment, the polypeptide comprises an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 7. In another embodiment, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 7.

[0073] In one embodiment, the polypeptide comprises the following amino acid sequence: i. the amino acid at position 3 is T; ii. the amino acid at position 5 is D; iii. the amino acid at position 25 is T; iv. the amino acid at position 26 is A; v. The amino acid at position 32 is S; vi. the amino acid at position 40 is V; vii. the amino acid at position 51 is A; viii. the amino acid at position 52 is A; ix. the amino acid at position 53 is S; x. the amino acid at position 57 is A; xi. the amino acid at position 58 is A; xii. the amino acid at position 63 is G; xiii. the amino acid at position 64 is G; xiv. the amino acid at position 70 is deleted; xv. the amino acid at position 73 is R; xvi. The amino acid at position 74 is S; xvii. the amino acid at position 75 is T; xviii. the amino acid at position 78 is A; xix. The amino acid at position 82 is A; xx. the amino acid at position 84 is T; xxi. the amino acid at position 88 is G; xxii. the amino acid at position 92 is L; xxiii. the amino acid at position 99 is A; xxiv. The amino acid at position 101 is H; xxv. The amino acid at position 110 is S; xxvi. The amino acid at position 114 is V; xxvii. The amino acid at position 124 is T; xxviii. The amino acid at position 131 is E; xxix. the amino acid at position 143 is H; xxx. The amino acid at position 148 is A; xxxi. The amino acid at position 149 is A; xxxii. the amino acid at position 167 is S; xxxiii. The amino acid at position 171 is E; xxxiv. The amino acid at position 186 is R; xxxv. The amino acid at position 193 is P; xxxvi. The amino acid at position 194 is S; xxxvii. The amino acid at position 197 is A; xxxviii. The amino acid at position 207 is A; xxxix. The amino acid at position 210 is R; xl. the amino acid at position 211 is R; xli. the amino acid at position 212 is G; xlii. the amino acid at position 215 is P; xliii. the amino acid at position 223 is V; xliv. the amino acid at position 234 is L; xlv. the amino acid at position 244 is D; xlvi. the amino acid at position 276 is I; xlvii. the amino acid at position 280 is L; xlviii. the amino acid at position 288 is E; xlix. the amino acid at position 291 is R; l. the amino acid at position 301 is V; li. the amino acid at position 304 is E; lii. the amino acid at position 305 is A; liii. the amino acid at position 311 is L; liv. the amino acid at position 312 is T; lv. The amino acid at position 313 is N, lvi. The amino acid at position 316 is R; lvii. the amino acid at position 328 is L; lviii. the amino acid at position 330 is G; lix. The amino acid at position 349 is A; lx. the amino acid at position 368 is V; lxi. the amino acid at position 370 is L; lxii. the amino acid at position 375 is L; lxiii. the amino acid at position 385 is C; lxiv. The amino acid at position 390 is R; lxv. the amino acid at position 397 is G; Numbering is with respect to amino acid positions in SEQ ID NO: 1 or SEQ ID NO: 96. In one embodiment, the polypeptide comprises an amino acid sequence having amino acid residues 2 to 397 of SEQ ID NO: 8. In another embodiment, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 8.

[0074] In one embodiment, the polypeptide comprises the following amino acid sequence: i. the amino acid at position 3 is T; ii. the amino acid at position 5 is N; iii. the amino acid at position 25 is Q; iv. the amino acid at position 26 is P; v. The amino acid at position 32 is A; vi. the amino acid at position 40 is I; vii. the amino acid at position 51 is P; viii. the amino acid at position 52 is V; ix. the amino acid at position 53 is S; x. the amino acid at position 57 is R; xi. the amino acid at position 58 is S; xii. the amino acid at position 63 is G; xiii. the amino acid at position 64 is E; xiv. The amino acid at position 70 is P; xv. the amino acid at position 73 is K; xvi. The amino acid at position 74 is E; xvii. the amino acid at position 75 is W; xviii. the amino acid at position 78 is G; xix. The amino acid at position 82 is E; xx. the amino acid at position 84 is S; xxi. The amino acid at position 88 is A; xxii. the amino acid at position 92 is M; xxiii. the amino acid at position 99 is A; xxiv. The amino acid at position 101 is W; xxv. The amino acid at position 110 is S; xxvi. The amino acid at position 114 is V; xxvii. The amino acid at position 124 is I; xxviii. The amino acid at position 131 is E; xxix. the amino acid at position 143 is R; xxx. The amino acid at position 148 is A; xxxi. The amino acid at position 149 is A; xxxii. the amino acid at position 167 is S; xxxiii. The amino acid at position 171 is E; xxxiv. The amino acid at position 186 is R; xxxv. The amino acid at position 193 is P; xxxvi. The amino acid at position 194 is P; xxxvii. The amino acid at position 197 is A; xxxviii. The amino acid at position 207 is V; xxxix. The amino acid at position 210 is K; xl. the amino acid at position 211 is K; xli. the amino acid at position 212 is G; xlii. the amino acid at position 215 is P; xliii. the amino acid at position 223 is A; xliv. the amino acid at position 234 is L; xlv. the amino acid at position 244 is D; xlvi. the amino acid at position 276 is I; xlvii. the amino acid at position 280 is L; xlviii. the amino acid at position 288 is E; xlix. the amino acid at position 291 is R; l. the amino acid at position 301 is I; li. the amino acid at position 304 is E; lii. the amino acid at position 305 is W; liii. the amino acid at position 311 is L; liv. the amino acid at position 312 is Q; lv. The amino acid at position 313 is N, lvi. The amino acid at position 316 is R; lvii. the amino acid at position 328 is F; lviii. the amino acid at position 330 is N; lix. The amino acid at position 349 is A; lx. the amino acid at position 368 is V; lxi. the amino acid at position 370 is L; lxii. the amino acid at position 375 is L; lxiii. the amino acid at position 385 is C; lxiv. The amino acid at position 390 is D; lxv. the amino acid at position 397 is A; Numbering is with respect to amino acid positions in SEQ ID NO: 1 or SEQ ID NO: 96. In one embodiment, the polypeptide comprises an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 9. In another embodiment, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 9.

[0075] In one embodiment, the polypeptide comprises the following amino acid sequence: i. the amino acid at position 3 is T; ii. the amino acid at position 5 is D; iii. the amino acid at position 25 is T; iv. the amino acid at position 26 is A; v. The amino acid at position 32 is A; vi. the amino acid at position 40 is V; vii. the amino acid at position 51 is A; viii. the amino acid at position 52 is A; ix. the amino acid at position 53 is W; x. the amino acid at position 57 is R; xi. the amino acid at position 58 is A; xii. the amino acid at position 63 is G; xiii. the amino acid at position 64 is G; xiv. The amino acid at position 70 is P; xv. the amino acid at position 73 is R; xvi. The amino acid at position 74 is S; xvii. the amino acid at position 75 is W; xviii. the amino acid at position 78 is G; xix. The amino acid at position 82 is A; xx. the amino acid at position 84 is T; xxi. the amino acid at position 88 is G; xxii. the amino acid at position 92 is L; xxiii. the amino acid at position 99 is A; xxiv. The amino acid at position 101 is H; xxv. The amino acid at position 110 is T; xxvi. The amino acid at position 114 is L; xxvii. The amino acid at position 124 is T; xxviii. The amino acid at position 131 is D; xxix. the amino acid at position 143 is D; xxx. The amino acid at position 148 is V; xxxi. The amino acid at position 149 is A; xxxii. the amino acid at position 167 is G; xxxiii. The amino acid at position 171 is D; xxxiv. the amino acid at position 186 is Q; xxxv. The amino acid at position 193 is P; xxxvi. The amino acid at position 194 is S; xxxvii. The amino acid at position 197 is A; xxxviii. The amino acid at position 207 is A; xxxix. The amino acid at position 210 is K; xl. the amino acid at position 211 is R; xli. the amino acid at position 212 is S; xlii. the amino acid at position 215 is E; xliii. the amino acid at position 223 is V; xliv. the amino acid at position 234 is L; xlv. the amino acid at position 244 is D; xlvi. the amino acid at position 276 is L; xlvii. the amino acid at position 280 is L; xlviii. the amino acid at position 288 is Q; xlix. the amino acid at position 291 is L; l. the amino acid at position 301 is I; li. the amino acid at position 304 is E; lii. the amino acid at position 305 is A; liii. the amino acid at position 311 is R; liv. the amino acid at position 312 is T; lv. The amino acid at position 313 is N, lvi. The amino acid at position 316 is P; lvii. the amino acid at position 328 is F; lviii. the amino acid at position 330 is G; lix. The amino acid at position 349 is G; lx. The amino acid at position 368 is A; lxi. the amino acid at position 370 is F; lxii. the amino acid at position 375 is L; lxiii. the amino acid at position 385 is C; lxiv. The amino acid at position 390 is D; lxv. the amino acid at position 397 is G; Numbering is with respect to amino acid positions in SEQ ID NO: 1 or SEQ ID NO: 96. In one embodiment, the polypeptide comprises an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 10. In another embodiment, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 10.

[0076] In one embodiment, the polypeptide comprises the following amino acid sequence: i. the amino acid at position 3 is Q; ii. the amino acid at position 5 is N; iii. the amino acid at position 25 is Q; iv. the amino acid at position 26 is P; v. The amino acid at position 32 is A; vi. the amino acid at position 40 is I; vii. the amino acid at position 51 is P; viii. the amino acid at position 52 is V; ix. the amino acid at position 53 is S; x. the amino acid at position 57 is R; xi. the amino acid at position 58 is S; xii. the amino acid at position 63 is G; xiii. the amino acid at position 64 is E; xiv. The amino acid at position 70 is P; xv. the amino acid at position 73 is K; xvi. The amino acid at position 74 is E; xvii. the amino acid at position 75 is W; xviii. the amino acid at position 78 is G; xix. The amino acid at position 82 is E; xx. the amino acid at position 84 is T; xxi. the amino acid at position 88 is G; xxii. the amino acid at position 92 is L; xxiii. the amino acid at position 99 is A; xxiv. The amino acid at position 101 is W; xxv. The amino acid at position 110 is T; xxvi. The amino acid at position 114 is V; xxvii. The amino acid at position 124 is T; xxviii. The amino acid at position 131 is E; xxix. the amino acid at position 143 is R; xxx. The amino acid at position 148 is A; xxxi. The amino acid at position 149 is A; xxxii. the amino acid at position 167 is S; xxxiii. The amino acid at position 171 is E; xxxiv. The amino acid at position 186 is R; xxxv. The amino acid at position 193 is P; xxxvi. The amino acid at position 194 is P; xxxvii. The amino acid at position 197 is A; xxxviii. The amino acid at position 207 is V; xxxix. The amino acid at position 210 is K; xl. the amino acid at position 211 is K; xli. the amino acid at position 212 is D; xlii. the amino acid at position 215 is P; xliii. the amino acid at position 223 is V; xliv. the amino acid at position 234 is L; xlv. the amino acid at position 244 is D; xlvi. the amino acid at position 276 is I; xlvii. the amino acid at position 280 is L; xlviii. the amino acid at position 288 is E; xlix. the amino acid at position 291 is R; l. the amino acid at position 301 is I; li. the amino acid at position 304 is E; lii. the amino acid at position 305 is W; liii. the amino acid at position 311 is L; liv. the amino acid at position 312 is Q; lv. The amino acid at position 313 is N, lvi. The amino acid at position 316 is R; lvii. the amino acid at position 328 is F; lviii. the amino acid at position 330 is N; lix. The amino acid at position 349 is A; lx. the amino acid at position 368 is V; lxi. the amino acid at position 370 is L; lxii. the amino acid at position 375 is L; lxiii. the amino acid at position 385 is C; lxiv. The amino acid at position 390 is D; lxv. the amino acid at position 397 is A; Numbering is with respect to amino acid positions in SEQ ID NO: 1 or SEQ ID NO: 96. In one embodiment, the polypeptide comprises an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 11. In another embodiment, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 11.

[0077] In one embodiment, the polypeptide comprises the following amino acid sequence: i. the amino acid at position 3 is T; ii. the amino acid at position 5 is D; iii. the amino acid at position 25 is T; iv. the amino acid at position 26 is P; v. The amino acid at position 32 is A; vi. the amino acid at position 40 is V; vii. the amino acid at position 51 is A; viii. the amino acid at position 52 is A; ix. the amino acid at position 53 is S; x. the amino acid at position 57 is A; xi. the amino acid at position 58 is S; xii. the amino acid at position 63 is G; xiii. the amino acid at position 64 is E; xiv. The amino acid at position 70 is P; xv. the amino acid at position 73 is R; xvi. The amino acid at position 74 is E; xvii. the amino acid at position 75 is W; xviii. the amino acid at position 78 is G; xix. The amino acid at position 82 is A; xx. the amino acid at position 84 is T; xxi. the amino acid at position 88 is G; xxii. the amino acid at position 92 is L; xxiii. the amino acid at position 99 is A; xxiv. The amino acid at position 101 is W; xxv. The amino acid at position 110 is S; xxvi. The amino acid at position 114 is V; xxvii. The amino acid at position 124 is T; xxviii. The amino acid at position 131 is E; xxix. the amino acid at position 143 is H; xxx. The amino acid at position 148 is A; xxxi. The amino acid at position 149 is A; xxxii. the amino acid at position 167 is S; xxxiii. The amino acid at position 171 is E; xxxiv. The amino acid at position 186 is R; xxxv. The amino acid at position 193 is P; xxxvi. The amino acid at position 194 is P; xxxvii. The amino acid at position 197 is A; xxxviii. The amino acid at position 207 is A; xxxix. The amino acid at position 210 is R; xl. the amino acid at position 211 is R; xli. the amino acid at position 212 is G; xlii. the amino acid at position 215 is P; xliii. the amino acid at position 223 is V; xliv. the amino acid at position 234 is L; xlv. the amino acid at position 244 is D; xlvi. the amino acid at position 276 is I; xlvii. the amino acid at position 280 is L; xlviii. the amino acid at position 288 is E; xlix. the amino acid at position 291 is R; l. the amino acid at position 301 is I; li. the amino acid at position 304 is G; lii. the amino acid at position 305 is W; liii. the amino acid at position 311 is L; liv. the amino acid at position 312 is T; lv. The amino acid at position 313 is N, lvi. The amino acid at position 316 is R; lvii. the amino acid at position 328 is F; lviii. the amino acid at position 330 is G; lix. The amino acid at position 349 is A; lx. the amino acid at position 368 is V; lxi. the amino acid at position 370 is L; lxii. the amino acid at position 375 is L; lxiii. the amino acid at position 385 is C; lxiv. The amino acid at position 390 is D; lxv. the amino acid at position 397 is A; Numbering is with respect to amino acid positions in SEQ ID NO: 1 or SEQ ID NO: 96. In one embodiment, the polypeptide comprises an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 12. In another embodiment, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 12.

[0078] In one embodiment, the polypeptide comprises the following amino acid sequence: i. the amino acid at position 3 is Q; ii. the amino acid at position 5 is N; iii. the amino acid at position 25 is Q; iv. the amino acid at position 26 is P; v. The amino acid at position 32 is A; vi. the amino acid at position 40 is I; vii. the amino acid at position 51 is P; viii. the amino acid at position 52 is V; ix. the amino acid at position 53 is S; x. the amino acid at position 57 is R; xi. the amino acid at position 58 is S; xii. the amino acid at position 63 is G; xiii. the amino acid at position 64 is E; xiv. The amino acid at position 70 is P; xv. the amino acid at position 73 is K; xvi. The amino acid at position 74 is E; xvii. the amino acid at position 75 is W; xviii. the amino acid at position 78 is G; xix. The amino acid at position 82 is E; xx. the amino acid at position 84 is T; xxi. the amino acid at position 88 is G; xxii. the amino acid at position 92 is L; xxiii. the amino acid at position 99 is A; xxiv. The amino acid at position 101 is W; xxv. The amino acid at position 110 is T; xxvi. The amino acid at position 114 is V; xxvii. The amino acid at position 124 is T; xxviii. The amino acid at position 131 is E; xxix. the amino acid at position 143 is R; xxx. The amino acid at position 148 is A; xxxi. The amino acid at position 149 is A; xxxii. the amino acid at position 167 is S; xxxiii. The amino acid at position 171 is E; xxxiv. The amino acid at position 186 is R; xxxv. The amino acid at position 193 is P; xxxvi. The amino acid at position 194 is P; xxxvii. The amino acid at position 197 is A; xxxviii. The amino acid at position 207 is A; xxxix. The amino acid at position 210 is K; xl. the amino acid at position 211 is K; xli. the amino acid at position 212 is D; xlii. the amino acid at position 215 is P; xliii. the amino acid at position 223 is V; xliv. the amino acid at position 234 is L; xlv. the amino acid at position 244 is D; xlvi. the amino acid at position 276 is I; xlvii. the amino acid at position 280 is L; xlviii. the amino acid at position 288 is E; xlix. the amino acid at position 291 is R; l. the amino acid at position 301 is I; li. the amino acid at position 304 is E; lii. the amino acid at position 305 is W; liii. the amino acid at position 311 is L; liv. the amino acid at position 312 is Q; lv. The amino acid at position 313 is N, lvi. The amino acid at position 316 is R; lvii. the amino acid at position 328 is F; lviii. the amino acid at position 330 is N; lix. The amino acid at position 349 is A; lx. the amino acid at position 368 is V; lxi. the amino acid at position 370 is L; lxii. the amino acid at position 375 is L; lxiii. the amino acid at position 385 is C; lxiv. The amino acid at position 390 is D; lxv. the amino acid at position 397 is A; Numbering is with respect to amino acid positions in SEQ ID NO: 1 or SEQ ID NO: 96. In one embodiment, the polypeptide comprises an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 13. In another embodiment, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 13.

[0079] In one embodiment, the polypeptide comprises the following amino acid sequence: i. the amino acid at position 3 is Q; ii. the amino acid at position 5 is N; iii. the amino acid at position 25 is Q; iv. the amino acid at position 26 is A; v. The amino acid at position 32 is S; vi. the amino acid at position 40 is V; vii. the amino acid at position 51 is P; viii. the amino acid at position 52 is V; ix. the amino acid at position 53 is S; x. the amino acid at position 57 is A; xi. the amino acid at position 58 is A; xii. the amino acid at position 63 is G; xiii. the amino acid at position 64 is E; xiv. The amino acid at position 70 is P; xv. the amino acid at position 73 is K; xvi. The amino acid at position 74 is E; xvii. the amino acid at position 75 is T; xviii. the amino acid at position 78 is G; xix. The amino acid at position 82 is E; xx. the amino acid at position 84 is T; xxi. the amino acid at position 88 is G; xxii. the amino acid at position 92 is L; xxiii. the amino acid at position 99 is A; xxiv. The amino acid at position 101 is H; xxv. The amino acid at position 110 is S; xxvi. The amino acid at position 114 is L; xxvii. The amino acid at position 124 is T; xxviii. The amino acid at position 131 is E; xxix. the amino acid at position 143 is H; xxx. The amino acid at position 148 is A; xxxi. The amino acid at position 149 is A; xxxii. the amino acid at position 167 is S; xxxiii. The amino acid at position 171 is E; xxxiv. The amino acid at position 186 is R; xxxv. The amino acid at position 193 is P; xxxvi. The amino acid at position 194 is S; xxxvii. The amino acid at position 197 is E; xxxviii. The amino acid at position 207 is A; xxxix. The amino acid at position 210 is K; xl. the amino acid at position 211 is K; xli. the amino acid at position 212 is G; xlii. the amino acid at position 215 is P; xliii. the amino acid at position 223 is V; xliv. the amino acid at position 234 is L; xlv. the amino acid at position 244 is D; xlvi. the amino acid at position 276 is I; xlvii. the amino acid at position 280 is L; xlviii. the amino acid at position 288 is E; xlix. the amino acid at position 291 is R; l. the amino acid at position 301 is I; li. the amino acid at position 304 is E; lii. the amino acid at position 305 is A; liii. the amino acid at position 311 is L; liv. the amino acid at position 312 is T; lv. The amino acid at position 313 is N, lvi. The amino acid at position 316 is K; lvii. the amino acid at position 328 is L; lviii. the amino acid at position 330 is N; lix. The amino acid at position 349 is A; lx. the amino acid at position 368 is V; lxi. the amino acid at position 370 is L; lxii. the amino acid at position 375 is L; lxiii. the amino acid at position 385 is C; lxiv. The amino acid at position 390 is R; lxv. the amino acid at position 397 is G; Numbering is with respect to amino acid positions in SEQ ID NO: 1 or SEQ ID NO: 96. In one embodiment, the polypeptide comprises an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 14. In another embodiment, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 14.

[0080] In one embodiment, the polypeptide comprises the following amino acid sequence: i. the amino acid at position 3 is T; ii. the amino acid at position 5 is D; iii. the amino acid at position 25 is Q; iv. the amino acid at position 26 is P; v. The amino acid at position 32 is A; vi. the amino acid at position 40 is V; vii. the amino acid at position 51 is P; viii. the amino acid at position 52 is V; ix. the amino acid at position 53 is S; x. the amino acid at position 57 is R; xi. the amino acid at position 58 is A; xii. the amino acid at position 63 is D; xiii. the amino acid at position 64 is E; xiv. The amino acid at position 70 is P; xv. the amino acid at position 73 is K; xvi. The amino acid at position 74 is E; xvii. the amino acid at position 75 is M; xviii. the amino acid at position 78 is G; xix. The amino acid at position 82 is E; xx. the amino acid at position 84 is T; xxi. the amino acid at position 88 is G; xxii. the amino acid at position 92 is L; xxiii. the amino acid at position 99 is S; xxiv. The amino acid at position 101 is H; xxv. The amino acid at position 110 is T; xxvi. The amino acid at position 114 is L; xxvii. The amino acid at position 124 is T; xxviii. The amino acid at position 131 is E; xxix. the amino acid at position 143 is H; xxx. The amino acid at position 148 is V; xxxi. The amino acid at position 149 is K; xxxii. the amino acid at position 167 is G; xxxiii. The amino acid at position 171 is E; xxxiv. the amino acid at position 186 is Q; xxxv. The amino acid at position 193 is P; xxxvi. The amino acid at position 194 is S; xxxvii. The amino acid at position 197 is E; xxxviii. The amino acid at position 207 is A; xxxix. The amino acid at position 210 is K; xl. the amino acid at position 211 is K; xli. the amino acid at position 212 is S; xlii. the amino acid at position 215 is E; xliii. the amino acid at position 223 is V; xliv. the amino acid at position 234 is I; xlv. the amino acid at position 244 is E; xlvi. the amino acid at position 276 is L; xlvii. the amino acid at position 280 is L; xlviii. the amino acid at position 288 is Q; xlix. the amino acid at position 291 is L; l. the amino acid at position 301 is I; li. the amino acid at position 304 is E; lii. the amino acid at position 305 is A; liii. the amino acid at position 311 is R; liv. the amino acid at position 312 is T; lv. The amino acid at position 313 is N, lvi. The amino acid at position 316 is K; lvii. the amino acid at position 328 is F; lviii. the amino acid at position 330 is N; lix. The amino acid at position 349 is G; lx. The amino acid at position 368 is A; lxi. the amino acid at position 370 is F; lxii. the amino acid at position 375 is L; lxiii. the amino acid at position 385 is C; lxiv. The amino acid at position 390 is E; lxv. the amino acid at position 397 is G; Numbering is with respect to amino acid positions in SEQ ID NO: 1 or SEQ ID NO: 96. In one embodiment, the polypeptide comprises an amino acid sequence having amino acid residues 2 to 398 of SEQ ID NO: 15. In another embodiment, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 15.

[0081] In one aspect disclosed herein, a polypeptide capable of hydrolyzing amide bonds in polyamides is provided, wherein the polypeptide comprises the amino acid sequence of amino acid residues 2-394 of SEQ ID NO: 88, or an amino acid sequence having greater than 75% sequence identity thereto. In one embodiment, the polypeptide has at least 80% sequence identity to SEQ ID NO: 88. In another embodiment, the polypeptide has at least 85% sequence identity to SEQ ID NO: 88. In another embodiment, the polypeptide has at least 90% sequence identity to SEQ ID NO: 88. In another embodiment, the polypeptide has at least 95% sequence identity to SEQ ID NO: 88. In one embodiment, the polypeptide comprises amino acid residues 2-392 of SEQ ID NO: 3. In one embodiment, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 3. In one embodiment, the polypeptide comprises amino acid residues 2-394 of SEQ ID NO: 63. In one embodiment, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 63. In one embodiment, the polypeptide comprises amino acid residues 2-395 of SEQ ID NO: 48. In one embodiment, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 48. In one embodiment, the polypeptide comprises amino acid residues 2 to 394 of SEQ ID NO: 123. In one embodiment, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:123.

[0082] In one aspect disclosed herein, a polypeptide capable of hydrolyzing amide bonds in polyamides is provided, wherein the polypeptide comprises the amino acid sequence of amino acid residues 2-391 of SEQ ID NO:45, or an amino acid sequence having at least 61% sequence identity thereto. In one embodiment, the polypeptide comprises amino acid residues 2-391 of SEQ ID NO:25. In one embodiment, the polypeptide comprises, or consists of, the amino acid sequence of SEQ ID NO:25. In one embodiment, the polypeptide comprises amino acid residues 2-391 of SEQ ID NO:22. In one embodiment, the polypeptide comprises, or consists of, the amino acid sequence of SEQ ID NO:22. In another embodiment, the polypeptide comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:45. In another embodiment, the polypeptide comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO:45. In another embodiment, the polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:45. In another embodiment, the polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:45. In another embodiment, the polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:45. In another embodiment, the polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 45. In one embodiment, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:45.

[0083] As used herein, the terms "peptide," "polypeptide," "protein," and "enzyme" are understood to refer to a chain of amino acids linked by peptide bonds, regardless of the number of amino acids forming the chain. Amino acids are typically represented by their one-letter or three-letter code according to the following nomenclature: A: alanine (Ala), C: cysteine ​​(Cys), D: aspartic acid (Asp), E: glutamic acid (Glu), F: phenylalanine (Phe), G: glycine (Gly), H: histidine (His), I: isoleucine (Ile), K: lysine (Lys), L: leucine (Leu), M: methionine (Met), N: asparagine (Asn), P: proline (Pro), Q: glutamine (Gln), R: arginine (Arg), S: serine (Ser), T: threonine (Thr), V: valine (Val), W: tryptophan (Trp), and Y: tyrosine (Tyr).

[0084] The terms "mutant" and "variant" may be used interchangeably herein and refer to a polypeptide that comprises an amino acid sequence derived from a wild-type or existing enzyme or from an ancestral sequence reconstruction process, and may further comprise modifications or alterations (e.g., substitutions, insertions, and / or deletions) at one or more (e.g., several) positions, and has enhanced activity in catalyzing the hydrolysis of nylon, nylon polymers, or nylon oligomers compared to an existing nylonase. Such variants may be obtained by various techniques well known in the art, illustrative examples of which include site-directed mutagenesis, random mutagenesis, and synthetic oligonucleotide construction. As used herein in reference to an amino acid residue or position, the terms "modified," "altered," "substituted," and the like typically mean that the amino acid at a particular position is modified compared to the amino acid in the wild-type or parent polypeptide.

[0085] Suitable substitutions may include the replacement of an amino acid residue with another selected from the standard 20 naturally occurring amino acid residues, rare naturally occurring amino acid residues (e.g., hydroxyproline, hydroxylysine, allohydroxylysine, 6-N-methyllysine, N-ethylglycine, N-methylglycine, N-ethylasparagine, allo-isoleucine, N-methylisoleucine, N-methylvaline, pyroglutamine, aminobutyric acid, ornithine, norleucine, norvaline), and non-naturally occurring amino acid residues, often synthetically produced (e.g., cyclohexylalanine). Preferably, the substitution involves the replacement of an amino acid residue with another selected from the standard 20 naturally occurring amino acid residues (G, P, A, V, L, I, M, C, F, Y, W, H, K, R, Q, N, E, D, S, and T). Modifications or alterations may be identified herein using the following terminology: Y197V indicates that the amino acid residue tyrosine (Y) at position 197 of the parent polypeptide sequence is substituted with valine (V). Y197V / I / M indicates that the amino acid residue tyrosine (Y) at position 197 of the parent sequence may be substituted with one of the following amino acids: valine (V), isoleucine (I), or methionine (M). Substitutions may be conservative or non-conservative. Examples of conservative substitutions will be familiar to those of skill in the art, and illustrative examples include substitutions within the group of basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine, asparagine, and threonine), hydrophobic amino acids (methionine, leucine, isoleucine, cysteine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, and serine).

[0086] Unless otherwise specified, positions disclosed in this application are numbered with reference to the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:96, both of which are 398 amino acids in length. In this context, the term "corresponding," as used in reference to an amino acid position, is intended to mean the amino acid position in a polypeptide sequence when that position is aligned with the equal or corresponding position in the sequence set forth in SEQ ID NO:1 or SEQ ID NO:96. For example, the amino acid residue designated X1 in the consensus sequence of SEQ ID NO:96 corresponds to the amino acid at position 3 of SEQ ID NO:96, and vice versa, while the amino acid residue designated X2 in the consensus sequence of SEQ ID NO:96 corresponds to the amino acid at position 5 of SEQ ID NO:96, and vice versa. In another example, the amino acid at position 360 of the consensus sequence of SEQ ID NO:96 corresponds to the amino acid at position 359 of SEQ ID NO:2 and SEQ ID NO:5, after alignment (see, e.g., Figure 9).

[0087] As used herein, the term "sequence identity" or "identity" refers to the number of matches (or fraction, expressed as a percentage) between two polypeptide sequences (identical amino acid residues). In a preferred embodiment, sequence identity is determined by comparing sequences when aligned to maximize overlap and identity while minimizing sequence gaps. Depending on the length of the two sequences, sequence identity may be determined using any of several mathematical global or local alignment algorithms known to those skilled in the art. Sequences of similar length may be aligned using a global alignment algorithm (e.g., the Needleman and Wunsch algorithm; Needleman and Wunsch, 1970), which optimally aligns the sequences over their entire length, while sequences of significantly different lengths are preferably aligned using a local alignment algorithm (e.g., the Smith and Waterman algorithm (Smith and Waterman, 1981) or the Altschul algorithm (Altschul et al., 1997; Altschul et al., 2005)). Alignment for purposes of determining percent amino acid sequence identity can be accomplished by any means available to those of skill in the art, illustrative examples of which include publicly available computer software such as that available at http: / / blast.ncbi.nlm.nih.gov / or http: / / www.ebi.ac.uk / Tools / emboss / . Those of skill in the art can readily determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.As used herein, % sequence identity typically refers to a value generated using pairwise sequence alignment to create an optimal global alignment of two sequences (e.g., using the Needleman-Wunsch algorithm), with all search parameters set to default values, e.g., scoring matrix=BLOSUM62, gap open=10, gap extension=0.5, end gap penalty=false, end gap open=10, and end gap extension=0.5.

[0088] The present disclosure also extends to compositions comprising the polypeptides described herein. The present disclosure also extends to nucleic acid sequences encoding the polypeptides described herein. The present disclosure also extends to expression vectors comprising the nucleic acid sequences described herein. The present disclosure also extends to host cells comprising the nucleic acid sequences or expression vectors described herein.

[0089] As used herein, the term "recombinant" typically refers to a nucleic acid construct, vector, polypeptide, or cell that is produced by genetic engineering.

[0090] As used herein, the term "expression" typically refers to any step involved in producing a polypeptide, for example, by transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0091] The term "expression cassette" refers to a nucleic acid construct that includes a coding region and, suitably, a regulatory region to which the coding region is operably linked.

[0092] The term "expression vector" typically refers to a DNA or RNA molecule that contains an expression cassette. Expression vectors can be linear or circular double-stranded DNA molecules.

[0093] As used herein, the terms "nucleic acid," "nucleic sequence," "polynucleotide," "oligonucleotide," and "nucleotide sequence" are used interchangeably and refer to a sequence of deoxyribonucleotides and / or ribonucleotides. A nucleic acid can be DNA (cDNA or gDNA), RNA, or a mixture of both. It can be in single-stranded or double-stranded form, or a mixture of both. It can be of recombinant, artificial, and / or synthetic origin and can contain modified nucleotides, including, for example, modified linkages, modified purine or pyrimidine bases, or modified sugars. Nucleic acids of the invention can be in isolated or purified form and are produced, isolated, and / or manipulated by techniques known per se in the art, such as cloning and expression of cDNA libraries, amplification, enzymatic synthesis, or recombinant technology. Nucleic acids can also be synthesized in vitro by well-known chemical synthesis techniques, such as those described by Belousov (1997) Nucleic Acids Res. 25:3440-3444.

[0094] The nucleic acid sequences disclosed herein may be suitably codon-optimized. Suitable methods for codon optimization are familiar to those skilled in the art, and illustrative examples are described in the textbook Sambrook et al. (Sambrook et al., 2001).

[0095] The nucleic acid sequences described herein may be suitably deduced from the amino acid sequences of the polypeptides described herein, and the codon usage may be adapted according to the host cell in which the nucleic acid will be transcribed.

[0096] In some embodiments, the nucleic acid sequences described herein may suitably include additional nucleotide sequences that can be used to cause or regulate expression of the polypeptide in a selected host cell or system, such as regulatory regions, i.e., promoters, enhancers, silencers, terminators, signal peptides, etc. Alternatively, or in addition, the nucleic acid sequences described herein may further include additional nucleotide sequences encoding fusion proteins, such as maltose binding protein (MBP) or glutathione S-transferase (GST), that can be used to support expression and / or solubility of the polypeptide.

[0097] As noted elsewhere herein, the present disclosure also extends to expression vectors and expression cassettes comprising the nucleic acid sequences described herein, optionally operably linked to one or more control sequences that direct expression of the nucleic acid sequence in a suitable host cell. Typically, the expression vector or cassette contains the nucleic acid sequence described herein operably linked to a control sequence, such as a transcription promoter and / or a transcription terminator. The control sequence may include a promoter recognized by a host cell or in vitro expression system for expression of a nucleic acid encoding a polypeptide described herein. The promoter typically includes a transcription control sequence that mediates expression of the polypeptide. The promoter may be any polynucleotide that exhibits transcriptional activity in a host cell, including mutant, truncated, and hybrid promoters, and may be suitably obtained from a gene encoding an extracellular or intracellular polypeptide, either homologous or heterologous to the host cell. The control sequence may also be a transcription terminator recognized by the host cell to terminate transcription. The terminator is typically operably linked to the 3' end of the nucleic acid encoding the polypeptide. Any terminator that is functional in the host cell may be used in this context. Typically, the expression vector or cassette contains a nucleic acid sequence described herein operably linked to a transcription promoter and a transcription terminator.

[0098] The term "vector" typically refers to a DNA molecule used as a vehicle to transfer recombinant genetic material into a host cell. Suitable vectors include plasmids, bacteriophages, viruses, fosmids, cosmids, and artificial chromosomes. A vector is typically a DNA sequence that contains an insert (heterologous nucleic acid sequence, transgene) and a larger sequence that serves as the "backbone" of the vector. The purpose of a vector to transfer genetic information to a host is typically to isolate, propagate, or express the insert in a target cell. Expression vectors (also called expression constructs) are specifically adapted for the expression of heterologous sequences in target cells and generally have a promoter sequence that drives the expression of a heterologous sequence encoding a polypeptide.

[0099] Generally, regulatory elements used in expression vectors include a transcription promoter, a ribosome binding site, a terminator, and an optional operator. Expression vectors may further include an origin of replication for autonomous replication in host cells, a selection marker, a limited number of useful restriction enzyme sites, and the potential for high copy number. Suitable expression vectors are familiar to those skilled in the art, and illustrative examples include cloning vectors, modified cloning vectors, plasmids, and viruses. Expression vectors that can provide appropriate levels of polypeptide expression in various hosts are also well known in the art. The choice of vector typically depends on the compatibility of the vector with the host cell into which the vector is introduced.

[0100] The present disclosure also extends to host cells comprising the nucleic acid sequences described herein. Host cells may be transformed, transfected, or transduced in a transient or stable manner. The nucleic acid, expression cassette, or vector is introduced into the host cell so that the nucleic acid, cassette, or vector is maintained as a chromosomal integrant or a self-replicating extrachromosomal vector. The term "host cell" encompasses any progeny of a parent host cell that is not identical to the parent host cell due to mutations that occur during replication. Host cells can be any cell useful in producing the variants of the invention, for example, a prokaryotic or eukaryotic organism. Prokaryotic host cells can be any gram-positive or gram-negative bacteria. Host cells can also be eukaryotic cells, for example, yeast, fungal, mammalian, insect, or plant cells. In certain embodiments, the host cell is selected from the group of Escherichia coli, Pseudomonas, Bacillus, Streptomyces, Trichoderma, Aspergillus, Saccharomyces, Pichia, Thermus, or Yarrowia.

[0101] The nucleic acids, expression cassettes, or expression vectors according to the invention may be introduced into host cells by any suitable method known to those skilled in the art, illustrative examples of which include electroporation, conjugation, transduction, competent cell transformation, protoplast transformation, protoplast fusion, biolistic "gene gun" transformation, PEG-mediated transformation, lipid-assisted transformation or transfection, chemical-mediated transfection, lithium acetate-mediated transformation, and liposome-mediated transformation.

[0102] In one embodiment, the host cell is a genetically modified host cell or microorganism. In this context, the host cell or microorganism may be genetically modified to enhance the expression of the polypeptide expressed therein and / or the activity of the host cell. For example, the polypeptides described herein may be used to complement a wild-type fungal or bacterial strain known to be capable of nylonase activity in order to improve and / or increase the nylonase activity of that strain.

[0103] The polypeptides disclosed herein are capable of hydrolyzing amide bonds in polyamides. In one embodiment, the polypeptides are capable of hydrolyzing polyamides. In another embodiment, the polypeptides are capable of hydrolyzing nylons, including nylon 6,6, as shown in the illustrative examples. Enzymes capable of digesting, hydrolyzing, or degrading nylons are broadly referred to as nylonases. Nylonases were first discovered in bacteria capable of digesting a by-product of nylon 6 production. Three nylonases were identified: 6-aminohexanoate-cyclic-dimer hydrolase (EI, NylA, UniProt:P13398), 6-aminohexanoate-dimer hydrolase (EII, NylB, UniProt:P07061), and 6-aminohexanoate-oligomer endohydrolase (EIII, NylC, UniProt:Q57326).

[0104] In some embodiments, the polypeptides disclosed herein have adipic acid mono- and di-N-alkylamide hydrolase activity. In some embodiments, the polypeptides disclosed herein are capable of hydrolyzing nylon 6,6 and nylon 6,6 oligomers. Methods for determining and measuring such activities, including adipic acid mono- and di-N-alkylamide hydrolase activity, are known to those of skill in the art, and illustrative examples are disclosed elsewhere herein.

[0105] The terms "nylon" or "nylon polymer" or "nylon oligomer" refer to a synthetic polymer composed of polyamides (repeating units joined by amide linkages). In one embodiment, the nylon or nylon polymer or nylon oligomer is nylon 6 (polycaprolactam). In another embodiment, the nylon or nylon polymer or nylon oligomer is nylon 6,6 (containing a diamine and a dicarboxylic acid). In one embodiment, the nylon or nylon polymer or nylon oligomer is a copolymer of different nylons comprised of nylon 6 and / or nylon 6,6. In another embodiment, the nylon or nylon polymer or nylon oligomer is a nylon blend of different nylons comprised of nylon 6 and / or nylon 6,6.

[0106] As used herein, the term "polymer" typically refers to a chemical substance or mixture of compounds whose structure is made up of multiple monomers (repeating units) linked by covalent chemical bonds. Within the context of the present invention, the term polymer includes natural or synthetic polymers composed of a single type of repeating unit (i.e., homopolymers) or a mixture of different repeating units (i.e., copolymers or heteropolymers).

[0107] Oligomers are low molecular weight polymers containing a small number of repeating units, and their physical properties typically depend on the chain length. As used herein, the term "nylon oligomer" refers to a nylon molecule containing a discrete number of repeating nylon monomer units. In one embodiment, the nylon oligomer contains up to a 50-mer of nylon. In another embodiment, the nylon oligomer contains up to a 40-mer of nylon. In another embodiment, the nylon oligomer contains up to a 30-mer of nylon. In another embodiment, the nylon oligomer contains up to a 20-mer of nylon. In another embodiment, the nylon oligomer contains a 20-mer, 19-mer, 18-mer, 17-mer, 16-mer, 15-mer, 14-mer, 13-mer, 12-mer, 11-mer, or 10-mer of nylon. In another embodiment, the nylon oligomer contains a 9-mer, 8-mer, 7-mer, or 6-mer of nylon. In another embodiment, the nylon oligomer is a nylon pentamer, tetramer, trimer, or dimer. In one embodiment, the nylon oligomer is a solubilized nylon oligomer. In one embodiment, the nylon oligomer is a water-soluble nylon oligomer. A solubilized nylon oligomer is one in which the oligomer is not in a solid form but is in solution (i.e., a fluid or liquid form). That is, the oligomer is dissolved in a liquid solution, usually through the addition of a solvent. In some embodiments, the oligomer is solubilized in an aqueous solution. In some embodiments, the oligomer is solubilized in a non-aqueous solution. In one embodiment, the solubilized nylon oligomer comprises a nylon decamer, nonamer, octamer, heptamer, or hexamer, pentamer, tetramer, trimer, and / or dimer. In one embodiment, the solubilized nylon oligomer comprises a nylon 6,6 decamer, nonamer, octamer, heptamer, or hexamer, pentamer, tetramer, trimer, and / or dimer. In one embodiment, the solubilized nylon oligomer comprises a nylon 6 decamer, nonamer, octamer, heptamer, or hexamer, pentamer, tetramer, trimer, and / or dimer. In one embodiment, the solubilized nylon oligomer contains a pentamer, tetramer, trimer, or dimer. In one embodiment, the solubilized nylon oligomer contains a nylon 6,6 pentamer, tetramer, trimer, or dimer.In one embodiment, the solubilizing nylon oligomer contains a nylon 6 pentamer, tetramer, trimer, or dimer. In some embodiments, the solubilizing nylon oligomer is a mixture of nylon decamers, nonamers, octamers, heptamers, or hexamers, pentamers, tetramers, trimers, and / or dimers. In some embodiments, the solubilizing nylon oligomer is a mixture of nylon 6,6 decamers, nonamers, octamers, heptamers, or hexamers, pentamers, tetramers, trimers, and / or dimers. In some embodiments, the solubilizing nylon oligomer is a mixture of nylon 6 decamers, nonamers, octamers, heptamers, or hexamers, pentamers, tetramers, trimers, and / or dimers. In one embodiment, the solubilizing nylon oligomer is a mixture of nylon pentamers, tetramers, trimers, or dimers. In one embodiment, the solubilized nylon oligomer is a mixture of nylon 6 pentamers, tetramers, trimers, or dimers. In one embodiment, the solubilized nylon oligomer is a mixture of nylon 6,6 pentamers, tetramers, trimers, or dimers. In one embodiment, the nylon oligomer is a tetramer. In one embodiment, the nylon oligomer is a trimer. In one embodiment, the nylon oligomer is a dimer. In another embodiment, the solubilized nylon oligomer contains a tetramer, trimer, or dimer. In one embodiment, the nylon oligomer is a nylon 6 tetramer. In one embodiment, the nylon oligomer is a nylon 6 trimer. In one embodiment, the nylon oligomer is a nylon 6 dimer. In another embodiment, the solubilized nylon oligomer contains a nylon 6 tetramer, trimer, or dimer. In one embodiment, the nylon oligomer is a nylon 6,6 tetramer. In one embodiment, the nylon oligomer is a nylon 6,6 trimer. In one embodiment, the nylon oligomer is a nylon 6,6 dimer. In another embodiment, the solubilizing nylon oligomer contains a nylon 6,6 tetramer, trimer, or dimer.

[0108] As used herein, the terms "nylon-containing material," "nylon-containing product," and the like are understood to refer to products, e.g., nylon products, containing at least one nylon in crystalline, semi-crystalline, or completely amorphous form. A nylon-containing material can refer to any article made from at least one nylon, e.g., nylon sheets, tubes, rods, profiles, shapes, films, large blocks, fibers, fabrics, and the like, containing at least one nylon and, optionally, other substances or additives, e.g., plasticizers, mineral, or organic fillers. In one embodiment, the nylon-containing material is a fabric or cloth comprising at least one nylon-containing fiber. In another embodiment, the nylon-containing material is a nylon compound or blend in the molten or solid state suitable for making a nylon product.

[0109] The nylon-containing material or product may be composed of nylon and one or more synthetic and / or natural materials. The nylon-containing material or product may be composed of nylon and another plastic, such as polyester, acrylic, polyurethane, polyethylene (PE), polypropylene (PP), polystyrene (PS), and polyvinyl chloride (PVC). The nylon-containing material or product may be composed of nylon and polyethylene terephthalate (PET). The nylon-containing material or product may be composed of nylon and natural materials, such as cotton, silk, cellulose, linen, and wool.

[0110] In another aspect, the disclosure provides a method of producing a polypeptide capable of hydrolyzing an amide bond in a polyamide, the method comprising: i) providing a polynucleotide described herein; ii) expressing the polynucleotide in a host cell under conditions sufficient to enable the host cell to produce the polypeptide; and iii) collecting the polypeptide produced by the host cell in ii).

[0111] In another aspect, the disclosure provides a method of hydrolyzing a nylon polymer or nylon oligomer, comprising exposing the nylon polymer or nylon oligomer to a polypeptide, composition, or host cell disclosed herein under conditions sufficient to convert the nylon-6,6 polymer to adipic acid and / or hexamethylenediamine.

[0112] In another aspect, the disclosure provides a method of degrading a nylon-containing product, the method comprising exposing the nylon-containing product to a polypeptide, composition, or host cell disclosed herein.

[0113] In one embodiment, the method disclosed herein comprises the steps of: i) chemically processing a nylon polymer, nylon oligomer, or nylon-containing product to produce nylon dimers, trimers, and tetramers; and ii) exposing the nylon dimers, trimers, tetramers, and pentamers produced in step (i) to a polypeptide, composition, or host cell disclosed herein under conditions sufficient to produce adipic acid and / or hexamethylenediamine.

[0114] In one embodiment, the nylon in the nylon polymer, nylon oligomer, or nylon-containing product is nylon 6 or nylon 6,6. In another embodiment, the nylon is nylon 6,6.

[0115] In another aspect, the disclosure provides a method of hydrolyzing a nylon 6,6 polymer, nylon 6,6 oligomer, or nylon 6,6-containing product, the method comprising exposing the nylon 6,6 polymer, nylon 6,6 oligomer, or nylon 6,6-containing product to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 73, 74, and 87, or an amino acid sequence having at least 70% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 73, 74, and 87. In one embodiment, the polypeptide comprises an amino acid sequence that is at least 80% sequence identical to the amino acid sequence of any one of SEQ ID NOs: 73, 74, and 87. In one embodiment, the polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of any one of SEQ ID NOs: 73, 74, and 87. In one embodiment, the polypeptide comprises an amino acid sequence that is at least 95% sequence identical to the amino acid sequence of any one of SEQ ID NOs: 73, 74, and 87. In one embodiment, the polypeptide comprises or consists of amino acid residues 2-392 of SEQ ID NO: 73. In one embodiment, the polypeptide comprises or consists of amino acid residues 2 to 392 of SEQ ID NO: 74. In one embodiment, the polypeptide comprises or consists of amino acid residues 2 to 392 of SEQ ID NO: 87. In one embodiment, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 73. In one embodiment, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 74. In one embodiment, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 87. In one embodiment, the polypeptide is a wild-type protein having NylB activity.

[0116] In another embodiment, the methods described herein further comprise recovering the adipic acid and / or hexamethylenediamine produced in step (ii).

[0117] In another aspect, the present disclosure provides compositions comprising adipic acid and / or hexamethylenediamine recovered by the methods disclosed herein.

[0118] In another aspect, the present disclosure provides a method for producing nylon polymers using the adipic acid and / or hexamethylenediamine compositions recovered by the methods disclosed herein.

[0119] In the context of the present disclosure, reference to increased or enhanced activity hydrolysis of amide bonds in polyamides refers to an increase in the ability of the polypeptides / engineered polypeptides and variants thereof to hydrolyze nylon, nylon polymers, or nylon oligomers compared to existing or wild-type NylB or Nylonase enzymes. In one embodiment, the activity of the polypeptides described herein is increased by at least about 1%. In one embodiment, the activity of the polypeptides described herein is increased by at least about 5%, preferably at least about 10%, preferably at least about 20%, preferably at least about 30%, preferably at least about 40%, preferably at least about 50%, preferably at least about 100%, preferably at least about 200%, preferably at least about 300%, preferably at least about 400%, preferably at least about 500%, preferably at least about 600%, preferably at least about 700%, preferably at least about 800%, preferably at least about 900%, or more preferably at least about 1,000% or more compared to existing or wild-type NylB or Nylonase enzymes.

[0120] As noted elsewhere herein, the inventors have engineered polypeptides with improved, increased, or enhanced hydrolytic enzyme activity, e.g., higher or broader activity, toward nylon polymers and nylon oligomers. These nylon oligomers can be solubilized nylon oligomers. The solubilized nylon oligomers can be nylon dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, 9amers, or 10amers. This disclosure is premised, at least in part, on the inventors' surprising discovery, based on ancestral sequence reconstructions of extant and ancestral variants of the NylB family, that the engineered polypeptides possess one or more increased or enhanced properties compared to one or more of the extant enzymes. This increased or enhanced activity is also observed with nylon 6,6 oligomeric substrates. For example, in certain embodiments, the engineered polypeptides disclosed herein have increased activity in hydrolyzing amide bonds in polyamides, such as an improved ability to hydrolyze amide bonds in nylon polyamides, as shown in the example provided herein. In certain embodiments, the engineered polypeptides disclosed herein have increased thermostability. This is a highly surprising finding, for reasons including that the temperature conditions to which a hypothetical ancestral enzyme may have been exposed would not be significantly different from the temperatures or conditions to which one or more corresponding existing enzymes are exposed. In certain embodiments, the engineered polypeptides disclosed herein are associated with increased recombinant expression in host cell systems, where such host cells have been modified by the insertion of a polynucleotide sequence encoding the enzyme. Currently known nylonase enzymes exhibit only low levels of enzyme expression in common industrial host organisms.

[0121] The polypeptides disclosed herein have excellent properties for use in industrial processes, with the aim of improving the activity of hydrolases, particularly those capable of hydrolyzing nylon, under conditions (i.e., conditions of heat, pH, and pressure) typically used in industrial degradation of nylon polymers and / or nylon-containing products. The polypeptides disclosed herein are capable of hydrolyzing amide bonds in polyamides (see Figure 1) and are particularly suitable for the degradation of nylon 6,6 polymers, nylon 6,6 oligomers, and / or nylon 6,6-containing materials or products. The engineered polypeptides described herein have adipic acid mono- and di-N-alkylamide hydrolase activity.

[0122] In the context of the present disclosure, reference to increased or enhanced activity may include one or more of the following: an increased ability of the polypeptide to hydrolyze amide bonds in polyamides, including nylons, such as nylon 6 and nylon 6,6, when compared to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 73-86; increased recombinant expression in a host cell system when compared to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 73-86; increased whole cell activity when compared to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 73-86; and increased thermostability when compared to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 73-86.

[0123] In one embodiment, the polypeptides disclosed herein that are capable of hydrolyzing amide bonds in polyamides exhibit increased recombinant expression in a host cell system as compared to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 73-86.

[0124] In another embodiment, the polypeptides disclosed herein that are capable of hydrolyzing amide bonds in polyamides exhibit increased thermal stability compared to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 73-86.

[0125] In another embodiment, the polypeptides disclosed herein that are capable of hydrolyzing amide bonds in polyamides comprise increased whole cell activity when compared to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 73-86. As used herein, the term whole cell activity typically refers to the ability of a polyamide to resemble a nylon polymer or nylon oligomer when expressed in a host cell system.

[0126] In one embodiment, the activity of the polypeptide in hydrolyzing amide bonds in polyamides disclosed herein or hydrolyzing nylon 6 oligomers or nylon 6,6 oligomers is similar to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 73-86. In one embodiment, the activity of a polypeptide described herein in hydrolyzing an amide bond in a polyamide disclosed herein or hydrolyzing a nylon 6 oligomer or a nylon 6,6 oligomer is increased by at least about 5%, preferably at least about 10%, preferably at least about 20%, preferably at least about 30%, preferably at least about 40%, preferably at least about 50%, preferably at least about 100%, preferably at least about 200%, preferably at least about 300%, preferably at least about 400%, preferably at least about 500%, preferably at least about 600%, preferably at least about 700%, preferably at least about 800%, preferably at least about 900%, or more preferably at least about 1,000%, or more, relative to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 73-86 or a wild-type NylB enzyme. In one embodiment, the activity of a polypeptide disclosed herein in hydrolyzing a nylon 6,6 oligomer is similar to that of a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 73-86.In one embodiment, the activity of a polypeptide described herein in hydrolyzing a nylon 6,6 oligomer disclosed herein is increased by at least about 5%, preferably at least about 10%, preferably at least about 20%, preferably at least about 30%, preferably at least about 40%, preferably at least about 50%, preferably at least about 100%, preferably at least about 200%, preferably at least about 300%, preferably at least about 400%, preferably at least about 500%, preferably at least about 600%, preferably at least about 700%, preferably at least about 800%, preferably at least about 900%, or more preferably at least about 1,000%, or more, relative to a polypeptide having the amino acid sequence of any one of SEQ ID NOS:73-86 or a wild-type NylB enzyme. Suitable methods for determining or measuring the enzymatic activity of a polypeptide are familiar to those of skill in the art, and illustrative examples are provided elsewhere herein. Other methods for measuring nylon hydrolysis are described in Kiumarsi and Parvinzadeh, 2010 J Appl Polymer Sci, 116:3140 and Gashti et al., 2013 Preparative Biochemistry & Biotechnology, 43:798, the contents of which are incorporated herein by reference in their entireties.In one embodiment, the activity of a polypeptide disclosed herein in hydrolyzing nylon 6,6 oligomers, including dimers, trimers, and tetramers, is increased by at least about 5%, preferably at least about 10%, preferably at least about 20%, preferably at least about 30%, preferably at least about 40%, preferably at least about 50%, preferably at least about 100%, preferably at least about 200%, preferably at least about 300%, preferably at least about 400%, preferably at least about 500%, preferably at least about 600%, preferably at least about 700%, preferably at least about 800%, preferably at least about 900%, or more preferably at least about 1,000%, or more, compared to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 73-86 or a wild-type NylB enzyme, as determined by a colorimetric assay or an LC-MC method using solubilized nylon 6,6 oligomers, including dimers, trimers, and tetramers, as a substrate.

[0127] The activity of a polypeptide in hydrolyzing amide bonds in the polyamides disclosed herein or hydrolyzing nylon 6 oligomers or nylon 6,6 oligomers can be assigned an absolute value or a value relative to the activity of a comparator (e.g., a polypeptide having the amino acid sequence of any one of SEQ ID NOS: 73-86 or a wild-type NylB enzyme). In one embodiment, the activity of a polypeptide is measured as the rate of monomer and / or oligomer release over time under appropriate conditions of temperature, pH, and buffer. In another embodiment, the activity of a polypeptide is measured as the rate of digestion of a substrate over time (i.e., measuring the rate of change in concentration or amount of substrate) under appropriate conditions of temperature, pH, and buffer.

[0128] The activity of a polypeptide in hydrolyzing amide bonds in polyamides or hydrolyzing nylon 6 or nylon 6,6 oligomers can be measured or assayed using purified enzymes, or the enzyme activity can be measured as a function of the activity of the enzyme when recombinantly expressed in a host cell system (also referred to herein as cellular catalytic activity or whole cell activity).

[0129] Advantageously, the polypeptides described herein exhibit increased or enhanced recombinant expression in host cells by at least about 5%, preferably at least about 10%, preferably at least about 20%, preferably at least about 30%, preferably at least about 40%, preferably at least about 50%, preferably at least about 100%, preferably at least about 200%, preferably at least about 300%, preferably at least about 400%, preferably at least about 500%, preferably at least about 600%, preferably at least about 700%, preferably at least about 800%, preferably at least about 900%, or more preferably at least about 1,000%, compared to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 73-86 or a wild-type NylB enzyme.

[0130] The polypeptides described herein exhibit increased thermostability, which is the ability to resist irreversible changes in enzymatic function or activity (i.e., denaturation) upon exposure to extreme temperatures. The polypeptides described herein retain their ability to hydrolyze amide bonds in polyamides or hydrolyze nylon 6 oligomers or nylon 6,6 oligomers after exposure to temperatures ranging from about 4°C to about 70°C, preferably from about 10°C to about 70°C, preferably from about 20°C to about 60°C, more preferably from about 35°C to about 55°C, even more preferably from about 40°C to about 50°C, and even more preferably about 45°C. In one embodiment, the polypeptides described herein exhibit activity at temperatures ranging from about 10°C to about 60°C, preferably from about 20°C to about 60°C, preferably from about 30°C to about 60°C, more preferably from about 40°C to about 60°C, even more preferably from about 40°C to about 50°C, or even more preferably about 45°C. Advantageously, the polypeptides described herein exhibit the ability to hydrolyze amide bonds in polyamides or hydrolyze nylon 6 oligomers or nylon 6,6 oligomers at temperatures ranging from about 10°C to about 80°C, preferably from about 20°C to about 70°C, preferably from about 30°C to about 60°C, more preferably from about 35°C to about 55°C, even more preferably from about 40°C to about 50°C, and even more preferably about 45°C. In one embodiment, the polypeptides described herein exhibit activity at temperatures ranging from about 10°C to about 70°C, preferably from about 20°C to about 60°C, preferably from about 30°C to about 60°C, more preferably from about 40°C to about 60°C, even more preferably from about 40°C to about 50°C, or even more preferably about 45°C. In one embodiment, the ability to hydrolyze amide bonds in polyamides or hydrolyze nylon 6 oligomers or nylon 6,6 oligomers can be measured at temperatures ranging from about 40°C to about 70°C, preferably from about 40°C to about 50°C, or even more preferably about 45°C. In another specific embodiment, the ability to hydrolyze amide bonds in polyamides or to hydrolyze nylon 6 oligomers or nylon 6,6 oligomers is measurable and remains measurable at temperatures of about 10°C to about 30°C, preferably about 15°C to about 28°C, which corresponds to the average temperature in the natural environment (room temperature).

[0131] In one embodiment, a polypeptide having the ability to hydrolyze amide bonds in polyamides or hydrolyze nylon 6 oligomers or nylon 6,6 oligomers is measurable at a temperature of about 10°C to about 60°C, preferably about 20°C to about 60°C, preferably about 30°C to about 60°C, more preferably about 40°C to about 60°C, even more preferably about 40°C to about 50°C, or even more preferably about 45°C, compared to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 73-86, with a hydrolysis rate of at least about 5%, preferably at least about 10%, preferably at least about 20%, preferably at least about 30%, preferably at least about 40%, preferably at least about 50%, preferably at least about 100%, preferably at least about 200%, preferably at least about 300%, preferably at least about 400%, preferably at least about 500%, preferably at least about 600%, preferably at least about 700%, preferably at least about 800%, preferably at least about 900%, or more preferably at least about 1,000%, or more.

[0132] In another specific embodiment, the polypeptide described herein has increased activity in hydrolyzing amide bonds in polyamides or hydrolyzing nylon 6 oligomers or nylon 6,6 oligomers at temperatures of about 10°C to about 70°C, preferably about 20°C to about 60°C, preferably about 30°C to about 60°C, preferably about 40°C to about 60°C, preferably about 40°C to about 50°C, or more preferably about 45°C, when compared to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 73-86 or a wild-type NylB enzyme. In one embodiment, the polypeptide described herein is capable of hydrolyzing amide bonds in polyamides or hydrolyzing nylon 6 oligomers or nylon 6,6 oligomers at about 20°C to about 70°C with at least about 5%, preferably at least about 10%, preferably at least about 20%, preferably at least about 30%, preferably at least about 40%, preferably at least about 50%, preferably at least about 100%, preferably at least about 200%, preferably at least about 300%, preferably at least about 400%, preferably at least about 500%, preferably at least about 600%, preferably at least about 700%, preferably at least about 800%, preferably at least about 900%, or more preferably at least about 1,000% or more, compared to the activity of a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 73-86 or a wild-type NylB enzyme.

[0133] In another embodiment, the polypeptide described herein has increased activity in hydrolyzing amide bonds in polyamides or hydrolyzing nylon 6 oligomers or nylon 6,6 oligomers at temperatures of about 10°C to about 60°C, preferably about 20°C to about 50°C, even more preferably about 20°C to about 40°C, or even more preferably about 40°C, compared to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 73-86 or a wild-type NylB enzyme. In one embodiment, the polypeptides described herein have an activity in hydrolyzing amide bonds in polyamides or hydrolyzing nylon 6 oligomers or nylon 6,6 oligomers at a temperature of about 10°C to about 40°C that is at least about 5%, preferably at least about 10%, preferably at least about 20%, preferably at least about 30%, preferably at least about 40%, preferably at least about 50%, preferably at least about 100%, preferably at least about 200%, preferably at least about 300%, preferably at least about 400%, preferably at least about 500%, preferably at least about 600%, preferably at least about 700%, preferably at least about 800%, preferably at least about 900%, or more preferably at least about 1,000% or more, compared to the activity of a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 73-86 or a wild-type NylB enzyme at the same temperature.

[0134] In one embodiment, the polypeptides described herein exhibit measurable activity in hydrolyzing amide bonds in polyamides or hydrolyzing nylon 6 oligomers or nylon 6,6 oligomers at least in the pH range of 5 to 11, preferably in the pH range of 6 to 10, more preferably in the pH range of 6.5 to 9, and even more preferably in the pH range of 7 to 8.

[0135] Advantageously, the thermostability of the polypeptides described herein is not significantly impaired compared to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 73-86 or a wild-type NylB enzyme. In some embodiments, the thermostability of the polypeptides described herein is improved when compared to the thermostability of a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 73-86 or a wild-type NylB enzyme. As used herein, the term "improved thermostability" or "increased thermostability" refers to an increased ability of an enzyme to resist changes in its chemical and / or physical structure at higher temperatures, more specifically at temperatures between 40°C and 70°C, compared to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 73-86 or a wild-type NylB enzyme. In one embodiment, the polypeptides described herein have an increased half-life at temperatures between 40°C and 70°C compared to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 73-86 or a wild-type NylB enzyme. The polypeptides described herein may exhibit a melting temperature (Tm) that is higher than or equal to that of a wild-type NylB enzyme or a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 73-86. In some embodiments, the polypeptides described herein exhibit improved thermostability at temperatures between 40°C and 70°C compared to a wild-type NylB enzyme or a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 73-86.

[0136] The thermal stability of a polypeptide can be evaluated by any suitable means known to those skilled in the art. For example, thermal stability can be evaluated by measuring the residual enzymatic activity of the polypeptide after incubation at various temperatures. The ability to undergo multiple rounds of hydrolysis at various temperatures can also be evaluated. Differential scanning fluorometry (DSF) can also be used to evaluate the thermal stability of a polypeptide. Circular dichroism can also be used to measure the thermal stability of a polypeptide described herein, including its melting temperature (Tm). The term "melting temperature (Tm)" is understood to mean the temperature at which a given protein is 50% denatured.

[0137] In one embodiment, the polypeptides described herein exhibit a melting temperature (Tm) of about 45°C to about 80°C, preferably about 50°C to about 75°C, and preferably about 52°C to about 75°C. In one embodiment, the polypeptides described herein exhibit a melting temperature (Tm) lower than the melting temperature (Tm) exhibited by a polypeptide having any one of the amino acid sequences of SEQ ID NOs: 73 to 86 or a wild-type NylB enzyme. In one embodiment, the polypeptides described herein exhibit a melting temperature (Tm) higher than the melting temperature (Tm) exhibited by a polypeptide having any one of the amino acid sequences of SEQ ID NOs: 73 to 86 or a wild-type NylB enzyme.

[0138] The present disclosure provides a method for producing a polypeptide capable of hydrolyzing amide bonds in polyamides or hydrolyzing nylon 6 oligomers or nylon 6,6 oligomers, comprising: a) providing a nucleic acid sequence as described herein; b) producing the polypeptide by expressing the nucleic acid sequence in a host cell culture; c) recovering the polypeptide produced in (b) from the host cell culture; The present invention also extends to methods including:

[0139] The present disclosure also extends to in vitro methods of producing a polypeptide described herein, comprising the steps of (a) contacting a nucleic acid, cassette, or vector of the invention with an in vitro expression system, and (b) recovering the produced polypeptide. In vitro expression systems are well known to those of skill in the art and are commercially available.

[0140] Suitable host cells are familiar to those of skill in the art, and illustrative examples include recombinant Bacillus, recombinant Escherichia coli (E. coli), recombinant Pseudomonas, recombinant Aspergillus, recombinant Trichoderma, recombinant Streptomyces, recombinant Saccharomyces, recombinant Pichia, recombinant Thermus, or recombinant Yarrowia. In one embodiment, the host cell is E. coli.

[0141] Host cells can be cultured in a nutrient medium suitable for polypeptide production using methods known to those skilled in the art. Suitable examples include culturing host cells in laboratory or industrial fermenters, using shake flask cultures or small- or large-scale fermentation (including continuous, batch, fed-batch, or solid-state fermentation) in a suitable medium under conditions that allow the enzyme to be expressed and / or isolated. Culturing typically occurs in an appropriate nutrient medium, prepared from a commercial supplier or according to a published composition (e.g., in the American Type Culture Collection catalog), or any other culture medium suitable for cell growth. If the polypeptide is expressed and / or secreted into the nutrient medium, the polypeptide can be used in the form of a cell / supernatant mixture or a crude cell lysate. Alternatively, the polypeptide can be recovered directly from the culture supernatant. Conversely, the polypeptide can be recovered from a cell lysate or after permeabilization of the host cell membrane. The polypeptide can be recovered using any suitable method known to those skilled in the art, illustrative examples of which include collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. Optionally, the polypeptide may be partially or completely purified by various procedures known in the art, including, but not limited to, heat shock, chromatography (e.g., ion exchange, affinity, hydrophobic, isoelectric focusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction to obtain a substantially pure polypeptide.

[0142] The polypeptides can be used in purified form, either alone or in combination with additional enzymes (e.g., PETase, MHETase, or nylonase) to catalyze enzymatic reactions involved in the degradation and / or recycling of nylon-containing materials. The polypeptides described herein can be in soluble form or in solid phase. In particular, they can be bound to cell membranes or lipid vesicles, or to synthetic supports, such as glass, plastic, polymer, filter membranes, in the form of beads, columns, plates, etc.

[0143] The present disclosure also extends to compositions comprising the polypeptides, nucleic acids, or host cells described herein.

[0144] The composition may be in liquid or dry form, e.g., powder form. In some embodiments, the composition is a lyophilizate. For example, the composition may include a polypeptide, a nucleic acid, and / or a host cell, and optionally an excipient and / or a reagent. Suitable excipients may include buffers commonly used in biochemistry, agents for adjusting pH, preservatives such as sodium benzoate, sodium sorbate, or sodium ascorbate, preservatives, protectants, or stabilizers such as starch, dextrin, gum arabic, salts, sugars such as sorbitol, trehalose, or lactose, glycerol, polyethylene glycol, polyethene glycol, polypropylene glycol, propylene glycol, divalent ions such as calcium, sequestering agents such as EDTA, reducing agents (e.g., beta-mercaptoethanol, dithiothreitol, ascorbic acid, tris(2-carboxyethyl)phosphine), amino acids, carriers such as solvents or aqueous solutions, etc.

[0145] In one embodiment, the composition comprises a polypeptide described herein (the polypeptide may be present in the composition in isolated or at least partially purified form). In one embodiment, the composition comprises the polypeptide described herein in an amount of about 0.1% to about 99.9% by weight, preferably about 0.1% to about 50% by weight, preferably about 0.1% to about 30% by weight, and preferably about 0.1% to about 5% by weight, based on the total weight of the composition. In a preferred embodiment, the composition comprises the polypeptide described herein in an amount of about 0.1 to about 5% by weight, based on the total weight of the composition. In another embodiment, the composition comprises the polypeptide described herein in an amount of about 0.1 to about 0.2% by weight, based on the total weight of the composition. The amount of polypeptide in the composition can be appropriately adapted by those skilled in the art depending, for example, on the nature and / or amount of the polyester-containing material to be degraded (hydrolyzed) and / or the presence or absence of any additional enzymes / polypeptides in the composition.

[0146] The compositions described herein may further comprise additional polypeptides that exhibit enzymatic activity, including but not limited to nylonase.

[0147] In one embodiment, the polypeptides described herein are solubilized in an aqueous medium with one or more excipients, e.g., excipients that suitably stabilize or protect the polypeptide from degradation. For example, the polypeptides described herein can be solubilized in water and then mixed with an excipient, e.g., glycerol, sorbitol, dextrin, starch, glycols such as propanediol, salts, etc. The resulting mixture can then be dried to obtain a powder. Methods for drying such mixtures are well known to those skilled in the art and include, but are not limited to, lyophilization, freeze-drying, spray drying, supercritical drying, downdraft evaporation, thin-layer evaporation, centrifugal evaporation, conveyor drying, fluidized-bed drying, drum drying, or any combination thereof.

[0148] In one embodiment, the composition comprises at least one host cell expressing a polypeptide described herein, or an extract thereof. By "cell extract" is meant any fraction obtained from a cell, such as a cell supernatant, cell debris, cell wall, DNA extract, enzyme or enzyme preparation, or any preparation derived from the cell by chemical, physical, and / or enzymatic treatment, that is essentially free of viable cells. Preferred extracts are those with enzymatic activity. The composition may comprise one or more host cells or extracts thereof containing a polypeptide described herein, and optionally one or more additional cells.

[0149] As noted elsewhere herein, the inventors have surprisingly found that the polypeptides described herein have greater activity in hydrolyzing amide bonds in polyamides or hydrolyzing nylon 6 oligomers or nylon 6,6 oligomers when compared to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 73-86 or a wild-type NylB enzyme. Accordingly, disclosed herein is a method for hydrolyzing a nylon polymer or nylon oligomer or nylon-containing product, the method comprising exposing the nylon polymer or nylon oligomer or nylon-containing product to a polypeptide, composition, or host cell described herein under conditions sufficient to convert the nylon polymer or nylon oligomer or nylon-containing product, adipic acid, and / or hexamethylenediamine.

[0150] The present disclosure extends to the use of the polypeptides, compositions, or host cells described herein to degrade nylon polymers or oligomers or nylon-containing products under aerobic or anaerobic conditions, and / or to recycle nylon-containing materials as plastic products made from or containing nylon, and / or to produce biodegradable plastic products containing nylon. Such methods are particularly useful for degrading nylon polymers or oligomers or nylon-containing products, including nylon 6,6 polymers or oligomers or nylon 6,6-containing products.

[0151] Advantageously, the nylon of the nylon-containing material or nylon-containing product is depolymerized to monomers and / or oligomers. In one embodiment, at least one nylon polymer or nylon oligomer is decomposed to obtain repolymerizable monomers and / or oligomers, which are advantageously reclaimed or recovered for further use.

[0152] In one embodiment, the nylon of the nylon-containing material or nylon-containing product is completely degraded.

[0153] As noted elsewhere herein, the nylon product may comprise at least one polyester selected from the group consisting of polyethylene terephthalate (PET), polylactic acid (PLA), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polyethylene terephthalate (PET), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), polycaprolactone (PCL), poly(ethylene adipate) (PEA), and combinations of any of the foregoing.

[0154] The time required to degrade a nylon-containing material or nylon-containing product can vary depending on the nylon-containing material or nylon-containing product itself (i.e., the nature and origin of the material / product, its composition, form, etc.), the type and amount of polypeptide used, and various process parameters (i.e., temperature, pH, additional agents, etc.) One skilled in the art can readily adapt the process parameters to polyester-containing materials.

[0155] Advantageously, the degradation process is carried out at a temperature of about 10° C. to about 70° C., preferably about 20° C. to about 60° C., preferably about 30° C. to about 60° C., more preferably about 40° C. to about 60° C., even more preferably about 40° C. to about 50° C., or even more preferably about 45° C. The temperature is typically kept below the inactivation temperature, which corresponds to the temperature at which the polypeptide is inactivated and / or at which the recombinant microorganism does not synthesize, produce, or release the polypeptide described herein.

[0156] In one embodiment, the nylon polymer, nylon oligomer, or nylon-containing product or material may be pretreated or preprocessed prior to contact with the polypeptide to physically alter its structure or solubility to increase the contact surface between the nylon and the enzyme. In one embodiment, the nylon polymer, nylon oligomer, or nylon-containing product or material may be pretreated or preprocessed such that the nylon is solubilized prior to contact with the polypeptide.

[0157] The monomers resulting from the depolymerization or decomposition process or method may be recovered, either sequentially or continuously, as appropriate. Depending on the starting nylon polymer, nylon oligomer, or nylon-containing material / product, a single type of monomer or several different types of monomers may be recovered.

[0158] The recovered monomer may be further purified and conditioned to a repolymerizable form using any suitable purification method, including, in combination or without combinations thereof, stripping processes, separation with aqueous solutions, vapor selective condensation, filtration and concentration of post-bioprocessing media, separation, distillation, vacuum evaporation, extraction, electrodialysis, adsorption, ion exchange, precipitation, crystallization, concentration and acid dehydration and precipitation, nanofiltration, acid-catalyzed treatment, semi-continuous or continuous mode distillation, solvent extraction, evaporative concentration, evaporative crystallization, liquid / liquid extraction, hydrogenation, azeotropic distillation processes, acid or thermally catalyzed lactamization, adsorption, column chromatography, simple vacuum distillation, and microfiltration.

[0159] The repolymerizable monomers can be used to synthesize new nylon polymers. Advantageously, nylon of the same nature is repolymerized. However, it is possible to mix the recovered monomers with other monomers, for example, to synthesize new nylon copolymers. Alternatively, the recovered monomers can be used as chemical intermediates to produce new chemical compounds of interest.

[0160] The present disclosure also extends to compounds including polypeptides, compositions, and / or host cells expressing said polypeptides or extracts thereof containing said polypeptides.

[0161] The present disclosure also extends to masterbatch compositions comprising the polypeptides, compositions, and / or host cells expressing said polypeptides or extracts thereof containing said polypeptides.

[0162] Advantageously, such compounds or masterbatch compositions described herein can be used to produce nylon-containing materials and / or plastic articles that include the polypeptides described herein.

[0163] In one embodiment, the resulting compound, masterbatch composition, or nylon article is a biodegradable plastic compound, masterbatch composition, or plastic article in accordance with at least one of the relevant standards and / or labels known by those skilled in the art, such as standard EN 13432, standard ASTM D6400, OK Biodegradation Soil (Vincotte label), OK Biodegradation Water (Vincotte label), OK Compost (Vincotte label), OK Home Compost (Vincotte label).

[0164] The polypeptides disclosed herein are suitable for a variety of applications, including industrial applications, illustrative examples of which include additives in detergents, compositions, textile production, electronics, and biomedical applications. For example, the polypeptides disclosed herein can be used in textile production, where they can be used as exonucleases to appropriately modify the properties of textile fibers.

[0165] The present invention will now be described with reference to the following examples which illustrate some preferred aspects of the invention, but it will be understood that the details of the following description of the invention do not supersede the generality of the preceding description of the invention. [Example]

[0166] Example 1 Ancestral sequence reconstruction The NylB sequences of P07061 from Flavobacterium species (strain K172) and P07062 from Flavobacterium species (strain K172) were used as query sequences for a blast search of the NCBI refseq protein database and the swissprot database using an E-value threshold of 10e-10.

[0167] Redundancy was removed to 90% sequence identity using CD-HIT (Fu et al., 2012), and nonredundant sequence datasets were aligned using the GINSI protocol in MAFFT with DASH structural homology enabled. One hundred independent iterations of maximum likelihood model parameterization and tree search were performed using empirical Bayesian ancestral sequence reconstruction in IQ-TREE. The maximum likelihood model was fitted as LG+F+R9 by the Bayesian Information Criterion. For all tree search iterations, 1000 iterations of ultrafast bootstrap approximation were performed. The tree search and bootstrap approximation converged within the default algorithm parameters for all iterations. Empirical Bayesian ancestral sequence reconstruction was further performed using the LG+G4 sequence evolution model in CodeML of the PAML software suite.

[0168] Indel events in the ancestral sequences generated by IQ-TREE and CodeML were modeled using a Jukes-Cantor-like equal-proportion model, which assumes equal probabilities of insertion and deletion, in the Ape package for R. Phylogenetic trees were visualized using GGtree in R. 172 ancestral and extant sequences were selected for synthesis based on their phylogenetic relationship to extant sequences with NylB activity (stage I sequences). These sequences (SEQ ID NOS: 2-72, see Table 1) spanned the complete evolutionary trajectory from the last common ancestor to extant NylBs, and were supported by high ultrafast bootstrap approximations (approximately 95%) and average posterior probabilities (approximately 80%).

[0169] Example 2 Protein expression and purification Plasmids were transformed into chemically competent E. cloni® cells (Lucigen) by heat shock, plated onto lysogeny broth (LB) agar supplemented with 100 μg / mL kanamycin, and incubated overnight at 37° C. A single colony was used to inoculate 2.2 mL of 1.5 mL LB medium supplemented with 100 μg / mL kanamycin into a 96-well deep-well block and grown at 1050 rpm at 37° C. to an OD of approximately 0.8, then induced with 1 mM IPTG and grown at 1050 rpm at room temperature (RT, 25° C.) for an additional 16 hours.

[0170] Cells were harvested by centrifugation at 2000×g for 15 minutes at room temperature and resuspended in lysis buffer (1× BugBuster® Protein Extraction Reagent (Merck-Millipore), 20 mM Tris, 300 mM NaCl, 1 U / ml Turbonuclease (Sigma) pH 8). The cell suspension was incubated for 20 minutes at room temperature with gentle shaking. The lysate was separated from insoluble cell debris by centrifugation at 2250×g for 1 hour at room temperature. The clarified lysate was then diluted with 100 μl of equilibration buffer (20 mM Tris, 300 mM NaCl, pH 8) and purified by nickel-charged IMAC using a 96-well HisPur™ Ni-NTA Spin Plate (ThermoFisher Scientific) equilibrated with equilibration buffer. The sample was washed three times with 250 μl of wash buffer (20 mM Tris, 300 mM NaCl, 10 mM imidazole, pH 8) and eluted with 250 μl of elution buffer (20 mM Tris, 300 mM NaCl, 150 mM imidazole, pH 8). All centrifugation steps after the addition of wash or elution buffer were at 1000 × g for 1 minute at RT. The eluate was stored at 4°C.

[0171] Example 3 Colorimetric assay for measuring activity and identifying candidate polypeptides with increased ability to hydrolyze amide bonds in polyamides 2 mM nylon 6,6 trimer in 100 μl of reaction buffer (45 mM NaHPO, 90 mM NaCl, pH 7.5) was pre-equilibrated to 40°C. The reaction was initiated by the addition of 100 μl of a 1:10 dilution of the eluate from the 96-well Ni-NTA purification in reaction buffer, followed by incubation at 40°C for 3 hours. The reaction was terminated by filtration through a 10 kDa MWCO centrifugal filter at 13,000 g for 15 minutes to remove the enzyme. 100 μl of the flow-through was added to 50 μl of a 0.01% solution of 2,4,6-trinitrobenzenesulfonic acid (TNBSA) in reaction buffer, and the absorbance was measured at 335 nm using an Epoch microplate spectrophotometer (BioTek).

[0172] Although many, but not all, of the variant sequences identified by ancestral sequence reconstruction exhibit the ability to hydrolyze amide bonds in polyamides, several variant polypeptides (SEQ ID NOS: 2-16) were surprisingly found to have an increased ability to hydrolyze amide bonds in polyamides compared to the extant sequences (Figure 3).

[0173] The polypeptides of SEQ ID NOs: 2 to 16 were also tested for their recombinant expression levels, and the expression yields are shown in Table 3.

[0174] Analysis of the sequences showed that many of the polypeptides had an increased ability to hydrolyze amide bonds in polyamides when compared to the existing sequences and showed high sequence identity represented by the consensus sequence of SEQ ID NO: 1 (see Figure 5). SEQ ID NO:1:

[0175] [ka]

[0176] (In the formula, X1~X 65 is any amino acid)

[0177] Example 4 thermal stability The thermal stability of the polypeptides disclosed herein was measured using differential scanning fluorimetry or circular dichroism (CD) using a Chirascan™ spectropolarimeter (Applied Photophysics). For differential scanning fluorimetry, 20 μl reactions containing 1× Protein Thermal Shift™ dye (ThermoFisher Scientific), 1× Protein Thermal Shift™ buffer (ThermoFisher Scientific), and 10 μM purified enzyme were prepared and transferred to a MicroAmp™ EnduraPlate™ optical 96-well clear reaction plate (ThermoFisher Scientific). Fluorescence was measured using a QuantStudio3 real-time PCR system (ThermoFisher Scientific) while heating the samples from 25 to 90°C at a rate of 0.05°C / sec. Data were analyzed using Protein Thermal Shift™ software, and a Boltzmann curve was fitted to the data to determine the T m was determined. For the CD method, the protein was diluted to 0.2 mg / mL in reaction buffer. The path length of the cuvette was 1 mm. CD at 222 nm was measured at 0.5°C intervals while the temperature was increased from 20°C to 90°C at 1°C / min.

[0178] The average Tms measured for the existing variants of SEQ ID NOS: 75, 76, 78-80, and 86 were 51.4, 49.2, 48.4, 55.1, 41.8, and 71.7, respectively. The polypeptides of SEQ ID NOS: 4 and 2 had average Tms of 75.0 and 71.1°C, respectively (Figure 4).

[0179] Example 5 Engineering polypeptides with increased activity To engineer variants with improved ability to hydrolyze amide bonds in polyamides, several highly active polypeptides from the first round of ancestral reconstruction in Example 1 were selected as a basis for further engineering. A second round of this engineering identified several new sequences (Phase II) that showed improved activity against nylon 6,6 oligomers in whole-cell assays compared to the extant sequence and SEQ ID NO:4 from the first-round ancestral reconstruction. Several variants exhibited high whole-cell activity. In Figure 7A, C4 corresponds to SEQ ID NO:90, D3 corresponds to the polypeptide comprising SEQ ID NO:91, E3 corresponds to the polypeptide comprising SEQ ID NO:95, F3 corresponds to the polypeptide comprising SEQ ID NO:92, and G3 corresponds to the polypeptide comprising SEQ ID NO:93.

[0180] Several candidate variants (including those with high whole-cell activity) were selected for assay of their specific enzymatic activity against nylon 6,6 dimer, trimer, and tetramer using LC-MS. The activities of various stage II variants are shown in Figure 7B.

[0181] Enzyme activity assays against nylon 6,6 oligomers (dimer, trimer, and tetramer) were performed in 200 μL reactions at 40°C using 20 μM purified enzyme. The reactions were incubated for 1 hour at 40°C and terminated by removing the enzyme using a 10 kDa MWCO centrifugal filter (15 minutes at 13,000 × g). Liquid chromatography-mass spectrometry (LC-MS) was used to analyze the reactions and compare them to control reactions containing no enzyme. Using the LC-MS method, several stage I enzymes were re-assayed in parallel with selected stage II variants. The activity of variant polypeptides containing SEQ ID NOs: 4, 48, 88, 63, 3, 89-94 compared to the native enzymes of SEQ ID NOs: 75-84 and 86 is shown in Figure 8. Notably, polypeptides containing SEQ ID NOs: 93, 90, 3, 63, 88, and 48 showed improved activity against nylon 6,6 oligomers.

[0182] Polypeptides containing SEQ ID NOs: 3 and 63 showed increased activity against nylon 6,6 dimer. Polypeptides containing SEQ ID NOs: 3, 63, 92, and 93 showed increased activity against nylon 6,6 trimer. Polypeptides containing SEQ ID NOs: 90, 3, 63, and 93 showed increased activity against nylon 6,6 tetramer.

[0183] Analysis of the Phase I and Phase II sequences showed that many of the polypeptides had an increased ability to hydrolyze amide bonds in polyamides when compared to the existing sequences and exhibited high sequence identity, which can be represented by consensus sequence II of SEQ ID NO:96 (below and Figure 10). Table 2 provides the amino acid substitutions at the indicated positions of the variant polypeptides, with numbering relative to the amino acid positions of SEQ ID NO:1 or SEQ ID NO:96. SEQ ID NO:96

[0184] [ka]

[0185] (In the formula, X1~X 79 is any amino acid)

[0186] Example 6 Stability of engineered enzymes For kinetic stability analysis, purified enzymes were incubated at 40°C, 50°C, 60°C, 70°C, or 80°C for 15 minutes before reaction with nylon 6,6 trimer. The data are shown in Figure 9. Previous heat shock exposure at 50°C to 60°C did not appear to result in significant loss of enzyme activity, although some variants showed a slight decline in enzyme activity at 60°C. A larger decrease in enzyme activity was observed for enzymes pre-exposed to a 70°C heat shock. After the 80°C heat shock, the enzyme appears to have lost its ability to convert nylon 6,6 trimer.

[0187] Example 7 Acid hydrolysis of nylon 6,6 oligomers with immobilized enzymes. The engineered polypeptide was immobilized onto an adsorption-based polymer resin at a 10% w / w enzyme loading. The immobilized enzyme was incubated with nylon 6,6 dimer, trimer, and tetramer. The reaction solution was sampled after 1 hour for analysis by LC-MS and compared to a control reaction containing no enzyme (time point 0). The data are shown in Figure 6.

[0188] Example 8 Further enzyme engineering Further rounds of enzyme engineering identified variant polypeptides that exhibited activity in hydrolyzing amide bonds in polyamides, including nylon 6,6 oligomers. Assays of enzyme activity against nylon 6,6 oligomers (dimers, trimers, and tetramers) were performed at 40°C for 2 hours. The reactions were terminated by removing the enzyme using a 10 kDa MWCO centrifugal filter (15 minutes, 13,000 × g). Ultra-high performance liquid chromatography (UHPLC) was used to analyze the reactions.

[0189] Polypeptide variants containing any one of the amino acid sequences of SEQ ID NOs: 97-123 were able to hydrolyze nylon 6,6 oligomers. The activity of some of these polypeptides in hydrolyzing nylon 6,6 trimers is demonstrated in Figure 11.

[0190] A significant proportion of the polypeptides capable of hydrolyzing nylon 6,6 oligomers shared the consensus sequence of SEQ ID NO:124 / consensus III. SEQ ID NO: 124

[0191] [ka]

[0192] (In the formula, X1~X 37 is any amino acid)

[0193] Example 9 Polypeptides capable of hydrolyzing nylon 6,6 oligomers Analysis of the polypeptides disclosed herein as being capable of hydrolyzing amide bonds in polyamides revealed significant sequence conservation, which can be represented as consensus sequence IV of SEQ ID NO: 125 (FIG. 13), which includes SEQ ID NOs: 2, 4-15, 89-95, and 97-122. SEQ ID NO: 125

[0194] [ka]

[0195] (In the formula, X1~X 107 is any amino acid)

[0196] Table 4 shows the amino acid substitutions at the indicated positions in the variant polypeptides, where the amino acid numbering is relative to the amino acid position in SEQ ID NO:1, SEQ ID NO:96, or SEQ ID NO:125.

[0197] The disclosures of all patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety.

[0198] The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.

[0199] Throughout this specification, the objective is to describe preferred embodiments of the invention without limiting the invention to any one embodiment or particular collection of features. Accordingly, those skilled in the art will appreciate that, in light of this disclosure, various modifications and changes can be made in the particular embodiments illustrated without departing from the scope of the present invention. All such modifications and changes are intended to be included within the scope of the appended claims.

[0200] [Table 1A]

[0201] Table 1B

[0202]

Table 1C

[0203]

Table 1D

[0204] Table 1E

[0205] Table 1F

[0206]

Table 1G

[0207] Table 1H

[0208]

Table 1I

[0209]

Table 1J

[0210] Table 1K

[0211]

Table 1L

[0212]

Table 1M

[0213]

Table 1N

[0214]

Table 10

[0215] [Table 1P]

[0216]

Table 1Q

[0217]

Table 1R

[0218]

Table 1S

[0219]

Table 1T

[0220]

Table 1U

[0221]

Table 1V

[0222]

Table 1W

[0223]

Table 1X

[0224]

Table 1Y

[0225]

Table 1Z

[0226]

Table 1AA

[0227]

Table 1AB

[0228]

Table 1AC

[0229]

Table 1AD

[0230]

Table 1AE

[0231]

Table 1AF

[0232] Table 2A

[0233] Table 2B

[0234] Table 3

[0235] Table 4A

[0236] Table 4B

[0237] Table 4C

Claims

1. A polypeptide capable of hydrolyzing amide bonds in polyamides, comprising the amino acid sequence of amino acid residues 2 to 398 of SEQ ID NO: 2, or an amino acid sequence having at least 70% sequence identity thereto.

2. 2. The polypeptide of claim 1, comprising the amino acid sequence of amino acid residues 2 to 398 of SEQ ID NO: 125, or an amino acid sequence having at least 80% sequence identity thereto.

3. 3. The polypeptide of claim 1 or claim 2, comprising the amino acid sequence of amino acid residues 2 to 398 of SEQ ID NO: 96, or an amino acid sequence having at least 80% sequence identity thereto.

4. 4. The polypeptide of any one of claims 1 to 3, comprising the amino acid sequence of amino acid residues 2 to 398 of SEQ ID NO: 1, or an amino acid sequence having at least 80% sequence identity thereto.

5. i. the amino acid at position 3 is Q or T; ii. the amino acid at position 5 is N or D; iii. the amino acid at position 7 is M or F; iv. the amino acid at position 9 is G or T; v. The amino acid at position 10 is S or F; vi. The amino acid at position 14 is P or A; vii. The amino acid at position 16 is Q or G; viii. The amino acid at position 17 is Q or E; ix. the amino acid at position 25 is Q or T; x. The amino acid at position 26 is A or P; xi. The amino acid at position 29 is N or S; xii. The amino acid at position 32 is A or S; xiii. The amino acid at position 39 is I or L; xiv. The amino acid at position 40 is I or V; xv. The amino acid at position 44 is N or Q; xvi. The amino acid at position 48 is G or A; xvii. The amino acid at position 51 is A or P; xviii. The amino acid at position 52 is A or V; xix. The amino acid at position 53 is S or W; xx. The amino acid at position 57 is A or R; xxi. The amino acid at position 58 is A or S; xxii. The amino acid at position 60 is R or A; xxiii. The amino acid at position 61 is D, A, or G; xxiv. The amino acid at position 63 is D or G; xxv. The amino acid at position 64 is G, E, or R; xxvi. The amino acid at position 67 is F or L; xxvii. The amino acid at position 70 is P or deleted; xxviii. The amino acid at position 72 is G or A; xxix. The amino acid at position 73 is R or K; xxx. The amino acid at position 74 is S or E; xxxi. The amino acid at position 75 is T, M, G, or W; xxxii. The amino acid at position 78 is A or G; xxxiii. The amino acid at position 82 is A or E; xxxiv. The amino acid at position 84 is S or T; xxxv. The amino acid at position 88 is A or G; xxxvi. The amino acid at position 92 is L or M; xxxvii. The amino acid at position 99 is A or S; xxxviii. The amino acid at position 101 is W or H; xxxix. The amino acid at position 110 is S or T; xl. The amino acid at position 114 is L or V; xli. The amino acid at position 123 is G or A; xlii. The amino acid at position 124 is I or T; xliii. The amino acid at position 131 is D or E; xliv. The amino acid at position 132 is R or D; xlv. The amino acid at position 138 is D or A; xlvi. The amino acid at position 143 is H, R, or D; xlvii. The amino acid at position 148 is A or V; xlviii. The amino acid at position 149 is A or K; xlix. The amino acid at position 155 is D or G; l. The amino acid at position 167 is S or G; li. The amino acid at position 171 is E or D; lii. The amino acid at position 173 is D or A; liii. The amino acid at position 178 is D or A; liv. The amino acid at position 179 is G or S; lv. The amino acid at position 180 is D or A; lvi. The amino acid at position 186 is R, E, or Q; lvii. The amino acid at position 188 is T or M; lviii. The amino acid at position 193 is A or P; lix. The amino acid at position 194 is S or P; lx. The amino acid at position 195 is D or P; lxi. The amino acid at position 197 is A or E; lxii. The amino acid at position 203 is R or Y; lxiii. The amino acid at position 206 is L or I; lxiv. The amino acid at position 207 is A or V; lxv. The amino acid at position 210 is R or K; lxvi. The amino acid at position 211 is R, K, or deleted; lxvii. The amino acid at position 212 is S, D, or G; lxviii. The amino acid at position 213 is D or A; lxix. The amino acid at position 215 is P or E; lxx. The amino acid at position 223 is A or V; lxxi. The amino acid at position 234 is L or I; lxxii. The amino acid at position 244 is D or E; lxxiii. The amino acid at position 249 is H or R; lxxiv. The amino acid at position 252 is Q or R; lxxv. The amino acid at position 258 is H or A; lxxvi. The amino acid at position 262 is I or V; lxxvii. The amino acid at position 276 is L or I; lxxviii. The amino acid at position 278 is A or C; lxxix. The amino acid at position 280 is L, A, or P; lxxx. The amino acid at position 288 is Q or E; lxxxi. The amino acid at position 291 is L or R; lxxxii. The amino acid at position 301 is I or V; lxxxiii. The amino acid at position 304 is E, A, or G; lxxxiv. The amino acid at position 305 is A or W; lxxxv. The amino acid at position 308 is A or D; lxxxvi. The amino acid at position 311 is L or R; lxxxvii. The amino acid at position 312 is Q or T; lxxxviii. The amino acid at position 313 is N or G; lxxxix. The amino acid at position 316 is R, K, or P; xc. The amino acid at position 321 is R or Q; xci. The amino acid at position 326 is K or H; xcii. The amino acid at position 327 is F or L; xciii. The amino acid at position 328 is F or L; xciv. The amino acid at position 330 is N or G; xcv. The amino acid at position 335 is S or N; xcvi. The amino acid at position 349 is A or G; xcvii. The amino acid at position 359 is I or V; xcviii. The amino acid at position 368 is A or V; xcix. The amino acid at position 370 is L or F; c. the amino acid at position 375 is L or E; ci. The amino acid at position 379 is D or A; cii. The amino acid at position 384 is L or E; ciii. The amino acid at position 385 is C, M, or N; civ. The amino acid at position 390 is D, E, or R; cv. The amino acid at position 394 is R or A; cvi. The amino acid at position 397 is G or A, and / or cvii. the amino acid at position 398 is G or is deleted; 5. The polypeptide of any one of claims 1 to 4, wherein the numbering is relative to the amino acid positions of SEQ ID NO: 1, SEQ ID NO: 96, or SEQ ID NO:

125.

6. i. the amino acid at position 3 is Q or T; ii. the amino acid at position 5 is N or D; iii. the amino acid at position 10 is S or F; iv. the amino acid at position 17 is Q or E; v. The amino acid at position 25 is Q or T; vi. The amino acid at position 26 is A or P; vii. The amino acid at position 29 is N or S; viii. The amino acid at position 32 is A or S; ix. the amino acid at position 39 is I or L; x. The amino acid at position 40 is I or V; xi. The amino acid at position 44 is N or Q; xii. The amino acid at position 51 is A or P; xiii. The amino acid at position 52 is A or V; xiv. The amino acid at position 53 is S or W; xv. The amino acid at position 57 is A or R; xvi. The amino acid at position 58 is A or S; xvii. The amino acid at position 63 is D or G; xviii. The amino acid at position 64 is G, E, or R; xix. The amino acid at position 67 is F or L; xx. The amino acid at position 70 is P or deleted; xxi. The amino acid at position 73 is R or K; xxii. The amino acid at position 74 is S or E; xxiii. The amino acid at position 75 is T, M, G, or W; xxiv. The amino acid at position 78 is A or G; xxv. The amino acid at position 82 is A or E; xxvi. The amino acid at position 84 is S or T; xxvii. The amino acid at position 88 is A or G; xxviii. The amino acid at position 92 is L or M; xxix. The amino acid at position 99 is A or S; xxx. The amino acid at position 101 is W or H; xxxi. The amino acid at position 110 is S or T; xxxii. The amino acid at position 114 is L or V; xxxiii. The amino acid at position 124 is I or T; xxxiv. The amino acid at position 131 is D or E; xxxv. The amino acid at position 143 is H, R, or D; xxxvi. The amino acid at position 148 is A or V; xxxvii. The amino acid at position 149 is A or K; xxxviii. The amino acid at position 167 is S or G; xxxix. The amino acid at position 171 is E or D; xl. The amino acid at position 186 is R, E, or Q; xli. The amino acid at position 188 is T or M; xlii. The amino acid at position 193 is A or P; xliii. The amino acid at position 194 is S or P; xliv. The amino acid at position 197 is A or E; xlv. The amino acid at position 207 is A or V; xlvi. The amino acid at position 210 is R or K; xlvii. The amino acid at position 211 is R, K, or deleted; xlviii. the amino acid at position 212 is S, D, or G; xlix. The amino acid at position 215 is P or E; l. The amino acid at position 223 is A or V; li. The amino acid at position 234 is L or I; lii. the amino acid at position 244 is D or E; liii. The amino acid at position 249 is H or R; liv. The amino acid at position 252 is Q or R; lv. The amino acid at position 258 is H or A; lvi. The amino acid at position 276 is L or I; lvii. The amino acid at position 280 is L or A; lviii. The amino acid at position 288 is Q or E; lix. The amino acid at position 291 is L or R; lx. The amino acid at position 301 is I or V; lxi. The amino acid at position 304 is E, A, or G; lxii. The amino acid at position 305 is A or W; lxiii. The amino acid at position 311 is L or R; lxiv. The amino acid at position 312 is Q or T; lxv. the amino acid at position 313 is N or G; lxvi. The amino acid at position 316 is R, K, or P; lxvii. The amino acid at position 326 is K or H; lxviii. The amino acid at position 327 is F or L; lxix. The amino acid at position 328 is F or L; lxx. The amino acid at position 330 is N or G; lxxi. The amino acid at position 349 is A or G; lxxii. The amino acid at position 359 is I or V; lxxiii. The amino acid at position 368 is A or V; lxxiv. The amino acid at position 370 is L or F; lxxv. The amino acid at position 375 is L or E; lxxvi. The amino acid at position 385 is C, M, or N; lxxvii. The amino acid at position 390 is D, E, or R; lxxviii. the amino acid at position 397 is G or A, and / or lxxix. the amino acid at position 398 is G or is deleted; 6. The polypeptide of any one of claims 1 to 5, wherein the numbering is relative to the amino acid positions of SEQ ID NO: 1 or SEQ ID NO:

96.

7. i. the amino acid at position 3 is Q or T; ii. the amino acid at position 5 is N or D; iii. the amino acid at position 25 is Q or T; iv. the amino acid at position 26 is A or P; v. The amino acid at position 32 is A or S; vi. The amino acid at position 40 is I or V; vii. The amino acid at position 51 is A or P; viii. The amino acid at position 52 is A or V; ix. The amino acid at position 53 is S or W; x. The amino acid at position 57 is A or R; xi. The amino acid at position 58 is A or S; xii. The amino acid at position 63 is D or G; xiii. The amino acid at position 64 is G, E, or R; xiv. The amino acid at position 70 is P or deleted; xv. The amino acid at position 73 is R or K; xvi. The amino acid at position 74 is S or E; xvii. The amino acid at position 75 is T, M, or W; xviii. The amino acid at position 78 is A or G; xix. The amino acid at position 82 is A or E; xx. The amino acid at position 84 is S or T; xxi. The amino acid at position 88 is A or G; xxii. The amino acid at position 92 is L or M; xxiii. The amino acid at position 99 is A or S; xxiv. The amino acid at position 101 is W or H; xxv. The amino acid at position 110 is S or T; xxvi. The amino acid at position 114 is L or V; xxvii. The amino acid at position 124 is I or T; xxviii. The amino acid at position 131 is D or E; xxix. The amino acid at position 143 is H, R, or D; xxx. The amino acid at position 148 is A or V; xxxi. The amino acid at position 149 is A or K; xxxii. The amino acid at position 167 is S or G; xxxiii. The amino acid at position 171 is E or D; xxxiv. The amino acid at position 186 is R, E, or Q; xxxv. The amino acid at position 193 is A or P; xxxvi. The amino acid at position 194 is S or P; xxxvii. The amino acid at position 197 is A or E; xxxviii. The amino acid at position 207 is A or V; xxxix. The amino acid at position 210 is R or K; xl. the amino acid at position 211 is R, K, or deleted; xli. The amino acid at position 212 is S, D, or G; xlii. The amino acid at position 215 is P or E; xliii. The amino acid at position 223 is A or V; xliv. The amino acid at position 234 is L or I; xlv. The amino acid at position 244 is D or E; xlvi. The amino acid at position 276 is L or I; xlvii. The amino acid at position 280 is L or A; xlviii. The amino acid at position 288 is Q or E; xlix. The amino acid at position 291 is L or R; l. The amino acid at position 301 is I or V; li. the amino acid at position 304 is E, A, or G; lii. The amino acid at position 305 is A or W; liii. The amino acid at position 311 is L or R; liv. The amino acid at position 312 is Q or T; lv. The amino acid at position 313 is N or G; lvi. The amino acid at position 316 is R, K, or P; lvii. The amino acid at position 328 is F or L; lviii. The amino acid at position 330 is N or G; lix. The amino acid at position 349 is A or G; lx. The amino acid at position 368 is A or V; lxi. The amino acid at position 370 is L or F; lxii. The amino acid at position 375 is L or E; lxiii. The amino acid at position 385 is C or N; lxiv. the amino acid at position 390 is D, E, or R; and / or lxv. the amino acid at position 397 is G or A; 7. The polypeptide of any one of claims 1 to 6, wherein the numbering is relative to the amino acid positions of SEQ ID NO: 1 or SEQ ID NO:

96.

8. i. the amino acid at position 3 is Q; ii. the amino acid at position 5 is N; iii. the amino acid at position 10 is S; iv. the amino acid at position 17 is E; v. the amino acid at position 25 is Q; vi. the amino acid at position 26 is A; vii. the amino acid at position 29 is S; viii. the amino acid at position 32 is S; ix. the amino acid at position 39 is L; x. the amino acid at position 40 is V; xi. the amino acid at position 44 is Q; xii. the amino acid at position 51 is P; xiii. the amino acid at position 52 is A; xiv. the amino acid at position 53 is S; xv. the amino acid at position 57 is A; xvi. The amino acid at position 58 is A; xvii. the amino acid at position 63 is G; xviii. the amino acid at position 64 is E; xix. The amino acid at position 67 is L; xx. The amino acid at position 70 is P; xxi. The amino acid at position 73 is K; xxii. the amino acid at position 74 is E; xxiii. the amino acid at position 75 is G; xxiv. The amino acid at position 78 is A; xxv. The amino acid at position 82 is E; xxvi. The amino acid at position 84 is S; xxvii. The amino acid at position 88 is G; xxviii. The amino acid at position 92 is L; xxix. the amino acid at position 99 is A; xxx. The amino acid at position 101 is H; xxxi. The amino acid at position 110 is S; xxxii. The amino acid at position 114 is V; xxxiii. the amino acid at position 124 is T; xxxiv. The amino acid at position 131 is E; xxxv. The amino acid at position 143 is D; xxxvi. The amino acid at position 148 is V; xxxvii. The amino acid at position 149 is A; xxxviii. The amino acid at position 167 is S; xxxix. The amino acid at position 171 is E; xl. the amino acid at position 186 is R; xli. the amino acid at position 188 is M; xlii. the amino acid at position 193 is P; xliii. the amino acid at position 194 is S; xliv. the amino acid at position 197 is E; xlv. The amino acid at position 207 is A; xlvi. The amino acid at position 210 is K; xlvii. the amino acid at position 211 is K; xlviii. the amino acid at position 212 is G; xlix. the amino acid at position 215 is P; l. the amino acid at position 223 is V; li. the amino acid at position 234 is L; lii. the amino acid at position 244 is D; liii. the amino acid at position 249 is R; liv. The amino acid at position 252 is R; lv. The amino acid at position 258 is A, lvi. The amino acid at position 276 is I; lvii. the amino acid at position 280 is L; lviii. the amino acid at position 288 is E; lix. The amino acid at position 291 is R; lx. The amino acid at position 301 is I; lxi. the amino acid at position 304 is E; lxii. the amino acid at position 305 is A; lxiii. the amino acid at position 311 is L; lxiv. The amino acid at position 312 is T; lxv. the amino acid at position 313 is N; lxvi. the amino acid at position 316 is R; lxvii. the amino acid at position 326 is H; lxviii. the amino acid at position 327 is L; lxix. The amino acid at position 328 is L; lxx. the amino acid at position 330 is N; lxxi. The amino acid at position 349 is A; lxxii. the amino acid at position 359 is V; lxxiii. the amino acid at position 368 is V; lxxiv. the amino acid at position 370 is L; lxxv. the amino acid at position 375 is L; lxxvi. the amino acid at position 385 is M; lxxvii. the amino acid at position 390 is R; lxxviii. the amino acid at position 397 is G; lxxix. The amino acid at position 398 is deleted; 8. The polypeptide of any one of claims 1 to 7, wherein the numbering is relative to the amino acid positions of SEQ ID NO: 1 or SEQ ID NO:

96.

9. 9. The polypeptide of claim 8, comprising or consisting of the amino acid sequence of SEQ ID NO:

93.

10. 8. The polypeptide of any one of claims 1 to 7, comprising the amino acid sequence of amino acid residues 2 to 394 of any one of SEQ ID NOs: 2, 4-15, 88-92, and 94-95.

11. A polypeptide according to any one of claims 1 to 7, consisting of any one of the amino acid sequences of SEQ ID NOs: 2, 4 to 15, 88 to 92, and 94 to 95.

12. A polypeptide capable of hydrolyzing amide bonds in polyamides, comprising the amino acid sequence of amino acid residues 2 to 394 of SEQ ID NO: 88, or an amino acid sequence having at least 75% sequence identity thereto.

13. 13. The polypeptide of claim 12, having at least 80% sequence identity to SEQ ID NO:

88.

14. 14. A polypeptide according to claim 12 or claim 13, having at least 90% sequence identity to SEQ ID NO:

88.

15. 15. A polypeptide according to any one of claims 12 to 14, having at least 95% sequence identity to SEQ ID NO:

88.

16. 14. The polypeptide of claim 12 or claim 13, comprising amino acid residues 2 to 394 of SEQ ID NO:

123.

17. 17. The polypeptide of claim 16, consisting of the amino acid sequence of SEQ ID NO:

123.

18. 14. The polypeptide of claim 12 or claim 13, comprising amino acid residues 2 to 392 of SEQ ID NO:

63.

19. 19. The polypeptide of claim 18, consisting of the amino acid sequence of SEQ ID NO:

63.

20. 14. The polypeptide of claim 12 or claim 13, comprising amino acid residues 2 to 392 of SEQ ID NO:

3.

21. 21. The polypeptide of claim 20, consisting of the amino acid sequence of SEQ ID NO:

3.

22. 22. The polypeptide of claim 1, which is capable of hydrolyzing nylon polymers.

23. 23. The polypeptide of claim 1, having adipic acid mono- and di-N-alkylamide hydrolase activity.

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

25. 25. The polypeptide of any one of claims 22 to 24, wherein the nylon polymer or nylon oligomer is a nylon 6 polymer, a nylon 6 oligomer, a nylon 6,6 polymer, or a nylon 6,6 oligomer.

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

27. 27. The polypeptide of claim 26, wherein the nylon 6,6 oligomer is a water-soluble nylon 6,6 oligomer.

28. 28. The polypeptide of claim 26 or claim 27, wherein the nylon 6,6 oligomer is selected from the group consisting of nylon 6,6 dimers, trimers, tetramers, pentamers, and hexamers.

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

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

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

32. 32. The polypeptide of any one of claims 27 to 31, wherein the nylon 6,6 oligomers can be hydrolyzed to produce adipic acid and hexamethylenediamine.

33. compared to a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 73-86, i. Increased recombinant expression in host cell lines; ii. Increased enzymatic activity, and / or iii. Increased thermal stability 33. The polypeptide of any one of claims 1 to 32, further comprising:

34. 34. The polypeptide of claim 33, having increased enzymatic activity when compared to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 73-86.

35. 35. A composition comprising a polypeptide according to any one of claims 1 to 34.

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

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

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

39. 1. A method for producing a polypeptide capable of hydrolyzing amide bonds in a polyamide, comprising: i. providing a polynucleotide according to claim 36; ii. producing the polypeptide by expressing the nucleic acid sequence in a host cell culture; iii. collecting the polypeptide produced in (ii) from the host cell culture.

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

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

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

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

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

45. 45. The method of claim 44, wherein the nylon 6,6 oligomer is a water soluble nylon 6,6 oligomer.

46. 46. ​​The method of any one of claims 42 to 45, wherein the nylon 6,6 oligomer is selected from the group consisting of nylon 6,6 dimers, trimers, tetramers, pentamers, and hexamers.

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

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

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

50. 1. A method for hydrolyzing a nylon 6,6 polymer, a nylon 6,6 oligomer, or a nylon 6,6-containing product, the method comprising the step of exposing the nylon 6,6 polymer, the nylon 6,6 oligomer, or the nylon 6,6-containing product to a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 73, 74, and 87, or an amino acid sequence having at least 70% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 73, 74, and 87.

51. 51. The method of claim 50, wherein the polypeptide comprises amino acid residues 2 to 392 of any one of SEQ ID NOs: 73, 74, and 87.

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

53. 53. A composition comprising the adipic acid and / or the hexamethylenediamine recovered by the method of claim 52.

54. 54. A method of producing a nylon polymer using the composition of claim 53.

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