Enzyme variants and uses thereof
Patent Information
- Application Number
- HK62026124242
- Authority / Receiving Office
- HK · HK
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2023-06-16
- Filing Date
- 2026-06-02
- Publication Date
- 2026-07-31
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Abstract
Description
W O 2 0 2 4 / 1 5 6 0 2 5 A 1 (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (43) International Publication Date 02 August 2024 (02.08.2024) WIPO PCT (51) International Patent Classification: C12N 9 / 80 (2006.01) C12Q 1 / 34 (2006.01) C08J 11 / 10 (2006.01) (21) International Application Number: PCT / AU2024 / 050035 (22) International Filing Date: 23 January 2024 (23.01.2024) (25) Filing Language: English (26) Publication Language: English (30) Priority Data: 2023900157 2023901918 23 January 2023 (23.01.2023) 16 June 2023 (16.06.2023) AU AU (71) Applicant: SAMSARA ECO PTY LIMITED [AU / AU]: 702 / 28-34 Clarke Street, Crows Nest, New South Wales 2065 (AU). (72) (74) (81) Inventors: SPENCE, Matthew; C / - Samsara Eco Pty Lim- ited, 702 / 28-34 Clarke Street, Crows Nest, New South Wales 2065 (AU). VONGSOUTHI, Vanessa; C / - Sam- sara Eco Pty Limited, 702 / 28-34 Clarke Street, Crows Nest, New South Wales 2065 (AU). MATTHEWS, Christo- pher Jonathan; C / - Samsara Eco Pty Limited, 702 / 28-34 Clarke Street, Crows Nest, New South Wales 2065 (AU). MATTHEWS, Dana Suzanne; C / -Samsara Eco Pty Limit- ed, 702 / 28-34 Clarke Street, Crows Nest, New South Wales 2065 (AU). TON, Jennifer Nhu Tuyet Nu; C / - Samsara Eco Pty Limited, 702 / 28-34 Clarke Street, Crows Nest, New South Wales 2065 (AU). JACKSON, Colin John; C / -Sam- sara Eco Pty Limited, 702 / 28-34 Clarke Street, Crows Nest. New South Wales 2065 (AU). Agent: DAVIES COLLISON CAVE PTY LTD; 1 Nichol- son Street, Melbourne, Victoria 3000 (AU). Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ. CA, CH. CL. CN. CO. CR. CU. CV. CZ, DE, DJ. DK, DM. DO. DZ, EC, EE, EG, ES, FI, GB. GD, GE, GH, GM. GT. HN, HR, HU, ID, IL, IN, IQ, IR, IS, IT, JM, JO, JP, KE, KG, KH, KN, KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, MG, MK, MN, MU, MW, MX, MY, MZ, NA, NG. NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, OA. RO. RS, RU, RW, SA, SC, SD, SE, SG, SK, SL. ST, SV. SY, TH. (10) International Publication Number WO 2024 / 156025 A1 TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, WS, ZA, ZM, ZW. (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, CV, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SC, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB. GR. HR, HU, IE, IS. IT. LT. LU, LV, MC, MЕ, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ. GW, KM, ML, MR, NE, SN, TD, TG). Published: with international search report (Art. 21(3)) with sequence listing part of description (Rule 5.2(a)) - in black and white; the international application as filed contained color or greyscale and is available for download from PATENTSCOPE (54) Title: ENZYME VARIANTS AND USES THEREOF (57) Abstract: The present disclosure relates generally to polypeptides capable of hydrolysing an amide bond in a polyamide, and more specifically to polypeptides comprising an amino acid sequence of amino acid residues 2-398 of SEQ ID NO: 2 or an amino acid sequence that has at least 70% sequence identity thereto; or polypeptides comprising an amino acid sequence of amino acid residues 2-394 of SEQ ID NO:88 or an amino acid sequence that has at least 75% sequence identity thereto. The present disclosure also extends to methods of using said polypeptides. In another embodiment, the polypeptides disclosed are capable of hydrolysing the nylon 6,6 oligomer to produce adipic acid and hexamethylenediamine. WO 2024 / 156025 1 PCT / AU2024 / 050035 ENZYME VARIANTS AND USES THEREOF
[0001] The present invention relates to novel synthetic enzymes, more particularly to enzymes that catalyse the hydrolysis of nylon polymers, and uses thereof. BACKGROUND
[0002] All references, including any patent or patent application cited in this specification are hereby incorporated by reference to enable full understanding of the invention. Nevertheless, such references are not to be read as constituting an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.
[0003] Global industrialization has had significant environmental impact, not least of which is an increase in the manufacture and reliance on plastic and plastic products. These plastics include a range of synthetic or semi-synthetic polymers, such as polyethylene terephthalate, PVC, polypropylene, polystyrene and polyamides.
[0004] Polyamide (also known generically as nylon) is a silk-like thermoplastic that can be melt-processed into fibres, films or shapes, including in combination with a variety of additives. Polyamides have many significant commercial applications, such as in clothing, the production of resins and molded shapes, in food packaging and industrial fibre and rope.
[0005] Whilst there is a growing effort to find suitable and environmentally sustainable alternatives to plastics, including nylon, including their manufacture and disposal, such products remain a significant problem and contribute to a vast majority of environmental pollutants. The environmental significance of this problem is attributed, at least in part, to the chemical nature of plastics, as they do not readily decompose in nature. Approaches to deal with the problem of plastic waste products have typically included incineration, disposal in landfill and mechanical disintegration. However, these approaches also have significant environmental impact. For instance, incineration of plastics produces potentially harmful byproducts that are released into the atmosphere; the decomposition rate of plastics in landfill is typically very slow and there is a risk that toxic materials will leach into groundwater; and mechanical disintegration is relatively expensive and inefficient and there is often limited use for its byproducts. WO 2024 / 156025 2 PCT / AU2024 / 050035
[0006] Nylon can take many decades to decompose, and while there are ways to recycle nylon, these methods require energy-intensive grinding and re-melting of the polymer. Depending on the nylon and the intended applications, the recycled nylon polymer may need to be mixed with virgin nylon, limiting its flexibility and sustainability.
[0007] More recently, biological (enzymatic) degradation of plastics has been considered as an alternative approach to reducing plastic waste accumulation. In the case of nylon, a strain of bacterium found in the waste water of a nylon factory was discovered to have unique enzymes (NylA, NylB and NylC) that could digest by-products of nylon 6 (a nylon homopolymer of caprolactam) manufacture.
[0008] Whilst enzymatic degradation of plastics such as nylon is an attractive alternative to alleviating the environmental impact of plastic waste products and their disposal, it has not yet seen widespread adoption, including because of its relative inefficiency, slow rate of enzymatic degradation and low levels of enzyme expression in common industrial host organisms. Hence, there remains an urgent need for improved methods and reagents for the enzymatic degradation of plastics, including nylon. SUMMARY OF THE INVENTION
[0009] In one disclosed herein, there is provided a ofaspect polypeptide capable hydrolysing an amide bond in a polyamide, wherein the polypeptide comprises an amino acid sequence of amino acid residues 2-398 of SEQ ID NO: 2 or an amino acid sequence that has at least 70% sequence identity thereto. In one embodiment, the polypeptide comprises an amino acid sequence that has at least 75% sequence identity of amino acid residues 2-398 of SEQ ID NO: 2. In one embodiment, the polypeptide comprises an amino acid sequence that has at least 80% sequence identity of amino acid residues 2-398 of SEQ ID NO: 2. In one embodiment, the polypeptide comprises an amino acid sequence that has at least 85% sequence identity of amino acid residues 2-398 of SEQ ID NO: 2. In one embodiment, the polypeptide comprises an amino acid sequence that has at least 90% sequence identity of amino acid residues 2-398 of SEQ ID NO: 2. In one embodiment, the polypeptide comprises an amino acid sequence that has at least 95% sequence identity of amino acid residues 2-398 of SEQ ID NO: 2. WO 2024 / 156025 3 PCT / AU2024 / 050035
[0010] In an embodiment, the polypeptide comprises an amino acid sequence having amino acid residues 2-398 of SEQ ID NO:125, or an amino acid sequence that has at least 80% sequence identity thereto
[0011] In an embodiment, the polypeptide comprises an amino acid sequence having amino acid residues 2-398 of SEQ ID NO:96, or an amino acid sequence that has at least 80% sequence identity thereto.
[0012] In an embodiment, the polypeptide comprises an amino acid sequence having amino acid residues 2-398 of SEQ ID NO:1, or an amino acid sequence that has at least 80% sequence identity thereto.
[0013] In one embodiment, the polypeptide comprises an amino acid sequence having amino acid residues 2-398 of SEQ ID NO: 125, or an amino acid sequence that has at least 80% sequence identity thereto comprises an amino acid sequence wherein 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 А 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; WO 2024 / 156025 PCT / AU2024 / 050035 4 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 А; 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; 1. 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; WO 2024 / 156025 5 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; Ivii. 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; Ix. the amino acid at position 195 is D or P; lxi. the amino acid at position 197 is A or E; Ixii. 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; Ixvii. the amino acid at position 212 is S, D or G; lxviii. the amino acid at position 213 is D or A; Ixix. 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; Ixxiv. 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; PCT / AU2024 / 050035 WO 2024 / 156025 6 PCT / AU2024 / 050035 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 deleted, wherein the numbering is relative to the amino acid positions of SEQ ID NO:1, SEQ ID NO:96 or SEQ ID NO:125.
[0014] In one embodiment, the polypeptide comprises an amino acid sequence having amino acid residues 2-398 of SEQ ID NO:96, or an amino acid sequence that has at least 80% sequence identity thereto comprises an amino acid sequence wherein 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; WO 2024 / 156025 7 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; PCT / AU2024 / 050035 WO 2024 / 156025 8 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; 1. 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; Ix. the amino acid at position 301 is I or V; lxi. the amino acid at position 304 is E, A or G; Ixii. the amino acid at position 305 is A or W; Ixiii. 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; Ixxii. the amino acid at position 359 is I or V; Ixxiii. the amino acid at position 368 is A or V; Ixxiv. 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; PCT / AU2024 / 050035 WO 2024 / 156025 9 PCT / AU2024 / 050035 lxxviii. the amino acid at position 397 is G or A; and / or Ixxix. the amino acid at position 398 is G or deleted, wherein the numbering is relative to the amino acid positions of SEQ ID NO:1 or SEQ ID NO:96.
[0015] In one embodiment, the polypeptide comprises an amino acid sequence having amino acid residues 2-398 of SEQ ID NO:96; amino acid residues 2-398 of SEQ ID NO:1; or an amino acid sequence that has at least 80% sequence identity thereto, wherein 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; WO 2024 / 156025 10 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; 1. 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; Iviii. 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; PCT / AU2024 / 050035 WO 2024 / 156025 11 PCT / AU2024 / 050035 Ixii. the amino acid at position 375 is L or E; Ixiii. 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 А, wherein the numbering is relative to the amino acid positions of SEQ ID NO:1 or SEQ ID NO:96.
[0016] In one embodiment, the polypeptide comprises or consists of the amino acid sequence having amino acid residues 2-397 of SEQ ID NO:2; the amino acid sequence having amino acid residues 2-398 of SEQ ID NO:15; the amino acid sequence having amino acid residues 2-398 of SEQ ID NO:5; the amino acid sequence having amino acid residues 2-398 of SEQ ID NO:10; the amino acid sequence having amino acid residues 2-397 of SEQ ID NO:8; the amino acid sequence having amino acid residues 2-398 of SEQ ID NO:4; the amino acid sequence having amino acid residues 2-398 of SEQ ID NO:12; the amino acid sequence having amino acid residues 2-397 of SEQ ID NO:91; the amino acid sequence having amino acid residues 2-397 of SEQ ID NO:92; the amino acid sequence having amino acid residues 2-397 of SEQ ID NO:93; the amino acid sequence having amino acid residues 2-397 of SEQ ID NO:94; the amino acid sequence having amino acid residues 2-397 of SEQ ID NO:95; the amino acid sequence having amino acid residues 2-398 of SEQ ID NO:14; the amino acid sequence having amino acid residues 2-398 of SEQ ID NO:89; the amino acid sequence having amino acid residues 2-398 of SEQ ID NO:6; the amino acid sequence having amino acid residues 2-398 of SEQ ID NO:90; the amino acid sequence having amino acid residues 2-398 of SEQ ID NO:9; the amino acid sequence having amino acid residues 2-398 of SEQ ID NO:7; the amino acid sequence having amino acid residues 2-398 of SEQ ID NO:11 or the amino acid sequence having amino acid residues 2-398 of SEQ ID NO:13.
[0017] 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.
[0018] In one aspect disclosed herein, there is provided a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the polypeptide comprises or consists of an amino acid sequence of amino acid residues 2-394 of SEQ ID NO:88 or an amino acid WO 2024 / 156025 PCT / AU2024 / 050035 12 sequence that has more 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.
[0019] In one embodiment, the polypeptide comprises the amino acid sequence having amino acid residues 2-394 of SEQ ID NO:123. In one embodiment, the polypeptide consists of the amino acid sequence of SEQ ID NO:123.
[0020] In another embodiment, the polypeptide comprises or consists of the amino acid sequence having amino acid residues 2-392 of SEQ ID NO:3; the amino acid sequence having amino acid residues 2-394 of SEQ ID NO:63; or the amino acid sequence having amino acid residues 2-398 of SEQ ID NO:48. In one embodiment, the polypeptide comprises the amino acid sequence of amino acid residues 2-394 of the amino acid sequence of SEQ ID NO:63.
[0021] 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.
[0022] In one aspect disclosed herein, there is provided a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the polypeptide comprises or consists of an amino acid sequence of amino acid residues 2-391 of SEQ ID NO:45 or an amino acid sequence that has 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. WO 2024 / 156025 PCT / AU2024 / 050035 13
[0023] In one embodiment, the polypeptide comprises or consists of the amino acid sequence having amino acid residues 2-391 of SEQ ID NO:25 or the amino acid sequence having amino acid residues 2-391 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.
[0024] In one embodiment, the polypeptides disclosed herein are capable of hydrolysing a nylon polymer. In another embodiment, the polypeptides disclosed herein has adipic acid mono- and di-N-alkyl amide hydrolase activity.
[0025] In another embodiment, the polypeptides disclosed herein are capable of hydrolysing 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 the 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 dimer, a trimer, a tetramer, a pentamer and hexamer of nylon 6,6. 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 an aqueous soluble nylon 6,6 oligomer.
[0026] In another embodiment, the polypeptides disclosed herein is capable of hydrolysing the nylon 6,6 oligomer to produce adipic acid and hexamethylenediamine.
[0027] In an embodiment, the polypeptides disclosed herein demonstrate increased recombinant expression in a host cell system; increased enzyme activity; and / or increased thermostability, when compared to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 73-86.
[0028] The present disclosure also extends to a composition comprising the polypeptide as described herein.
[0029] The present disclosure also extends to a nucleic acid sequence encoding the polypeptide described herein.
[0030] The present disclosure also extends to an expression vector comprising the nucleic acid sequence described herein. WO 2024 / 156025 PCT / AU2024 / 050035 14
[0031] The present disclosure also extends to a host cell comprising the nucleic acid sequence or the expression vector described herein.
[0032] In another aspect, the present disclosure provides a method of producing a polypeptide capable of hydrolysing an amide bond in a polyamide, the method comprising: i) providing the polynucleotide described herein; ii) expressing the polynucleotide in a host cell under conditions sufficient to allow the host cell to produce the polypeptide; and iii) collecting the polypeptide produced by the host cell in ii).
[0033] In another aspect, the present disclosure provides a method of hydrolysing nylon polymer or nylon oligomer, the method comprising exposing the nylon polymer or nylon oligomer to the polypeptide, the composition or the host cell disclosed herein under conditions sufficient to convert the nylon-6,6 polymer to adipic acid and / or hexamethylenediamine.
[0034] In another aspect, the present disclosure provides a method of degrading a nylon- containing product, the method comprising exposing the nylon-containing product to the polypeptide, the composition or the host cell as disclosed herein.
[0035] In an embodiment, the methods disclosed herein comprises i) chemical processing of nylon polymer, nylon oligomer or nylon-containing product to generate nylon oligomers, ii) exposing the nylon oligomers generated in step (i) to the polypeptide, the composition or the host cell as disclosed herein, under conditions sufficient to produce adipic acid and / or hexamethylenediamine. In one embodiment, the nylon oligomers are nylon dimers, nylon trimers, nylon tetramers, nylon pentamers and nylon hexamers. In another embodiment, the nylon oligomers are aqueous soluble nylon oligomers.
[0036] In an embodiment, the nylon in the nylon polymer, nylon oligomer or the nylon- containing product is a nylon 6 or a nylon 6,6. In another embodiment, the nylon is a nylon 6,6.
[0037] In another aspect, the present disclosure provides a method of hydrolysing a nylon 6,6 polymer, a nylon 6,6 oligomer or a nylon 6,6-comprising product, the method comprising exposing the nylon 6,6 polymer, nylon 6,6 oligomer or a nylon 6,6 comprising 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 that has at least 70% sequence identity to the amino acid sequence WO 2024 / 156025 PCT / AU2024 / 050035 15 of any one of SEQ ID NOs: 73,74 and 87. In an embodiment, the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 73, 74 and 87.
[0038] In another embodiment, the methods described herein further comprise recovering the adipic acid and / or the hexamethylenediamine produced in step (ii).
[0039] In another aspect, the present disclosure provides a composition comprising the adipic acid and / or the hexamethylenediamine recovered by the methods disclosed herein.
[0040] In another aspect, the present disclosure provides a method of producing a nylon polymer using the composition of adipic acid and / or the hexamethylenediamine recovered by the methods disclosed herein. BRIEF DESCRIPTION OF THE FIGURES
[0041] Figure 1 shows the enzymatic hydrolysis of nylon 6,6 oligomers (A). The red dashed lines in (B) indicate the amide bonds that are hydrolysed by the polypeptides disclosed herein.
[0042] Figure 2 shows the amino acid sequences of nylonase NylB, P07061 and P07062.
[0043] Figure 3 shows whole-cell activity assays demonstrating the activity of variant polypeptides having amino acid sequence of SEQ ID NOs:2-15 and 88 in hydrolysing an amide bond in a polyamide, in comparison to a selection of extant / ancestral NylB polypeptides. A colorimetric assay is used to detect free amines in solution (absorbance at 335 nm). Increased concentration of free amine groups in the reaction solution corresponds to hydrolysis of Nylon-6,6 trimer substrate to hexamethylenediamine and adipic acid.
[0044] Figure 4 shows the thermostability of variants A6 (SEQ ID NO:4) and C2 (SEQ ID NO:2) as determined by Circular Dichroism, in comparison to extant 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.
[0045] Figure 5 shows an alignment of polypeptides of SEQ ID NOs:2, 4-15.
[0046] Figure 6 shows the activity of immobilised enzyme of SEQ ID NO:4 on the nylon 6,6 dimer, trimer and tetramer when incubated with the immobilised enzyme for 1 hour at WO 2024 / 156025 PCT / AU2024 / 050035 16 40 °C. Conversion of the substrate was quantified by LC-MS and compared to a control (no enzyme at time 0 hour).
[0047] Figure 7 shows activity of variant polypeptides generated from further engineering (Phase II). High-throughput (crude / whole-cell activity) screening of candidate nylon 6,6 enzymes (compared to Genl_Top (SEQ ID NO: 4) and no enzyme control) against nylon 6,6 oligomers. The candidates appearing to have the highest whole-cell activity is highlighted (A). Purified candidate enzymes were analysed for activity against nylon 6,6, dimers, trimers and tetramers using LC-MS (B). Y-axis values correspond to % normalised conversion of nylon 6,6 oligomers in comparison to no-enzyme control.
[0048] Figure 8 shows refined LC-MS assay comparing the whole-cell activity of top candidates from Phase 1 (SEQ ID NOs 4, 48, 88, 63 and 4) and Phase II (SEQ ID NOs: 89, 90, 91, 92, 93, 94) in comparison to extant sequences (SEQ ID NOs 75, 76, 79, 80, 81, 82, 83, 84, 86) on nylon 6,6, dimers (A) trimers (B) and tetramers (C). Y-axis values correspond to nylon 6,6 oligomer peak area as measured using LC-MS.
[0049] Figure 9 shows the kinetic stability of top candidates from Gen 2 (SEQ ID NOs: 89, 90, 91, 92, 93, 95) on nylon 6,6, trimer at 40-80°C. Y-axis values correspond to % normalised conversion of nylon 6,6 trimer in comparison to no-enzyme control.
[0050] Figure 10 shows an alignment of polypeptides of SEQ ID NOs:2, 4-15, 89, and 91- 95, with consensus II (SEQ ID NO:96).
[0051] Figure 11 shows the activity of SEQ ID NOs: 100-115 and 121-123 in comparison to extant sequence of SEQ ID NO: 75 on nylon 6,6, trimers. A no-enzyme control is included.
[0052] Figure 12 shows an alignment of SEQ ID NOs: 2, 4-15, 89, 91-95, and 97-122 in generating consensus sequence of consensus IV (SEQ ID NO:125). DETAILED DESCRIPTION OF THE INVENTION
[0053] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred 摘要 本公開總體上涉及能够水解聚醯胺中的醯胺鍵的多肽，更具體地涉及包含 SEQ ID NO：2 的氨 基酸殘基 2-398 的氨基酸序列或與其具有至少 70%序列同一性的氨基酸序列的多肽；或包含 SEQ ID NO：88 的氨基酸殘基 2-394 的氨基酸序列或與其具有至少 75%序列同一性的氨基酸 序列的多肽。本公開還延伸至使用所述多肽的方法。在另一種實施方案中，所公開的多肽能 够水解尼龍 6,6 寡聚物以産生己二酸和六亞甲基二胺。
Claims
THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:
1. A polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the polypeptide comprises an amino acid sequence of amino acid residues 2-398 of SEQ ID NO: 2 or an amino acid sequence that has at least 70% sequence identity thereto.
2. The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence of amino acid residues 2-398 of SEQ ID NO: 125 or an amino acid sequence that has at least 80% sequence identity thereto.
3. The polypeptide of claim 1 or claim 2, wherein the polypeptide comprises an amino acid sequence of amino acid residues 2-398 of SEQ ID NO: 96 or an amino acid sequence that has at least 80% sequence identity thereto.
4. The polypeptide of any one of claims 1-3, wherein the polypeptide comprises an amino acid sequence of amino acid residues 2-398 of SEQ ID NO: 1 or an amino acid sequence that has at least 80% sequence identity thereto.
5. The polypeptide of any one of claims 1-4, wherein 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;1. 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;Iv. the amino acid at position 180 is D or A;Ivi. the amino acid at position 186 is R, E or Q;Ivii. the amino acid at position 188 is T or M;Iviii. 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;Ixi. the amino acid at position 197 is A or E;Ixii. the amino acid at position 203 is R or Y;Ixiii. the amino acid at position 206 is L or I;Ixiv. the amino acid at position 207 is A or V;Ixv. the amino acid at position 210 is R or K;Ixvi. the amino acid at position 211 is R, K or deleted;Ixvii. the amino acid at position 212 is S, D or G;Ixviii. the amino acid at position 213 is D or A;Ixix. the amino acid at position 215 is P or E;Ixx. the amino acid at position 223 is A or V;Ixxi. the amino acid at position 234 is L or I;Ixxii. the amino acid at position 244 is D or E;Ixxiii. the amino acid at position 249 is H or R;Ixxiv. the amino acid at position 252 is Q or R;Ixxv. the amino acid at position 258 is H or A;Ixxvi. the amino acid at position 262 is I or V;Ixxvii. the amino acid at position 276 is L or I;Ixxviii. the amino acid at position 278 is A or C;Ixxix. the amino acid at position 280 is L, A or P;Ixxx. the amino acid at position 288 is Q or E;Ixxxi. the amino acid at position 291 is L or R;Ixxxii. the amino acid at position 301 is I or V;Ixxxiii. the amino acid at position 304 is E, A or G;Ixxxiv. the amino acid at position 305 is A or W;Ixxxv. the amino acid at position 308 is A or D;Ixxxvi. the amino acid at position 311 is L or R;Ixxxvii. the amino acid at position 312 is Q or T;Ixxxviii. the amino acid at position 313 is N or G;Ixxxix. 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 deleted, 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. The polypeptide of any one of claims 1-5, wherein 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;1. 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;Iv. the amino acid at position 258 is H or A;Ivi. the amino acid at position 276 is L or I;Ivii. the amino acid at position 280 is L or A;Iviii. 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;Ixi. the amino acid at position 304 is E, A or G;Ixii. the amino acid at position 305 is A or W;Ixiii. the amino acid at position 311 is L or R;Ixiv. the amino acid at position 312 is Q or T;Ixv. the amino acid at position 313 is N or G;Ixvi. the amino acid at position 316 is R, K or P;Ixvii. the amino acid at position 326 is K or H;Ixviii. the amino acid at position 327 is F or L;Ixix. the amino acid at position 328 is F or L;Ixx. the amino acid at position 330 is N or G;Ixxi. the amino acid at position 349 is A or G;Ixxii. the amino acid at position 359 is I or V;Ixxiii. the amino acid at position 368 is A or V;Ixxiv. the amino acid at position 370 is L or F;Ixxv. the amino acid at position 375 is L or E;Ixxvi. the amino acid at position 385 is C, M or N;Ixxvii. the amino acid at position 390 is D, E or R;Ixxviii. the amino acid at position 397 is G or A; and / orIxxix. the amino acid at position 398 is G or deleted, wherein the numbering is relative to the amino acid positions of SEQ ID NO:1 or SEQ ID NO:96.
7. The polypeptide of any one of claims 1-6; wherein 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; 1. 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; Iv. the amino acid at position 313 is N or G; Ivi. the amino acid at position 316 is R, K or P; Ivii. the amino acid at position 328 is F or L; Iviii. 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;Ixi. the amino acid at position 370 is L or F;Ixii. the amino acid at position 375 is L or E;Ixiii. the amino acid at position 385 is C or N;Ixiv. the amino acid at position 390 is D, E or R; and / orIxv. the amino acid at position 397 is G or A, wherein the numbering is relative to the amino acid positions of SEQ ID NO:1 or SEQ ID NO:96.
8. The polypeptide of any one of claims 1-7, wherein 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;1. 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;Iv. the amino acid at position 258 is A;Ivi. the amino acid at position 276 is I;Ivii. the amino acid at position 280 is L;Iviii. 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;Ixi. the amino acid at position 304 is E;Ixii. the amino acid at position 305 is A;Ixiii. the amino acid at position 311 is L;Ixiv. the amino acid at position 312 is T;Ixv. the amino acid at position 313 is N;Ixvi. the amino acid at position 316 is R;Ixvii. the amino acid at position 326 is H;Ixviii. the amino acid at position 327 is L;Ixix. the amino acid at position 328 is L;Ixx. the amino acid at position 330 is N;Ixxi. the amino acid at position 349 is A;Ixxii. the amino acid at position 359 is V ;Ixxiii. the amino acid at position 368 is V ;Ixxiv. the amino acid at position 370 is L;Ixxv. the amino acid at position 375 is L;Ixxvi. the amino acid at position 385 is M;Ixxvii. the amino acid at position 390 is R;Ixxviii. the amino acid at position 397 is G; andIxxix. the amino acid at position 398 is deleted, wherein the numbering is relative to the amino acid positions of SEQ ID NO:1 or SEQ ID NO:96.
9. The polypeptide of claim 8, comprising or consisting of the amino acid sequence of SEQ ID NO: 93.
10. The polypeptide of any one of claims 1-7, comprising the amino acid sequence of amino acid residues 2-394 of any one of SEQ ID NOs: 2, 4-15, 88-92 and 94-95.
11. The polypeptide of any one of claims 1-7, consisting of the amino acid sequence of any one of SEQ ID NOs: 2, 4-15, 88-92 and 94-95.
12. A polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the polypeptide comprises an amino acid sequence of amino acid residues 2-394 of SEQ ID NO: 88 or an amino acid sequence that has at least 75% sequence identity thereto.
13. The polypeptide of claim 12 having at least 80% sequence identity to SEQ ID NO:88.
14. The polypeptide of claim 12 or claim 13, having at least 90% sequence identity to SEQ ID NO:88.
15. The polypeptide of any one of claims 12-14, having at least 95% sequence identity to SEQ ID NO:88.
16. The polypeptide of claim 12 or claim 13, comprising amino acid residues 2-394 of SEQ ID NO: 123.
17. The polypeptide of 16, consisting of amino acid sequence of SEQ ID NO: 123.
18. The polypeptide of claim 12 or claim 13, comprising amino acid residues 2-392 of SEQ ID NO:63.
19. The polypeptide of 18, consisting of amino acid sequence of SEQ ID NO:63.
20. The polypeptide of claim 12 or claim 13, comprising amino acid residues 2-392 of SEQ ID NO:3.
21. The polypeptide of 20, consisting of amino acid sequence of SEQ ID NO:3.
22. The polypeptide of any one of claims 1-21, wherein the polypeptide is capable of hydrolysing a nylon polymer.
23. The polypeptide of any one of claims 1-22, wherein the polypeptide has adipic acid mono- and di-N-alkyl amide hydrolase activity.
24. The polypeptide of claim 23, wherein the nylon polymer is a nylon oligomer.
25. The polypeptide of any one of claims 22-24, wherein the nylon polymer or nylon oligomer is a nylon 6 polymer, a nylon 6 oligomer, a nylon 6,6 polymer or a nylon 6,6 oligomer.
26. The polypeptide of claim 25, wherein the nylon polymer or the nylon oligomer is a nylon 6,6 polymer or a nylon 6,6 oligomer.
27. The polypeptide of claim 26, wherein the nylon 6,6 oligomer is an aqueous soluble nylon 6,6 oligomer.
28. The polypeptide of claim 26 or claim 27, wherein the nylon 6,6 oligomer is selected from the group consisting of a dimer, a trimer, a tetramer, a pentamer and hexamer of nylon 6,6.
29. The polypeptide of claim 28, wherein the nylon 6,6 oligomer is a nylon 6,6 trimer.
30. The polypeptide of claim 28, wherein the nylon 6,6 oligomer is a nylon 6,6 dimer.
31. The polypeptide of claim 28, wherein the nylon 6,6 oligomer is a nylon 6,6 tetramer.
32. The polypeptide of any one of claims 27-31, wherein the polypeptide is capable of hydrolysing the nylon 6,6 oligomer to produce adipic acid and hexamethylenediamine.
33. The polypeptide of any one of claims 1-32, further comprising: i. increased recombinant expression in a host cell system; ii. increased enzyme activity; and / or iii. increased thermostability, when compared to a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 73-86.
34. The polypeptide of claim 33, having increased enzyme activity when compared to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 73-86.
35. A composition comprising the polypeptide of any one of claims 1-34.
36. A polynucleotide comprising a nucleic acid sequence encoding the polypeptide of any one of claims 1-34.
37. An expression vector comprising the polynucleotide of claim 36.
38. A host cell comprising the polynucleotide of claim 36, or the expression vector of claim 37.
39. A method of producing a polypeptide capable of hydrolysing an amide bond in a polyamide, the method comprising: i. providing the polynucleotide of claim 36; ii. expressing the nucleic acid sequence in a host cell culture, thereby producing the polypeptide; and iii. collecting the polypeptide produced in (ii) from the host cell culture.
40. A method of hydrolysing nylon polymer or nylon oligomer, the method comprising exposing the nylon polymer or nylon oligomer to the polypeptide of any one of claims 1 to 34, the composition of claim 35 or the host cell of claim 38 under conditions sufficient to convert the nylon-6, 6 polymer to adipic acid and / or hexamethylenediamine.
41. A method of degrading a nylon-containing product, the method comprising exposing the nylon-containing product to the polypeptide of any one of claims 1 to 34, the composition of claim 35 or the host cell of claim 38.
42. The method of claim 40 or claim 41, comprising: i. chemical processing of nylon polymer, nylon oligomer or nylon-containing product to generate nylon oligomers; ii. exposing the nylon oligomers generated in step (i) to the polypeptide of any one of claims 1 to 34, the composition of claim 35 or the host cell of claim 38, under conditions sufficient to produce adipic acid and / or hexamethylenediamine.
43. The method of any one of claims 40-42, wherein the nylon in the nylon polymer, nylon oligomer or the nylon-containing product is nylon 6 or nylon 6,6.
44. The method of claim 43, wherein the nylon is nylon 6,6.
45. The method of claim 44, wherein the nylon 6,6 oligomer is an aqueous soluble nylon 6,6 oligomer.
46. The method of any one of claims 42-45, wherein the nylon 6,6 oligomer is selected from the group consisting of a dimer, a trimer, a tetramer, a pentamer and hexamer of nylon 6,6.
47. The method of claim 46, wherein the nylon 6,6 oligomer is a nylon 6,6 trimer.
48. The method of claim 46, wherein the nylon 6,6 oligomer is a nylon 6,6 dimer.
49. The method of claim 46, wherein the nylon 6,6 oligomer is a nylon 6,6 tetramer.
50. A method of hydrolysing a nylon 6,6 polymer, a nylon 6,6 oligomer or a nylon 6,6- comprising product, the method comprising exposing the nylon 6,6 polymer, nylon 6,6 oligomer or nylon 6,6 comprising product to a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 73, 74 and 87, or an amino acid sequence that has at least 70% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 73,74 and 87.
51. The method of claim 50, wherein the polypeptide comprises the amino acid residues 2- 392 of any one of SEQ ID NO:73, 74 and 87.
52. The method of any one of claims 42-49, further comprising recovering the adipic acid and / or the hexamethylenediamine produced in step (ii).
53. A composition comprising the adipic acid and / or the hexamethylenediamine recovered by the method of claim 52.
54. A method of producing a nylon polymer using the composition of claim 53.