Modified nylon hydrolase and use thereof

Abstract

Provided is a chemical recycling method for nylon using an enzyme reaction instead of depolymerization. A mutant, which is a protein that is the following (4), (5), or (6) and in which an amino acid corresponding to at least one selected from among D304, R52, D99, and G111 of the sequence of SEQ ID NO: 1 is mutated, is used. (4) is a protein composed of the amino acid sequence of SEQ ID NO: 1. (5) is a protein composed of a sequence obtained by deleting, substituting, or adding 1-35 amino acids in the amino acid sequence of the protein described in (4), and having 6-aminohexanoic acid oligomer endo-type hydrolase (NylC) activity. (6) is a protein composed of a sequence that shares a sequence identity of at least 90% with the protein described in (4), and having NylC activity.

Description

Modified nylon hydrolase and its use

[0001] The present invention relates to a modified nylon-hydrolyzing enzyme and its use. The present invention also relates to the pretreatment of a composite material containing nylon. The pretreatment of the present invention is useful for chemical recycling of nylon using an enzymatic reaction.

[0002] Nylon, a type of polyamide, is a material with excellent heat resistance, oil resistance, chemical resistance, fatigue resistance, creep resistance, and toughness. While nylon is widely used in clothing and everyday items, 35-40% of total demand is for automobiles and other vehicles, where it is widely used in engine compartment parts, intake system parts, fuel system parts, airbags, and more. Nylon is also used in fishing nets and other fishing gear, for which recycling is strongly encouraged, with a view to reducing marine plastic waste. However, nylon is not very biodegradable, and no universal recycling method has been established. Therefore, currently, most nylon waste is incinerated or disposed of in landfills.

[0003] Three enzymes with different decomposition modes for nylon are known from microorganisms: NylA (6-aminohexanoate-cyclic dimer hydrolase), NylB (6-aminohexanoate-dimer hydrolase), and NylC (6-aminohexanoate oligomer endohydrolase). Among these, NylC, derived from the soil bacterium Arthrobacter sp. KI72 (encoded on the plasmid pOAD2), was modified with four amino acid substitutions (122G, 130Y, 36A, and 263Q) (NylC-GYAQ). This enzyme possesses high activity and converts high molecular weight nylon into oligomers, hence the name nylon hydrolase (Non-Patent Documents 1-4). Recently, it has been reported that heat resistance was improved by amino acid substitution at position 111 (Non-patent Document 5).

[0004] On the other hand, NylC is expressed as an inactive precursor (36 kDa), which is autocatalytically cleaved at Asn266 / Thr267 to separate into a 27 kDa α-chain and a 9 kDa β-chain. X-ray crystallography has revealed that NylC forms a donut-shaped quaternary structure consisting of four heterodimeric monomers (Non-Patent Document 4).

[0005] In addition, limited chemical degradation of nylon-6 with formic acid has been studied to reduce the molecular weight and improve the degradation rate by NylC-GYAQ. However, since NylC-GYAQ cannot degrade dimers, which remain in the system, it has been investigated to degrade nylon by using NylB, which can degrade linear dimers, in combination with nylon degradation, aiming for complete degradation to monomers (Patent Document 1).

[0006] On the other hand, nylon is widely used as a composite material. Nylon fibers are resistant to friction and have excellent strength, but are weak to heat, so they can be blended with polyester fibers, which compensate for this weakness, to create an excellent fabric. Tires are also made stronger by adding organic fiber cords such as nylon. Generally, technology for recycling waste plastics can be used for single-material items. When recycling composite materials, it is necessary to first break them down (separate them) into their individual components, which is time-consuming and costly.

[0007] International Publication WO2023 / 167314 (PCT / JP2023 / 008043) Patent Application No. 2024-191239 (unpublished at the time of filing this application) Patent Application No. 2024-143286 (unpublished at the time of filing this application) Patent Application No. 2025-97087 (unpublished at the time of filing this application)

[0008] Ohki T, Wakitani Y, Takeo M, Yasuhira K, Shibata N, Higuchi Y, et al. Mutational analysis of 6‐aminohexanoate‐dimer hydrolase: Relationship between nylon oligomer hydrolytic and esterolytic activities. FEBS Lett. 2006;580: 5054-5058.Negoro S, Shibata N, Tanaka Y, Yasuhira K, Shibata H, Hashimoto H, et al. Three-dimensional Structure of Nylon Hydrolase and Mechanism of Nylon-6 Hydrolysis. J Biol Chem. 2012;287: 5079 -5090.Negoro S, Kawashima Y, Shibata N, Kobayashi T, Baba T, Lee Y, et al. Mutations affecting the internal equilibrium of the reaction catalyzed by 6‐aminohexanoate‐dimer hydrolase. FEBS Lett. 2016;590: 3133-3143.Negoro S, Shibata N, Kato D, Tanaka Y, Yasuhira K, Nagai K, et al. X‐ray crystallographic and mutational analysis of the NylC precursor: catalytic mechanism of autocleavage and substrate hydrolysis of nylon hydrolase. FEBS J. 2023;290: 3400-3421.Bell EL, Rosetto G, Ingraham MA, Ramirez KJ, Lincoln C, Clarke RW, et al.Natural diversity screening, assay development, and characterization of nylon-6 enzymatic depolymerization. Nat Commun. 2024;15: 1-17.

[0009] One method for processing waste plastics is chemical recycling. In chemical recycling, collected waste plastics are generally first sorted, crushed, washed, and any foreign matter removed, and then depolymerized to chemically break them down into raw materials or intermediate materials. These intermediate materials are then purified and polymerized to produce new plastic products. For nylon-6, it would be desirable to establish a chemical recycling method that uses enzyme reactions instead of depolymerization.

[0010] One aspect of the present invention is to provide a modified NylC with improved decomposition properties that can be applied to chemical recycling methods for nylon-6, and to provide a process for increasing the decomposition rate when decomposing with NylC. Considering future application to actual products, the decomposition rate is preferably 80% or more for a single material consisting of nylon alone, and if other materials (e.g., cellulosic materials such as polyester or cotton) are contained, the decomposition rate is preferably 80% or more relative to the nylon-6 contained.

[0011] On the other hand, to fully activate NylC expressed as a precursor, the precursor must be incubated at 37°C for 24 hours. Some mutant enzymes containing amino acid substitutions do not self-cleave, making it impossible to confirm the effect of the amino acid substitution.

[0012] Another objective of the present invention is to provide a modified NylC that does not require a process for self-cleavage.

[0013] Furthermore, for composite materials containing nylon, if nylon can be selectively oligomerized (Patent Document 3), enzymes such as NylC (Patent Documents 1 and 2) can then be used, which is desirable.

[0014] An object of one aspect of the present invention is to provide a method for selectively decomposing nylon.

[0015] The present invention provides the following: [1] A protein that is (4), (5), or (6) below, wherein at least one amino acid mutation corresponding to D304, R52, D99, and G111 in the sequence of SEQ ID NO: 1 has been made: (4) a protein consisting of the amino acid sequence of SEQ ID NO: 1; (5) a protein consisting of the amino acid sequence of the protein described in (4) with 1 to 36 amino acids deleted, substituted, or added, and having 6-aminohexanoic acid oligomer endohydrolase (NylC) activity; (6) a protein consisting of a sequence that has 90% or more sequence identity with the protein described in (4) and has NylC activity. [2] The protein described in 1, wherein the mutation is at least one mutation selected from D304A, R52Y, D99A, and G111A or G111T. [3] The mutation is at least two mutations selected from D304A, R52Y, D99A, and G111A. The protein according to 1 or 2. [4] The protein according to any one of 1 to 3, wherein the mutations correspond to at least two selected from R52Y and D304A, R52Y and D99A, D99A and D304A, and G111A and D304A. [5] A method for producing a nylon degradation product, comprising the steps of contacting nylon with 10% or more hydrochloric acid or sulfuric acid at 300 to 1200 g / L to obtain a liquid containing degraded nylon; and adding an alkali to the obtained liquid to adjust the pH to 6 to 8 to obtain a neutral liquid containing degraded nylon. [6] The production method according to 5, comprising the step of treating the obtained neutral liquid with at least one of 6-aminohexanoic acid dimer hydrolase (NylB) and NylC.[7] A protein having a structure in which the N-terminus of a polypeptide (a1), (a2), or (a3) ​​below is linked to the C-terminus of a polypeptide (b1), (b2), or (b3) below: (a1) a polypeptide having a sequence of 19 to 260 of SEQ ID NO: 1; (a2) a polypeptide consisting of the sequence of the polypeptide (a1) in which one or more amino acids have been deleted, substituted, or added, and which is capable of functioning as an α-subunit of NylC; (a3) ​​a polypeptide consisting of a sequence having 70% or more sequence identity with the polypeptide (a1) and which is capable of functioning as an α-subunit of NylC; (b1) a polypeptide having a sequence of 267 to 355 of SEQ ID NO: 1; (b2) a polypeptide consisting of the sequence of the polypeptide (b1) in which one or more amino acids have been deleted, substituted, or added, and which is capable of functioning as a β-subunit of NylC; (b3) A polypeptide having a sequence identity of 70% or more with the polypeptide of (b1), and capable of functioning as a β-subunit of NylC. [8] The protein of 7, having a structure in which the N-terminus of a polypeptide of the following (a1'), (a2'), or (a3') is linked to the C-terminus of a polypeptide of the following (b1'), (b2'), or (b3'): (a1') a polypeptide having the sequence of positions 19 to 260 of SEQ ID NO: 1; (a2') a polypeptide having the sequence of the polypeptide of (a1') in which 1 to 24 amino acids have been deleted, substituted, or added, and capable of functioning as an α-subunit of NylC; (a3') a polypeptide having a sequence identity of 90% or more with the polypeptide of (a1'), and capable of functioning as an α-subunit of NylC; (b1') a polypeptide having the sequence of positions 267 to 355 of SEQ ID NO: 1; (b2') A polypeptide having a sequence in which 1 to 9 amino acids are deleted, substituted, or added in the polypeptide described in (b1'), and capable of functioning as a β subunit of NylC; (b3') A polypeptide having a sequence that has 90% or more sequence identity with the polypeptide described in (b1'), and capable of functioning as a β subunit of NylC.[9] The protein according to any one of items 7 or 8, which is (1), (2), or (3) below: (1) a protein having the sequence of amino acids 1 to 333 of SEQ ID NO: 2; (2) a protein consisting of the sequence of the polypeptide according to item (1) in which one or more amino acids have been deleted, substituted, or added, and which has NylC activity; (3) a protein consisting of a sequence that has 70% or more sequence identity with the polypeptide according to item (1) and which has NylC activity.

[10] The protein according to any one of items 7 to 9, which is (1'), (2'), or (3') below: (1') a protein having the sequence of amino acids 1 to 333 of SEQ ID NO: 2; (2') a protein consisting of the sequence of the polypeptide according to item (1') in which 1 to 34 amino acids have been deleted, substituted, or added, and which has NylC activity; (3') a protein consisting of a sequence that has 90% or more sequence identity with the polypeptide according to item (1') and which has NylC activity.

[11] The protein according to any one of claims 7 to 10, wherein the protein has been mutated to at least one mutation selected from T1S, T1C, F35L, D38A, M39D, P98Q, R123Y, D170A, G182A, T203A, and K260A in SEQ ID NO: 2.

[12] The protein according to any one of claims 7 to 11, wherein the protein has been mutated to F35L and P98Q in SEQ ID NO: 2.

[13] The production method according to claim 6, wherein NylC is the protein according to any one of claims 1 to 4 and 7 to 12.

[14] The production method according to claim 13, wherein the nylon-degraded product is from a nylon-containing composite material, wherein the nylon-containing composite material comprises any one selected from polyester, wool, linen, and cotton.

[15] A method for chemically recycling nylon, comprising all of the steps of the production method according to any one of claims 5, 6, 13, and 14.

[16] The protein according to any one of 1 to 4, further comprising a mutation corresponding to at least one mutation selected from P27A, P27Q, P28A, F134W, F301L, L331D, L331E, F134W, and L139R in SEQ ID NO: 1.

[0016] The present invention also provides the following: [1] A protein that is the following (4), (5), or (6): (4) a protein having the amino acid sequence of amino acids 1 to 355 of SEQ ID NO: 1, wherein at least one amino acid selected from M50, R52, D99, G111, L139, D304, and L331 is substituted; (5) a protein having the amino acid sequence of the protein according to (4), wherein one or more amino acids are deleted, substituted, or added, with the proviso that amino acids corresponding to G122, Y130, A36, Q263, F134, L137, Y146, K189, N219, N266, D306, and D308 of SEQ ID NO: 1 and the amino acids corresponding to said at least one substitution are maintained, and the amino acid corresponding to T267 of SEQ ID NO: 1 may be maintained or substituted with S or C, and the protein has NylC activity; (6) A protein having NylC activity, which consists of an amino acid sequence having 70% or more sequence identity with the protein described in (4), provided that the amino acids corresponding to G122, Y130, A36, Q263, F134, L137, Y146, K189, N219, N266, D306, and D308 of SEQ ID NO: 1 and the amino acids corresponding to at least one of the substitutions are maintained, and the amino acid corresponding to T267 is either maintained or may be substituted with S or C. [2] The protein according to 1, which is a protein of the following (4'), (5'), or (6'): (4') a protein having the amino acid sequence of amino acids 1 to 355 of SEQ ID NO: 1, with at least one substitution selected from M50I, R52Y, D99A, G111A, L139R, D304A, L331D, and L331E; (5') a protein consisting of a sequence in which 1 to 36 amino acids have been deleted, substituted, or added in the amino acid sequence of the protein according to (4'), and which has NylC activity; (6') a protein consisting of a sequence having 70% or more sequence identity with the protein according to (4'), and which has NylC activity. [3] The protein according to 1, which has at least two substitutions selected from M50I, R52Y, D99A, G111A, L139R, D304A, L331D, and L331E.[4] The protein according to 1, wherein at least two substitutions selected from R52Y and D304A, R52Y and D99A, D99A and D304A, and G111A and D304A are made. [5] A protein having a structure in which the N-terminus of a polypeptide of the following (a1), (a2), or (a3) ​​is linked to the C-terminus of a polypeptide of the following (b1), (b2), or (b3): ​​(a1) a polypeptide having the sequence of amino acids 19 to 260 of SEQ ID NO: 1; (a2) a polypeptide having the amino acid sequence of the polypeptide of (a1) in which one or more amino acids have been deleted, substituted, or added, with the proviso that the amino acids corresponding to F134, L137, Y146, K189, and N219 of SEQ ID NO: 1 may be maintained, and the amino acid corresponding to T267 of SEQ ID NO: 1 may be maintained or substituted with S or C, and which is capable of functioning as the α-subunit of NylC; (a3) A polypeptide that has an amino acid sequence that has 70% or more sequence identity with the polypeptide described in (a1), with the proviso that amino acids corresponding to F134, L137, Y146, K189, and N219 of SEQ ID NO: 1 may be maintained, and that is capable of functioning as an α-subunit of NylC; (b1) A polypeptide having the sequence of 267 to 355 of SEQ ID NO: 1; (b2) A polypeptide that has the amino acid sequence of the polypeptide described in (b1), with one or more amino acids deleted, substituted, or added, with the proviso that amino acids corresponding to D306 and D308 of SEQ ID NO: 1 may be maintained, and the amino acid corresponding to T267 of SEQ ID NO: 1 may be maintained or substituted with S or C, and that is capable of functioning as a β-subunit of NylC; (b3) A polypeptide having an amino acid sequence that has 70% or more sequence identity with the polypeptide described in (b1), with the proviso that the amino acids corresponding to D306 and D308 in SEQ ID NO: 1 may be maintained, and the amino acid corresponding to T267 in SEQ ID NO: 1 may be maintained or substituted with S or C, and that is capable of functioning as a β subunit of NylC.[6] The protein according to 5, having a structure in which the N-terminus of a polypeptide (a1'), (a2'), or (a3') below is linked to the C-terminus of a polypeptide (b1'), (b2'), or (b3') below: (a1') a polypeptide having a sequence of amino acids 19 to 260 of SEQ ID NO: 1; (a2') a polypeptide consisting of a sequence in which 1 to 24 amino acids have been deleted, substituted, or added in the polypeptide (a1'), and which is capable of functioning as the α-subunit of NylC; (a3') a polypeptide consisting of a sequence having 90% or more sequence identity with the polypeptide (a1'), and which is capable of functioning as the α-subunit of NylC; (b1') a polypeptide having a sequence of amino acids 267 to 355 of SEQ ID NO: 1; (b2') A polypeptide having a sequence in which 1 to 9 amino acids are deleted, substituted, or added in the polypeptide described in (b1'), and capable of functioning as a β subunit of NylC; (b3') A polypeptide having a sequence that has 90% or more sequence identity with the polypeptide described in (b1'), and capable of functioning as a β subunit of NylC. [7] The protein according to 5, which is (1), (2), or (3) below: (1) a protein having the sequence of amino acids 1 to 333 of SEQ ID NO: 2; (2) a protein having NylC activity, which is the polypeptide according to (1) with one or more amino acids deleted, substituted, or added, provided that the amino acids corresponding to D40, D42, F206, L208, Y217, K260, and N290 of SEQ ID NO: 2 may be maintained, and the amino acid corresponding to T1 of SEQ ID NO: 2 may be maintained or substituted with S or C; (3) a protein having NylC activity, which is a sequence having 70% or more sequence identity with the polypeptide according to (1), provided that the amino acids corresponding to D40, D42, F206, L208, Y217, K260, and N290 of SEQ ID NO: 2 may be maintained, and the amino acid corresponding to T1 of SEQ ID NO: 2 may be maintained or substituted with S or C.[8] The protein according to 7, which is the following (1'), (2'), or (3'): (1') a protein having the sequence of amino acids 1 to 333 of SEQ ID NO: 2; (2') a protein consisting of the sequence of the polypeptide according to (1') in which 1 to 34 amino acids have been deleted, substituted, or added, and which has NylC activity; (3') a protein consisting of a sequence having 90% or more sequence identity with the polypeptide according to (1') and which has NylC activity. [9] The protein according to 5, in which the protein has a substitution of at least one amino acid selected from the group consisting of M50I, R52Y, D99A, G111A, L139R, D304A, L331D, and L331E of SEQ ID NO: 1.

[10] The protein according to 5, wherein at least two amino acids selected from those corresponding to M50I, R52Y, D99A, G111A, L139R, D304A, L331D, and L331E in SEQ ID NO: 1 are substituted.

[11] A method for producing a nylon-6 degradation product, comprising the steps of contacting nylon with 10% or more hydrochloric acid or sulfuric acid to obtain a solution containing degraded nylon-6; adding an alkali to the obtained solution to adjust the pH to 6 to 8, and diluting as desired to obtain a neutral solution containing 1 to 50 mg / mL of degraded nylon-6.

[12] The production method according to 11, comprising the step of treating the obtained neutral solution with at least one of 6-aminohexanoic acid dimer hydrolase (NylB) and NylC.

[13] The production method according to 12, wherein NylC is the protein according to any one of 1 to 10.

[14] A method for producing a nylon decomposition product from a composite material containing nylon, wherein the composite material containing nylon contains any one selected from polyester, wool, linen, and cotton.

[15] A method for chemically recycling nylon, comprising all of the steps of the production method according to any one of items 11 to 14.

[0017] The present invention also provides the following: [1] A protein that is the following (4), (5), or (6): (4) a protein having the amino acid sequence of amino acids 1 to 355 of SEQ ID NO: 1, wherein at least one amino acid selected from M50, R52, D99, G111, L139, D304, and L331 is substituted; (5) a protein having the amino acid sequence of the protein according to (4), wherein one or more amino acids are deleted, substituted, or added, with the proviso that amino acids corresponding to G122, Y130, A36, Q263, F134, L137, Y146, K189, N219, N266, D306, and D308 of SEQ ID NO: 1 and the amino acids corresponding to said at least one substitution are maintained, and the amino acid corresponding to T267 of SEQ ID NO: 1 may be maintained or substituted with S or C, and the protein has NylC activity; (6) A protein having NylC activity, which consists of an amino acid sequence having 70% or more sequence identity with the protein described in (4), provided that the amino acids corresponding to G122, Y130, A36, Q263, F134, L137, Y146, K189, N219, N266, D306, and D308 of SEQ ID NO: 1 and the amino acids corresponding to at least one of the substitutions are maintained, and the amino acid corresponding to T267 is either maintained or may be substituted with S or C. [2] The protein according to 1, which is a protein of the following (4'), (5'), or (6'): (4') a protein having the amino acid sequence of amino acids 1 to 355 of SEQ ID NO: 1, with at least one substitution selected from M50I, R52Y, D99A, G111A, L139R, D304A, L331D, and L331E; (5') a protein consisting of a sequence in which 1 to 36 amino acids have been deleted, substituted, or added in the amino acid sequence of the protein according to (4'), and which has NylC activity; (6') a protein consisting of a sequence having 70% or more sequence identity with the protein according to (4'), and which has NylC activity. [3] The protein according to 1, which has at least two substitutions selected from M50I, R52Y, D99A, G111A, L139R, D304A, L331D, and L331E.[4] The protein according to 1, wherein at least two substitutions selected from R52Y and D304A, R52Y and D99A, D99A and D304A, and G111A and D304A are made. [5] A protein having a structure in which the N-terminus of a polypeptide of the following (a1), (a2), or (a3) ​​is linked to the C-terminus of a polypeptide of the following (b1), (b2), or (b3): ​​(a1) a polypeptide having the sequence of amino acids 19 to 260 of SEQ ID NO: 1; (a2) a polypeptide having the amino acid sequence of the polypeptide of (a1) in which one or more amino acids have been deleted, substituted, or added, with the proviso that the amino acids corresponding to F134, L137, Y146, K189, and N219 of SEQ ID NO: 1 may be maintained, and the amino acid corresponding to T267 of SEQ ID NO: 1 may be maintained or substituted with S or C, and which is capable of functioning as the α-subunit of NylC; (a3) A polypeptide that has an amino acid sequence that has 70% or more sequence identity with the polypeptide described in (a1), with the proviso that amino acids corresponding to F134, L137, Y146, K189, and N219 of SEQ ID NO: 1 may be maintained, and that is capable of functioning as an α-subunit of NylC; (b1) A polypeptide having the sequence of 267 to 355 of SEQ ID NO: 1; (b2) A polypeptide that has the amino acid sequence of the polypeptide described in (b1), with one or more amino acids deleted, substituted, or added, with the proviso that amino acids corresponding to D306 and D308 of SEQ ID NO: 1 may be maintained, and the amino acid corresponding to T267 of SEQ ID NO: 1 may be maintained or substituted with S or C, and that is capable of functioning as a β-subunit of NylC; (b3) A polypeptide having an amino acid sequence that has 70% or more sequence identity with the polypeptide described in (b1), with the proviso that the amino acids corresponding to D306 and D308 in SEQ ID NO: 1 may be maintained, and the amino acid corresponding to T267 in SEQ ID NO: 1 may be maintained or substituted with S or C, and that is capable of functioning as a β subunit of NylC.[6] The protein according to 5, having a structure in which the N-terminus of a polypeptide (a1'), (a2'), or (a3') below is linked to the C-terminus of a polypeptide (b1'), (b2'), or (b3') below: (a1') a polypeptide having a sequence of amino acids 19 to 260 of SEQ ID NO: 1; (a2') a polypeptide consisting of a sequence in which 1 to 24 amino acids have been deleted, substituted, or added in the polypeptide (a1'), and which is capable of functioning as the α-subunit of NylC; (a3') a polypeptide consisting of a sequence having 90% or more sequence identity with the polypeptide (a1'), and which is capable of functioning as the α-subunit of NylC; (b1') a polypeptide having a sequence of amino acids 267 to 355 of SEQ ID NO: 1; (b2') A polypeptide having a sequence in which 1 to 9 amino acids are deleted, substituted, or added in the polypeptide described in (b1'), and capable of functioning as a β subunit of NylC; (b3') A polypeptide having a sequence that has 90% or more sequence identity with the polypeptide described in (b1'), and capable of functioning as a β subunit of NylC. [7] The protein according to 5, which is (1), (2), or (3) below: (1) a protein having the sequence of amino acids 1 to 333 of SEQ ID NO: 2; (2) a protein having NylC activity, which is the polypeptide according to (1) with one or more amino acids deleted, substituted, or added, provided that the amino acids corresponding to D40, D42, F206, L208, Y217, K260, and N290 of SEQ ID NO: 2 may be maintained, and the amino acid corresponding to T1 of SEQ ID NO: 2 may be maintained or substituted with S or C; (3) a protein having NylC activity, which is a sequence having 70% or more sequence identity with the polypeptide according to (1), provided that the amino acids corresponding to D40, D42, F206, L208, Y217, K260, and N290 of SEQ ID NO: 2 may be maintained, and the amino acid corresponding to T1 of SEQ ID NO: 2 may be maintained or substituted with S or C.[8] The protein according to 7, which is the following (1'), (2'), or (3'): (1') a protein having the sequence of amino acids 1 to 333 of SEQ ID NO: 2; (2') a protein consisting of the sequence of the polypeptide according to (1') in which 1 to 34 amino acids have been deleted, substituted, or added, and which has NylC activity; (3') a protein consisting of a sequence having 90% or more sequence identity with the polypeptide according to (1') and which has NylC activity. [9] The protein according to 5, in which the protein has a substitution of at least one amino acid selected from the group consisting of M50I, R52Y, D99A, G111A, L139R, D304A, L331D, and L331E of SEQ ID NO: 1.

[10] The protein according to 5, wherein at least two amino acids selected from those corresponding to M50I, R52Y, D99A, G111A, L139R, D304A, L331D, and L331E in SEQ ID NO: 1 are substituted.

[11] A method for producing a nylon-6 degradation product, comprising the steps of: contacting nylon-6 with 10% or more hydrochloric acid or sulfuric acid to obtain a solution containing degraded nylon-6; adding an alkali to the obtained solution to adjust the pH to 6 to 8, and diluting as desired to obtain a neutral solution containing 1 to 50 mg / mL of degraded nylon-6; and allowing at least one of 6-aminohexanoic acid dimer hydrolase (NylB) and NylC to act on the obtained neutral solution.

[12] The production method according to 11, wherein NylC is the protein according to any one of 1 to 10.

[13] A method for chemically recycling nylon-6, comprising all of the steps defined in 11 or 12.

[0018]

[14] A method for producing a degradation product of nylon-66, comprising the steps of: contacting nylon-66 with 10% or more hydrochloric acid or sulfuric acid to obtain a liquid containing degraded nylon-6; adding an alkali to the obtained liquid to adjust the pH to 6 to 8, and diluting if desired to obtain a neutral liquid containing 1 to 50 mg / mL of degraded nylon-66; and allowing at least one of NylB and NylC to act on the obtained neutral liquid.

[15] The production method according to 14, wherein NylC is the protein described in any one of 1 to 10.

[16] A method for chemically recycling nylon-6, comprising all of the steps defined in 14 or 15.

[0019] One aspect of the present invention provides a highly active nylon hydrolase. One aspect of the present invention provides a nylon hydrolase that does not require treatment for self-cleavage. One aspect of the present invention provides a method for decomposing nylon-6, which has a high decomposition rate (monomerization rate) of nylon-6 and is applicable to chemical recycling techniques for nylon-6. One aspect of the present invention provides a method for decomposing nylon from a composite material containing nylon.

[0020] Evaluation of NOM decomposition activity using various mutants. When NOM was treated with various mutants, two spots, ALD and Ahx, appeared on TLC as shown in the figure, and each was quantified. When evaluating ALD decomposition activity, two spots, ALD (substrate) and Ahx (product), appeared. In this case, a strong Ahx spot is promising, while a weaker ALD spot is preferable. Comparison of various properties of the engineered cpGYAQ protein - 1. The cpGYAQ protein exhibited a Tm value 2°C higher than p2-GYAQ. Comparison of various properties of the engineered cpGYAQ protein - 2. The cpGYAQ protein exhibited substrate recognition equivalent to p2-GYAQ. Comparison of various properties of the engineered cpGYAQ protein - 3. The cpGYAQ protein exhibited enzymatic activity equivalent to p2-GYAQ toward Aco (Ahx cyclic oligomer). Comparison of various properties of the engineered cpGYAQ protein - 4. Monomerization rate of nylon-6 after acid treatment and enzymatic reaction. The cpGYAQ protein exhibited enzymatic hydrolysis activity on nylon-6 equivalent to that of p2-GYAQ. DNY (NylB): 6-aminohexanoic acid oligomer exohydrolase, G&B: p2-GYAQ+DNY, cpG&D: cpGYAQ+DNY. a. Appearance of the reaction system after 72 hours of enzymatic reaction. b. Results of TLC analysis. A: Ahx (6-aminohexanoic acid) monomer, B: Ahx dimer, C: Ahx oligomer, 1: nylon only, 2: GYAQ, 3: GYAQ&DNY, 4: DNY. Photographs of the inside of a flask before and after limit hydrolysis of a used fishing net with 18% hydrochloric acid. Before hydrochloric acid treatment (left), and after 2 hours of hydrochloric acid treatment (right). Hydrochloric acid treatment and enzymatic reaction on a fishing net in which the net was completely dissolved by hydrochloric acid treatment. Nylon-6 was efficiently decomposed to produce Ahx by treatment with hydrochloric acid and the use of two enzymes. Examination of the enzyme reaction conditions for nylon-6. TLC showed oligomer spots in the early stages of the enzyme reaction, but the monomer spots became stronger over time. After 3 hours of enzyme reaction, no spots other than the monomer were observed at any enzyme concentration.A: Ahx monomer, B: Ahx dimer, C: Ahx oligomer, 1: nylon only, 2: enzyme concentration 1.0 mg / mL, 3: enzyme concentration 0.5 mg / mL, 4: enzyme concentration 0.1 g / mL. Photographs of the inside of a flask before and after limit hydrolysis of an airbag (coated) with 18% hydrochloric acid. Before hydrochloric acid treatment (left) and after 2 hours of hydrochloric acid treatment (right). The airbag was completely dissolved by hydrochloric acid treatment. a. Appearance of the reaction system after 72 hours of enzyme reaction. b. Results of TLC analysis. A: 66MU, B: 1.5er, C: HMD, 1: nylon only, 2: GYAQ, 3: GYAQ & DNY, 4: DNY. Photographs of the inside of a flask before and after limit hydrochloric acid hydrolysis of an airbag (uncoated) with 18% hydrochloric acid. Before hydrochloric acid treatment (left) and after 2 hours of hydrochloric acid treatment (right). The airbag was completely dissolved by hydrochloric acid treatment. a. Appearance of the reaction system after 72 hours of enzyme reaction. b. Results of TLC analysis. A: 66MU, B: 1.5er, C: HMD, 1: nylon only, 2: GYAQ, 3: GYAQ & DNY, 4: DNY. Summary of verification results using products. Acid treatment and the Nyl series reaction process were confirmed to show a high monomerization rate for airbags. They were also confirmed to be effective for actual products. The amino acid sequence of NylC-GYAQ (SEQ ID NO: 1) and the amino acid sequence of cp-NylC obtained in the examples of this specification (SEQ ID NO: 2). In the sequence of SEQ ID NO: 1, squares represent M50, R52, D99, G111, L139, D304, and L331, respectively; underlines represent A36, G122, Y130, and Q263, respectively; and shading represents F134, L137, Y146, K189, N219, N266, D306, and D308, respectively. Italics represent T267. Wavy underlines represent the β-strand portion. In the sequence of SEQ ID NO: 2, squares represent D38, L65, M121, R123, D170, G182, and L210, respectively; underlines represent A107, G193, and Y201, respectively; and shading represents D40, D42, F206, L208, Y217, K260, and N290, respectively. Italics indicate T1. Wavy underlines indicate the β-strand portion.The sequence of SEQ ID NO:5 differs from the NylB-DNY (NylB-NylB' hybrid enzyme with G181D / H266N / D370Y substitutions) described in the paper in that the first amino acid, methionine M, is omitted. Photographs of the reaction system after 24 hours of enzymatic reaction: 1: PA on the front, polyester on the back; 2: 100% PA; 3: 40% PA, 60% polyester; 4: 53% PA, 47% ester. Photographs of polyester-nylon-containing fabric before and after acid treatment. TLC analysis before and after enzymatic reaction: A: Ahx monomer; B: Ahx dimer; C: Ahx oligomer. 1: PA on the front, polyester on the back; 2: 100% nylon; 3: 40% nylon, 60% polyester; 4: 53% nylon, 47% ester. Photographs of the reaction system after 24 hours of enzymatic reaction. 1: 90% wool, 10% nylon; 2: 84% cotton, 16% nylon; 3: 68% cotton, 32% nylon; 4: 70% cotton, 30% nylon. Natural material - TLC analysis of nylon-containing fabric before and after acid treatment. A: Ahx monomer; B: Ahx dimer; C: Ahx oligomer. 1: 90% wool, 10% nylon; 2: 84% cotton, 16% nylon; 3: 68% cotton, 32% nylon; 4: 70% cotton, 30% nylon. D: 66MU; E: 66-N1.5mer; F: HMD monomerization rate before and after enzymatic reaction. Photo of reaction system after reaction. Photo of tire sample before and after acid treatment. TLC analysis before and after enzymatic reaction. A: 6,6-MU, B: 6,6-N 1.5mer, C: HMD, 1: Sample 1, 2: Sample 2 Monomerization rate before and after enzyme reaction Photos of tire sample before and after acid treatment LC-MS analysis of the solution portion after the reaction Degradation activity by a combination of self-cleaving GYAQ mutant and DNY enzyme Degradation activity by a combination of self-cleaving GYAQ mutant and DNY enzyme Degradation activity by a combination of self-cleaving GYAQ mutant and DNY enzyme.

[0021] The following abbreviations may be used in the present specification, claims, and drawings: 66MU: Nylon-66 monomer unit (see formula below) HMD: Hexamethylenediamine (1,6-diaminohexane) Ahx: 6-aminohexanoic acid (also called ε-aminocaproic acid) ALD: 6-aminohexanoate linear dimer Aco: 6-aminohexanoic acid cyclic oligomer (Ahx cyclic oligomer) NOM: Nylon oligomer mixture NylB: Exo-type nylon hydrolase. Also called 6-aminohexanoate dimer hydrolase. NylC: Endo-type nylon hydrolase. Also called 6-aminohexanoate oligomer endo-hydrolase. p2-NylC: NylC from Arthrobacter sp. KI72. Unless otherwise specified, this is the NylC that is referred to when simply referring to NylC. Hyb-24: A mutant in which several amino acid substitutions have been made in the hybrid enzyme between NylB and NylB'. Sometimes simply referred to as DNY. NylC-GYAQ: A quadruple mutant in which 122G, 130Y, 36A, and 263Q substitutions have been made in p2-NylC. Sometimes simply referred to as GYAQ. PET: Polyethylene terephthalate (sometimes referred to as polyethylene terephthalate)

[0022] In the present specification, claims, and drawings, unless otherwise specified, amino acids are represented by a single letter of the alphabet, specifically, A is alanine, L is leucine, R is arginine, K is lysine, N is asparagine, M is methionine, D is aspartic acid, F is phenylalanine, C is cysteine, P is proline, Q is glutamine, S is serine, E is glutamic acid, T is threonine, G is glycine, W is tryptophan, H is histidine, Y is tyrosine, I isoleucine, and V is valine.

[0023] Furthermore, when an amino acid substitution in a protein or enzyme is represented by a mutant using a character string consisting of a single letter and a number followed by another letter, the leftmost letter indicates the amino acid before the mutation, the central number indicates the position of the amino acid, and the rightmost letter indicates the amino acid after the mutation, meaning that the leftmost amino acid has been substituted with the rightmost amino acid. For example, M50I indicates that methionine at position 50 in the amino acid sequence has been substituted with isoleucine. Note that, in the present invention, the term "amino acid" is sometimes used to mean "amino acid residue," but this is clear to those skilled in the art.

[0024] In proteins or enzymes, specific amino acids in an amino acid sequence may be represented by a single letter and a number. For example, M50 refers to the 50th amino acid in the amino acid sequence, M. Similarly, 50I refers to the 50th amino acid in the amino acid sequence being substituted with I, regardless of the original amino acid.

[0025] I. Mutant Enzyme (Embodiment 1) This embodiment relates to a mutant enzyme further having a mutation in NylC-GYAQ (SEQ ID NO: 1), more specifically, a mutant enzyme in which at least one amino acid selected from amino acids 50, 52, 99, 111, 139, 304, and 331 in a protein having an amino acid sequence of amino acids 1 to 355 of SEQ ID NO: 1 has been substituted, for example, a mutant enzyme in which at least one amino acid selected from D304, R52, D99, and G111 of SEQ ID NO: 1 has been mutated.

[0026] Three enzymes with different decomposition patterns are known to be derived from microorganisms: NylA (6-aminohexanoate-cyclic dimer hydrolase, EC 3.5.2.12), NylB (EC 3.5.1.46), and NylC (EC 3.5.1.117).

[0027] NylC catalyzes the following reaction:

[0028]

[0029] This enzyme is involved in the degradation of nylon-6 oligomers. It degrades linear or cyclic poly(6-aminohexanoyl) oligomers with a degree of polymerization of 3 or more by endo-cleavage, usually to oligomers with lengths of 2 or more (2 ≤ x < n).

[0030] The mutant enzyme of this embodiment has higher activity or superior thermostability compared to the original NylC-GYAQ (SEQ ID NO: 1). In one embodiment, such a mutant enzyme is any one of the proteins (4), (5), and (6) below: (4) A protein having the amino acid sequence of amino acids 1 to 355 of SEQ ID NO: 1, in which at least one amino acid selected from M50, R52, D99, G111, L139, D304, and L331 is substituted; (5) A protein having the amino acid sequence of the protein described in (4), in which one or more amino acids are deleted, substituted, or added, and which satisfies (i) and (ii), and which has NylC activity; (i) Amino acids corresponding to G122, Y130, A36, Q263, F134, L137, Y146, K189, N219, N266, D306, and D308 of SEQ ID NO: 1, and the amino acids corresponding to said at least one substitution, are maintained. (ii) the amino acid corresponding to T267 in SEQ ID NO: 1 may be maintained or substituted with S or C. (6) a protein having NylC activity, which has an amino acid sequence that has 70% or more sequence identity with the protein described in (4), provided that (iii) and (iV) are satisfied; (iii) the amino acids corresponding to G122, Y130, A36, Q263, F134, L137, Y146, K189, N219, N266, D306, and D308 in SEQ ID NO: 1, and the amino acids corresponding to at least one of the substitutions, are maintained. (iv) the amino acid corresponding to T267 may be maintained or substituted with S or C.

[0031] Another embodiment of the mutant enzyme having higher activity or superior thermostability compared to the original NylC-GYAQ (SEQ ID NO: 1) is a mutant enzyme of any one of the following proteins (4), (5), and (6), in which at least one amino acid selected from D304, R52, D99, and G111 in SEQ ID NO: 1 has been mutated: (4) a protein consisting of the amino acid sequence of SEQ ID NO: 1; (5) a protein consisting of the amino acid sequence of the protein described in (4) in which one or several, preferably 1 to 36, amino acids have been deleted, substituted, or added, and which has 6-aminohexanoic acid oligomer endohydrolase (NylC) activity; (6) a protein consisting of a sequence having 70% or more, preferably 90% or more, sequence identity to the protein described in (4) and which has NylC activity. In addition, when referring to NylC-GYAQ (SEQ ID NO: 1), the term "mutant enzyme" refers to an enzyme in which at least one amino acid selected from D304, R52, D99, and G111 in the sequence of SEQ ID NO: 1 has been mutated, unless otherwise specified.

[0032] In SEQ ID NO: 1, the following amino acids, F134, L137, Y146, K189, N219, N266, D306, and D308, are important for self-cleavage and substrate recognition. It is also important that the nucleophilic residue of this enzyme, amino acid 267, is T (although activity is slightly reduced, S or C may also be used) (Non-Patent Document 4). In SEQ ID NO: 1, G122, Y130, A36, and Q263 are the amino acids that are important for NylC derived from Arthrobacter sp. KI72. p2 This corresponds to four substitutions from the nucleotide sequence: D122G, H130Y, D36A, and E263Q.

[0033] Thus, in the mutant, the amino acids corresponding to G122, Y130, A36, Q263, F134, L137, Y146, K189, N219, N266, D306, and D308 of SEQ ID NO: 1 may be maintained. Preferably, the amino acids corresponding to G122, Y130, A36, Q263, L137, Y146, K189, N219, N266, D306, and D308 of SEQ ID NO: 1 are maintained. In addition, in the mutant, the amino acid corresponding to T267 of SEQ ID NO: 1 may be maintained or substituted with S or C.

[0034] With respect to amino acids and mutations, the term "corresponding" refers to a position corresponding to the nth position in the original sequence, but where one or more amino acids are deleted, substituted, or added to the original sequence, resulting in a shift in position such that it is no longer the nth position, in the resulting sequence. A "corresponding amino acid" refers to, for example, position 122 in the reference sequence of SEQ ID NO: 1, but where one or more amino acids are deleted, substituted, or added to SEQ ID NO: 1, the position may be shifted so that it is no longer the 122nd position, but refers to the amino acid at the position corresponding to position 122 in SEQ ID NO: 1 in the resulting sequence. Similarly, a "corresponding mutation" refers to, for example, an R52Y mutation in the reference sequence of SEQ ID NO: 1, but where one or more amino acids are deleted, substituted, or added to SEQ ID NO: 1, the position may be shifted so that it is no longer the 52nd position, but refers to a mutation from R to Y at the position corresponding to position 52 in SEQ ID NO: 1 in the resulting sequence.

[0035] The "at least one substitution" refers to all of "at least one mutation selected from M50I, R52Y, D99A, G111A, L139R, D304A, L331D, and L331E" present in the reference sequence (4) of SEQ ID NO: 1. For example, if the protein (4) has only the M50I mutation among M50I, R52Y, D99A, G111A, L139R, D304A, L331D, and L331E, then "the at least one mutation" in (5) refers to the amino acid corresponding to position 50 being mutated to I, and if the protein (4) has the M50I and R52Y mutations, then "the at least one mutation" in (5) refers to the amino acid corresponding to position 50 being I and the amino acid corresponding to position 52 being Y. Similarly, when referring to "the at least two mutations," etc., it refers to all mutations such as "at least two mutations selected from M50I, R52Y, D99A, G111A, L139R, D304A, L331D, and L331E" that the reference sequence (4) of SEQ ID NO: 1 has. The same applies to other items such as (6).

[0036] Those skilled in the art can appropriately identify corresponding amino acids, corresponding positions, and corresponding amino acid substitutions by, for example, aligning the two sequences in an optimal manner.

[0037] With respect to M50, R52, D99, G111, L139, D304, and L331, the amino acid after substitution is not particularly limited as long as the desired activity is maintained.

[0038] Specifically, M50, i.e., the amino acid corresponding to methionine (M) at position 50 in the sequence of SEQ ID NO: 1, may be substituted with isoleucine (I) or an amino acid with properties similar to those of isoleucine, or may be substituted with an amino acid with properties different from those of isoleucine.

[0039] In one embodiment, the amino acid corresponding to methionine at position 50 in SEQ ID NO: 1 is substituted with isoleucine or an amino acid with properties similar to isoleucine. Examples of amino acids with properties similar to isoleucine include the following: hydrophobic amino acids: alanine, valine, glycine, leucine, phenylalanine, proline, tryptophan, and tyrosine; neutral amino acids: alanine, asparagine, cysteine, glutamine, glycine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; and branched-chain amino acids: valine and leucine.

[0040] R52, i.e., the amino acid corresponding to arginine (R) at position 52 in SEQ ID NO: 1, may be substituted with tyrosine (Y) or an amino acid with properties similar to tyrosine, or with an amino acid with properties different from tyrosine.

[0041] In one embodiment, the amino acid corresponding to arginine at position 52 in SEQ ID NO: 1 is substituted with tyrosine or an amino acid with properties similar to tyrosine. Examples of amino acids with properties similar to tyrosine include: hydrophobic amino acids such as alanine, valine, glycine, isoleucine, leucine, phenylalanine, proline, and tryptophan; neutral amino acids such as alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, and valine; and aromatic ring-containing amino acids such as tryptophan and phenylalanine.

[0042] D99, i.e., the amino acid corresponding to aspartic acid (D) at position 99 in the sequence of SEQ ID NO: 1, may be substituted with alanine (A) or an amino acid with properties similar to alanine, or with an amino acid with properties different from alanine.

[0043] In one embodiment, the amino acid corresponding to aspartic acid at position 99 in SEQ ID NO: 1 is substituted with alanine or an amino acid with properties similar to alanine. Examples of amino acids with properties similar to alanine include the hydrophobic amino acids valine, glycine, isoleucine, leucine, phenylalanine, proline, tryptophan, and tyrosine; and the neutral amino acids asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0044] In one embodiment, G111, i.e., the amino acid corresponding to glycine (G) at position 111 in SEQ ID NO: 1, may be substituted with alanine or an amino acid having properties similar to alanine, or may be substituted with an amino acid having properties different from alanine. In another embodiment, G111 may be substituted with threonine.

[0045] In one embodiment, the amino acid corresponding to glycine at position 111 in SEQ ID NO: 1 is substituted with alanine or an amino acid with properties similar to alanine. Examples of amino acids with properties similar to alanine include the hydrophobic amino acids valine, isoleucine, leucine, phenylalanine, proline, tryptophan, and tyrosine; and the neutral amino acids asparagine, cysteine, glutamine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0046] L139, i.e., the amino acid corresponding to leucine (L) at position 139 in the sequence of SEQ ID NO: 1, may be substituted with arginine (R) or an amino acid with properties similar to arginine, or may be substituted with an amino acid with properties different from arginine.

[0047] In one embodiment, the amino acid corresponding to leucine at position 139 in SEQ ID NO: 1 is substituted with arginine or an amino acid with properties similar to arginine. Examples of amino acids with properties similar to arginine include the following: non-hydrophobic amino acids, such as asparagine, aspartic acid, glutamic acid, glutamine, lysine, serine, threonine, cysteine, and histidine; hydrophilic amino acids, such as asparagine, aspartic acid, glutamic acid, glutamine, lysine, serine, and threonine; and basic amino acids, such as lysine and histidine.

[0048] D304, i.e., the amino acid corresponding to aspartic acid (D) at position 304 in the sequence of SEQ ID NO: 1, may be substituted with alanine (A) or an amino acid with properties similar to alanine, or with an amino acid with properties different from alanine.

[0049] In one embodiment, the amino acid corresponding to aspartic acid at position 304 in SEQ ID NO: 1 is substituted with alanine or an amino acid with properties similar to alanine. Examples of amino acids with properties similar to alanine include the hydrophobic amino acids valine, glycine, isoleucine, leucine, phenylalanine, proline, tryptophan, and tyrosine; and the neutral amino acids asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0050] L331, i.e., the amino acid corresponding to leucine (L) at position 331 in the sequence of SEQ ID NO: 1, may be substituted with aspartic acid (D) or an amino acid with properties similar to aspartic acid, or with an amino acid with properties different from aspartic acid.

[0051] In one embodiment, the amino acid corresponding to leucine at position 331 in SEQ ID NO: 1 is substituted with aspartic acid or an amino acid with properties similar to aspartic acid. Examples of amino acids with properties similar to aspartic acid include: non-hydrophobic amino acids such as arginine, asparagine, glutamic acid, glutamine, lysine, serine, threonine, cysteine, and histidine; hydrophilic amino acids such as arginine, asparagine, glutamic acid, glutamine, lysine, serine, and threonine; and acidic amino acid such as glutamic acid.

[0052] Furthermore, the amino acid corresponding to leucine (L) at position 331 in the sequence of SEQ ID NO: 1 may be substituted with glutamic acid (E) or an amino acid with properties similar to glutamic acid, or with an amino acid with properties different from glutamic acid.

[0053] In one embodiment, the amino acid corresponding to leucine at position 331 in SEQ ID NO: 1 is substituted with glutamic acid or an amino acid with properties similar to glutamic acid. Examples of amino acids with properties similar to glutamic acid include: non-hydrophobic amino acids such as arginine, aspartic acid, asparagine, glutamine, lysine, serine, threonine, cysteine, and histidine; hydrophilic amino acids such as arginine, aspartic acid, asparagine, glutamine, lysine, serine, and threonine; and acidic amino acid such as aspartic acid.

[0054] In one embodiment, the mutant enzyme is a protein of (4'), (5'), or (6') below: (4') A protein having the amino acid sequence of amino acids 1 to 355 of SEQ ID NO: 1, in which at least one amino acid selected from M50I, R52Y, D99A, G111A, L139R, D304A, L331D, and L331E is substituted; (5') A protein consisting of a sequence in which 1 to 36 amino acids have been deleted, substituted, or added in the amino acid sequence of the protein described in (4'), and having NylC activity; (6') A protein consisting of a sequence having 70% or more sequence identity with the protein described in (4'), and having NylC activity.

[0055] In another embodiment, the mutant enzyme is one in which the mutation corresponds to at least one selected from D304A, R52Y, D99A, and G111A or G111T.

[0056] In one aspect, the mutation of the mutant enzyme further having a mutation in NylC-GYAQ is a protein having at least two mutations selected from M50I, R52Y, D99A, G111A, L139R, D304A, L331D, and L331E, or a protein having high sequence identity with such a protein. That is, it is any of the following: (4") A protein consisting of the amino acid sequence of amino acids 1 to 355 of SEQ ID NO: 1, and having at least two mutations selected from M50I, R52Y, D99A, G111A, L139R, D304A, L331D, and L331E; (5") A protein having the amino acid sequence of the protein described in (4"), in which one or more amino acids have been deleted, substituted, or added, provided that the amino acids corresponding to 122G, 130Y, 36A, and 263Q of SEQ ID NO: 1 and the amino acids corresponding to the at least two mutations are maintained, and having NylC activity; (6") A protein having a sequence identity of 90% or more to the protein described in (4"), provided that the amino acids corresponding to 122G, 130Y, 36A, and 263Q of SEQ ID NO: 1 and the amino acids corresponding to the at least two mutations are maintained, and having NylC activity.

[0057] In another embodiment, the mutant enzyme is one in which the mutations correspond to at least two selected from D304A, R52Y, D99A, and G111A.

[0058] In one aspect, the mutation of the mutant enzyme further having a mutation in NylC-GYAQ is a protein having at least two mutations selected from R52Y and D304A, R52Y and D99A, D99A and D304A, and G111A and D304A. That is, it is any of the following: (4'") A protein consisting of the amino acid sequence of amino acids 1 to 355 of SEQ ID NO: 1, and having at least two mutations selected from R52Y and D304A, R52Y and D99A, D99A and D304A, and G111A and D304A; (5'") A protein having the amino acid sequence of the protein set forth in (4'"), in which one or more amino acids have been deleted, substituted, or added, provided that the amino acids corresponding to 122G, 130Y, 36A, and 263Q of SEQ ID NO: 1 and the amino acids corresponding to the at least two mutations are maintained, and the protein has NylC activity; (6'") A protein having a sequence identity of 90% or more to the protein set forth in (4'"), provided that the amino acids corresponding to 122G, 130Y, 36A, and 263Q of SEQ ID NO: 1 and the amino acids corresponding to the at least two mutations are maintained, and the protein has NylC activity.

[0059] In another aspect, the mutant enzyme has mutations corresponding to at least two mutations selected from R52Y and D304A, R52Y and D99A, D99A and D304A, and G111A and D304A.

[0060] In another aspect, the following may be considered: Preferably, the mutation is not a mutation corresponding to G111W, F, E, K, N, P, Q, I, S, or M. Also, preferably, the mutation is not a mutation corresponding to F134A and Y. Furthermore, preferably, the mutation is not a mutation corresponding to Y146, G188, W192, P300, M305A, D, or E. Furthermore, preferably, the mutation is not a mutation corresponding to the combination of G90A and D304A, or the combination of V92A and D304A.

[0061] For the self-cleaving GYAQ, mutations that increase the decomposition activity for each substrate are summarized below. The amino acid positions are the corresponding positions in SEQ ID NO: 1. (1) Nylon-6 substrates Mutations that enhance Nom degradation activity: D99A_G111T, G111A_D304A Mutations that enhance ALD degradation activity: G111D, F134W, P27Q_D304A, R52Y_D99A, R52Y_D304A, D99A_D304A, G111D_D304A Mutations that enhance 6-Nylon degradation activity: Increased Ahx production capacity: R52Y, D99A, G111A, F134W, R52Y_D99A, R52Y_D304A, D99A_D304A Increased ALD production capacity: P27A, P27Q, P28A, F301L

[0062] (2) Mutations that enhance the degradation activity of nylon-66MU, a nylon-66 substrate: D99A, G111A, G111T, G111L, G111H, G111C, L139R, D304A, L331D, L331E, R52Y_D99A, D99A_D304A, G111A_D304A, G111A_L331D, G111A_L331E Mutations that enhance the degradation activity of nylon-66: G111A, G111T, F134W, D304A, R52Y_D99A, R52Y_G111T, G90A_G111T, V92A_G111T, D99A_G111T, D99A_D304A, G111T_D304A, D99A_G111A_D304A

[0063] (3) Mutations that increase thermostability (above 90°C): G111Y, D304A, R52Y_D304A, D99A_G111T, G111A_D304A, G111T_D304A, R52Y_G111A_D304A, D99A_G111A_D304A

[0064] Furthermore, from the viewpoint of improving enzyme activity, preferred mutations include P27A, P27Q, P28A, R52Y, D99A, G111D, G111R, G111A, G111T, G111L, G111H, G111C, F134W, L139R, F301L, D304A, L331D, L331E, P27Q_D304A, R52Y_D99A, R52Y_G111T, R52Y_D304A, G90A_G111T, V92A_G111T, D99A_G111T, D99A_D304A, G111A_D304A, G111A_L331D, G111A_L331E, Examples include G111D_D304A, G111T_D304A, R52Y_G111A_D304A, and D99A_G111A_D304A.

[0065] One of the most preferred embodiments of the mutant enzyme is a protein that is (iv), (v), or (vi) below, which has mutations corresponding to G111A and D304 in SEQ ID NO: 1: (iv) a protein consisting of the amino acid sequence of SEQ ID NO: 1; (v) a protein consisting of a sequence in which one or several, preferably 1 to 36, amino acids have been deleted, substituted, or added in the amino acid sequence of the protein described in (iv), and which has NylC activity; (vi) a protein consisting of a sequence that has 70% or more, preferably 90% or more, sequence identity with the protein described in (iv), and which has NylC activity.

[0066] This embodiment provides an enzyme with improved decomposition ability for nylon-6, nylon-66, and copolymers thereof, approximately 5 to 10 times that of the original GYAQ enzyme (SEQ ID NO: 1).

[0067] This embodiment also provides a polynucleotide encoding a mutant enzyme further mutated in NylC-GYAQ. That is, a polynucleotide encoding any one of the proteins (4), (5), (6), (4'), (5'), (6'), (4"), (5"), (6"), (4'"), (5'"), and (6'") above is provided. Such a polynucleotide can be incorporated into an appropriate vector to express the desired enzyme in E. coli or the like.

[0068] In the present invention, unless otherwise specified, the term "NylC activity" refers to the activity of endo-degrading cyclic or linear Ahx oligomers having a degree of polymerization of 3 or more.

[0069] In the present invention, when referring to amino acid sequence identity, unless otherwise specified, it refers to sequence identity of at least 50%, for example, 60% or more, 70% or more, preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, even more preferably 91% or more, even more preferably 92% or more, even more preferably 93% or more, even more preferably 94% or more, even more preferably 95% or more, even more preferably 96% or more, even more preferably 97% or more, even more preferably 97.5% or more, even more preferably 98% or more, even more preferably 98.5% or more, even more preferably 99% or more, even more preferably 99.1% or more, even more preferably 99.4% or more, and even more preferably 99.7% or more.

[0070] In the present invention, "identity" of amino acid sequences is defined as a value measured by BLAST of NCBI (http: / / www.ncbi.nlm.nih.gov / ), unless otherwise specified. When comparing amino acid sequences with BLAST, Blastp can be used with the default settings as the algorithm. The measurement results are quantified as positives or identities.

[0071] In the description of the present invention, when a protein or amino acid sequence is referred to as a "sequence in which one or more amino acids are deleted, substituted, or added," the number of amino acids to be substituted, etc. is not particularly limited, unless otherwise specified, for any protein, as long as the protein consisting of that amino acid sequence has the desired function, but may be approximately 1-250, 1-200, 1-150, 1-100, 1-50, 1-40, 1-38, 1-37, 1-35, 1-30, 1-20, 1-15, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1, or may be even greater, as long as the substitutions are with amino acids with similar properties. Means for preparing proteins with such amino acid sequences are well known to those skilled in the art.

[0072] Amino acids with similar properties refer to amino acids with similar physical properties such as hydropathy, charge, pKa, and solubility, and include, for example, the following: Hydrophobic amino acids: alanine, valine, glycine, isoleucine, leucine, phenylalanine, proline, tryptophan, tyrosine; non-hydrophobic amino acids: arginine, asparagine, aspartic acid, glutamic acid, glutamine, lysine, serine, threonine, cysteine, histidine; hydrophilic amino acids: arginine, asparagine, aspartic acid, glutamic acid, glutamine, lysine, serine, threonine; acidic amino acids: aspartic acid, glutamic acid; basic amino acids: lysine, arginine, histidine; neutral amino acids: alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine; sulfur-containing amino acids: methionine, cysteine; aromatic ring-containing amino acids: tyrosine, tryptophan, phenylalanine; Branched chain amino acids: valine, leucine, isoleucine.

[0073] II. Circular permutation (Embodiment 2) This embodiment relates to NylC modified by circular permutation (circular permutation). NylC is originally expressed as an inactive precursor (36 kDa).

[0074]

[0075] The precursor is autocatalytically cleaved at Asn266 / Thr267 to separate into a 27 kDa α-chain and a 9 kDa β-chain.

[0076]

[0077] Furthermore, four heterodimer structures consisting of an α chain and a β chain assemble to form a doughnut-shaped three-dimensional structure, which expresses enzymatic activity.

[0078]

[0079] Conventional NylC requires an incubation process at 37°C for 24 hours after enzyme purification to allow complete self-cleavage. In addition, some amino acid substitutions do not self-cleave, making it impossible to confirm the effect of the amino acid substitution.

[0080] In this embodiment, NylC is cleaved by cleavage, linking the N-terminus of the α-chain polypeptide to the C-terminus of the β-chain polypeptide. Such a cleaved enzyme does not require further processing to promote autocleavage and, unexpectedly, can have a higher Tm value than the parent enzyme.

[0081] Specifically, this embodiment provides a protein having a structure in which the α chain, i.e., the N-terminus of a polypeptide that is (a1), (a2), or (a3) ​​below, is linked to the C-terminus of the β chain, i.e., the C-terminus of a polypeptide that is (b1), (b2), or (b3) below: (a1) a polypeptide having the sequence of positions 19 to 260 of SEQ ID NO: 1; (a2) a polypeptide having the amino acid sequence of the polypeptide described in (a1), in which one or more amino acids are deleted, substituted, or added, with the proviso that the amino acids corresponding to F134, L137, Y146, K189, and N219 of SEQ ID NO: 1 may be maintained, and the amino acid corresponding to T267 of SEQ ID NO: 1 may be maintained or substituted with S or C, and which is capable of functioning as the α-subunit of NylC; (a3) ​​a sequence having 70% or more sequence identity with the polypeptide described in (a1), with the proviso that the amino acids corresponding to F134, L137, Y146, K189, and N219 of SEQ ID NO: 1 may be maintained, and which is capable of functioning as the α-subunit of NylC; (b1) a polypeptide having the sequence of positions 267 to 355 of SEQ ID NO: 1; (b2) A polypeptide that is capable of functioning as a β subunit of NylC, which is a sequence of the polypeptide described in (b1) in which one or more amino acids have been deleted, substituted, or added, with the proviso that the amino acids corresponding to D306 and D308 in SEQ ID NO: 1 may be maintained, and the amino acid corresponding to T267 in SEQ ID NO: 1 may be maintained or substituted with S or C; (b3) A polypeptide that is capable of functioning as a β subunit of NylC, which is a sequence that has 70% or more sequence identity with the polypeptide described in (b1), with the proviso that the amino acids corresponding to D306 and D308 in SEQ ID NO: 1 may be maintained, and the amino acid corresponding to T267 in SEQ ID NO: 1 may be maintained or substituted with S or C.

[0082] When the N-terminus of one polypeptide is said to be "linked" to the C-terminus of another polypeptide, this includes cases where the N-terminus is directly linked to the C-terminus, and cases where the C-terminus is linked via a spacer of 1 to 10 amino acids.

[0083] Being able to function as an α subunit or a β subunit means that it can exhibit the desired enzymatic activity when combined with the other subunit.

[0084] In one embodiment, the cp-modified enzyme is the protein according to claim 5, having a structure in which the N-terminus of a polypeptide (a1'), (a2'), or (a3') below is linked to the C-terminus of a polypeptide (b1'), (b2'), or (b3') below: (a1') a polypeptide having a sequence of 19 to 260 of SEQ ID NO: 1; (a2') a polypeptide consisting of a sequence in which 1 to 24 amino acids have been deleted, substituted, or added in the polypeptide (a1'), and which is capable of functioning as the α-subunit of NylC; (a3') a polypeptide consisting of a sequence having 90% or more sequence identity with the polypeptide (a1'), and which is capable of functioning as the α-subunit of NylC; (b1') a polypeptide having a sequence of 267 to 355 of SEQ ID NO: 1; (b2') A polypeptide having a sequence in which 1 to 9 amino acids are deleted, substituted, or added in the polypeptide described in (b1'), and capable of functioning as a β subunit of NylC; (b3') A polypeptide having a sequence that has 90% or more sequence identity with the polypeptide described in (b1'), and capable of functioning as a β subunit of NylC.

[0085] In one embodiment, the cpd enzyme is a protein of (1), (2), or (3) below: (1) a protein having the sequence of amino acids 1 to 333 of SEQ ID NO: 2; (2) a protein having NylC activity, which is composed of the polypeptide of (1) with one or more amino acids deleted, substituted, or added, provided that the amino acids corresponding to D40, D42, F206, L208, Y217, K260, and N290 of SEQ ID NO: 2 may be maintained, and the amino acid corresponding to T1 of SEQ ID NO: 2 may be maintained or substituted with S or C; (3) a protein having NylC activity, which is composed of a sequence having 70% or more sequence identity with the polypeptide of (1), provided that the amino acids corresponding to D40, D42, F206, L208, Y217, K260, and N290 of SEQ ID NO: 2 may be maintained, and the amino acid corresponding to T1 of SEQ ID NO: 2 may be maintained or substituted with S or C.

[0086] In one embodiment, the cpd enzyme is a protein of the following (1'), (2'), or (3'): (1') a protein having a sequence of 1 to 333 of SEQ ID NO: 2; (2') a protein having NylC activity, which consists of a sequence in which 1 to 34 amino acids are deleted, substituted, or added in the polypeptide described in (1'); (3') a protein having NylC activity, which consists of a sequence having 90% or more sequence identity with the polypeptide described in (1').

[0087] The sequence listing shows the amino acid sequence of the cp-modified NylC obtained in the Examples herein as SEQ ID NO: 2. In SEQ ID NO: 2, D40, D42, F206, L208, Y217, K260, and N290 correspond to amino acids important for self-cleavage and substrate recognition in NylC-GYAQ (SEQ ID NO: 1). T1 (which may be replaced by S or C, although the activity may be slightly reduced) corresponds to amino acid 267, the nucleophilic residue in NylC-GYAQ (SEQ ID NO: 1). A107, G193, and Y201 in SEQ ID NO: 2 correspond to A36, G122, and Y130 in NylC-GYAQ (SEQ ID NO: 1). D38, L65, M121, R123, D170, G182, and L210 in SEQ ID NO: 2 correspond, in that order, to D304, L331, M50, R52, D99, G111, and L139 in NylC-GYAQ (SEQ ID NO: 1). The portion 1 to 89 in SEQ ID NO: 2 is the β-strand.

[0088] In one aspect, such cp'ed NylC may further comprise at least one mutation selected from the mutations described in the first embodiment above, i.e., amino acids corresponding to M50, R52, D99, G111, L139, D304, L331, and L331 of SEQ ID NO: 1. Such cp'ed NylC may also comprise at least two mutations selected from amino acids corresponding to M50I, R52Y, D99A, G111A, L139R, D304A, L331D, and L331E of SEQ ID NO: 1.

[0089] In another aspect, the cp-modified NylC may be a cp-modified mutant enzyme having a mutation corresponding to at least one mutation selected from T1S, T1C, F35L, D38A, M39D, P98Q, R123Y, D170A, G182A, T203A, and K260A in SEQ ID NO: 2, with or without the mutations described in the previous paragraph. Unless otherwise specified, a reference to a cp-modified mutant enzyme refers to a cp-modified enzyme having a mutation corresponding to at least one mutation selected from T1S, T1C, F35L, D38A, M39D, P98Q, R123Y, D170A, G182A, T203A, and K260A in SEQ ID NO: 2. The cp-modified mutant enzyme may also have mutations corresponding to F35L and P98Q in SEQ ID NO: 2.

[0090] Regarding the cp enzyme, the mutations that increase the decomposition activity for each substrate are summarized below. The amino acid positions are the corresponding positions in SEQ ID NO: 2, and the positions in parentheses are the corresponding positions in the sequence of SEQ ID NO: 1. (1) Nylon-6 substrates Mutations that increase the decomposition activity of Nom: T203(132)A Mutations that increase the decomposition activity of ALD: T203(132)A Mutations that increase the decomposition activity of nylon-6: D170(99)A, F35(301)L, T1(267)S, T1(267)C

[0091] (2) Mutations that enhance the degradation activity of nylon-66MU, a nylon-66 substrate: D38(304)A, D170(99)A, G182(111)A, M39(305)D, T1(267)C. Mutations that enhance the degradation activity of nylon-66: D38(304)A, R123(52)Y, D170(99)A, G182(111)A, P98(27)Q, F35(301)L, F35L_P98Q.

[0092] (3) Mutation K260(189)A that increases thermostability Although no overall improvement in thermostability was observed, this mutation resulted in a Tm value of nearly 90 degrees.

[0093] In addition, preferred mutations from the viewpoint of improving enzyme activity include D38(304)A, R123(52)Y, D170(99)A, G182(111)A, M39(305)D, P98(27)Q, F35(301)L, T203(132)A, T1(267)S, T1(267)C, and F35L_P98Q.

[0094] This embodiment provides an enzyme with improved degrading ability for nylon-6, nylon-66, and their copolymers compared to the original cp enzyme (SEQ ID NO: 2).

[0095] This embodiment also provides a polynucleotide encoding cp-modified NylC. That is, a polynucleotide encoding a protein having a structure in which the N-terminus of the α-chain and the C-terminus of the β-chain are linked together. Such a polynucleotide can be incorporated into an appropriate vector to express the desired enzyme in E. coli or the like.

[0096] III. Use of Hydrochloric Acid or Sulfuric Acid (Embodiment 3) This embodiment relates to the treatment of nylon (also called polyamide) using hydrochloric acid or sulfuric acid. Unless otherwise specified, nylon in this embodiment may be in the form of a composite material in which nylon is combined with other materials.

[0097] More specifically, the present embodiment provides a method for producing a nylon decomposition product, which includes the following steps:

[0098] A step of adding nylon to 10% or more hydrochloric acid or sulfuric acid and mixing to obtain a low molecular weight nylon solution; a step of adding an alkali to the obtained low molecular weight nylon solution to adjust the pH, and optionally diluting with a solvent to obtain a neutral nylon solution of 1 to 50 mg / mL.

[0099] The treatment method of this embodiment is suitable for pretreatment of nylon decomposition methods using NylC. Note that although the following explanation will be given using nylon-6 as an example of nylon, the explanation also applies to other nylons, such as nylon-66.

[0100] More specifically, the present embodiment provides a method for producing a degradation product of nylon-6, comprising the following steps:

[0101] A method for producing a nylon degradation product includes the steps of: contacting nylon with 10% or more hydrochloric acid or sulfuric acid at a concentration of 100 to 1500 g / L, preferably 300 to 1200 g / L, to obtain a solution containing degraded nylon; and adding an alkali to the obtained solution to adjust the pH to 6 to 8 to obtain a neutral solution containing degraded nylon. This production method may also include the steps of diluting the solution obtained by acid treatment to a concentration suitable for enzyme treatment, as described below: adding and mixing nylon-6 with 10% or more hydrochloric acid or sulfuric acid at a concentration of 100 to 1500 g / L, preferably 300 to 1200 g / L, to obtain a degraded nylon solution; adding an alkali to the obtained degraded nylon solution to adjust the pH, and optionally diluting with a solvent to obtain a neutral nylon solution of 1 to 50 mg / mL; and treating the obtained nylon solution with at least one of NylB and NylC.

[0102] (Depolymerization) In the step of adding and mixing nylon-6 with hydrochloric acid or sulfuric acid at a concentration sufficient to decompose nylon to the intended level to obtain a depolymerized nylon solution, nylon-6 is contacted with hydrochloric acid or sulfuric acid at a concentration sufficient to dissolve nylon-6, thereby depolymerizing nylon-6. The nylon-6 used in this step may be mechanically pulverized.

[0103] When an acid is used, the acid acts as a catalyst to hydrolyze nylon and reduce its molecular weight.

[0104] The degree of degradation of the molecular weight of nylon-6 can be appropriately determined, but can be, for example, such that the monomerization rate (sometimes referred to as decomposition rate) represented by the following formula is 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or approximately 100%.

[0105]

[0106] If the monomerization rate is 50% or more, nylon-6 is water-soluble, and the subsequent enzymatic reaction can achieve monomerization in nearly 100% yield.

[0107] When hydrochloric acid is used, it can be used at a concentration that can decompose nylon to the intended extent, specifically, 6% or more, 9% or more, 10% or more, 15% or more, 17% or more, for example, 18%. Regardless of other conditions, the concentration of hydrochloric acid can be 50% or less, or may be 40% or less.

[0108] When sulfuric acid is used, it can be used at a concentration that can decompose nylon to the intended extent, specifically, 15% or more, 25% or more, 35% or more, 40% or more, 45% or more, for example, 50%. Regardless of other conditions, the sulfuric acid concentration can be 60% or less, or may be 55% or less.

[0109] This step may be carried out under heat or pressure, but is not particularly limited as long as the nylon-6 is depolymerized to a predetermined degree. The temperature is, for example, 50 to 160°C, preferably 60 to 140°C, more preferably 80 to 130°C, and even more preferably 100 to 125°C.

[0110] The concentration of nylon-6 in the acid solution is not particularly limited as long as nylon-6 is depolymerized to a predetermined degree. For example, 1 part by weight of nylon-6 can be used in an acid solution of 0.1 to 100 parts by weight, 0.2 to 50 parts by weight, 0.3 to 30 parts by weight, 0.5 to 20 parts by weight, or 1 to 10 parts by weight.

[0111] The time required for this step may be any time sufficient to fully decompose the nylon, and may be 0.5 hours or more, 1 hour or more, 2 hours or more, 12 hours or more, 24 hours or more, or 72 hours or more, depending on whether heating is used or not. Furthermore, since a prolonged acid treatment time is expected to cause side reactions such as deamination and polymerization to proceed, regardless of other conditions, the time required for this step is preferably 100 hours or less, more preferably 50 hours or less, and may also be 48 hours or less, 32 hours or less, or 24 hours or less.

[0112] In one embodiment, this step involves adding and mixing 1 part by weight of nylon-6 with 1 to 10 parts by weight of 10 to 40% hydrochloric acid or 20 to 55% sulfuric acid, and reacting the mixture at 80 to 130°C for 1 to 32 hours to obtain a low molecular weight nylon solution.

[0113] Particularly preferred embodiments include the following. Sufficient oligomerization can be achieved under these conditions: Nylon-6, regardless of other treatment conditions, feed concentration: 100 to 1000 g / L; Acid: 9 to 27% hydrochloric acid, regardless of other treatment conditions, temperature: 100 to 150°C, regardless of other treatment conditions, treatment time: 1.5 to 32 hours; Nylon-66, regardless of other treatment conditions, feed concentration: 50 to 750 g / L; Acid: 9 to 27% hydrochloric acid, regardless of other treatment conditions, temperature: 110 to 150°C, regardless of other treatment conditions, treatment time: 1.5 to 32 hours

[0114] One preferred embodiment of the treatment conditions for nylon-6, nylon-66, and copolymers thereof is as follows: Charge concentration: 300 to 1200 g / L, regardless of other treatment conditions Acid: 9 to 27% hydrochloric acid, preferably 14 to 22% hydrochloric acid, more preferably 15 to 21% hydrochloric acid, and even more preferably 16 to 20% hydrochloric acid, regardless of other treatment conditions Temperature: 110 to 150°C, preferably 113 to 140°C, more preferably 115 to 130°C, and even more preferably 117 to 125°C, regardless of other treatment conditions Time: 1.5 to 32 hours, preferably 1.6 to 16 hours, more preferably 1.7 to 8 hours, and even more preferably 1.8 to 4 hours, regardless of other treatment conditions

[0115] One of the most preferred embodiments is as follows. Under these conditions, almost complete water-soluble oligomerization can be achieved. Nylon-6: Charge concentration: 1000 g / L, or twice the amount of nylon charged. Acid: 18% hydrochloric acid. Temperature: 120°C. Time: 2 hours. Nylon-66: Charge concentration: 400 g / L, or twice the amount of nylon charged. Acid: 18% hydrochloric acid. Temperature: 120°C. Time: 2 hours.

[0116] (Neutralization) The solution containing the depolymerized nylon-6 is adjusted to a pH level by adding an alkali so that the enzyme used in the subsequent enzymatic reaction step can fully exhibit its activity.

[0117] The alkali can be used in the form of a solution, for example, an aqueous solution of a hydroxide of a Group 1 or Group 2 element, such as sodium hydroxide or potassium hydroxide. In one embodiment, the alkaline aqueous solution has a concentration of 5 to 15 N, or may be 7 to 13 N, or may be 8 to 12 N. The pH of the alkaline aqueous solution can be 11.0 to 13.5.

[0118] In one embodiment the pH is adjusted to between 6 and 8, preferably between 6.8 and 7.8, more preferably between 7.1 and 7.5, for example 7.3.

[0119] The solution containing the depolymerized nylon-6 is adjusted to a concentration that will fully activate the enzyme used in the subsequent enzymatic reaction step, preferably using a phosphate buffer containing glycerol, more specifically Buffer A (10% glycerol containing 20 mM phosphate buffer, pH 7.3).

[0120] In one embodiment, the solution containing the depolymerized nylon-6 is adjusted to, for example, 0.5 to 100 mg / mL, 1 to 50 mg / mL, preferably 3 to 25 mg / mL, more preferably 5 to 15 mg / mL, for example, 10 mg / mL.

[0121] (Processing of Composite Materials) This embodiment is also suitable as a method for producing nylon decomposition products from composite materials containing nylon.

[0122] In one embodiment, the nylon-containing composite material is in the form of a fabric containing nylon and any one selected from various chemical fibers or natural materials, such as polyester, wool, linen, and cotton. When used in clothing products, fabrics are sometimes called textiles.

[0123] In one embodiment, the composite material containing nylon is in the form of a tire. Generally, a tire structure is broadly divided into tread, shoulder, sidewall, and bead sections, and is composed of components such as a rubber layer, belt, carcass, and bead wire, and multiple materials are used. The materials used in tires can be divided into rubber, compounding agents, and structural materials. Examples of rubber include natural rubber, styrene-butadiene rubber, butadiene rubber, isoprene rubber, and butyl rubber. Examples of compounding agents include carbon black, silica, oil, resin, antioxidant, zinc oxide, stearic acid, vulcanization accelerator, and sulfur. Examples of structural materials include nylon, polyester (e.g., PET), polyethylene naphthalate (PEN), aramid, rayon, and steel. Nylon, polyester (e.g., PET), and the like are typically used in tire carcasses (the cord layer that forms the tire's framework).

[0124] (Enzyme Reaction) When the pretreatment is followed by an enzymatic reaction, an enzyme having nylon-6 decomposition activity is added to a solution of a predetermined pH containing degraded nylon-6 in the enzymatic reaction step to hydrolyze nylon. The nylon hydrolyzate may be a dimer or monomer of Ahx.

[0125] In this embodiment, at least one of NylB and NylC is used, and preferably both NylB and NylC are used.

[0126] The temperature in this step can be appropriately set depending on the enzyme used, for example, 20 to 50°C, 25 to 47°C, or 30 to 45°C.

[0127] The concentration of the enzyme is not particularly limited as long as the target monomerization rate can be achieved. When multiple enzymes are used, the concentration of each enzyme can be, for example, 0.001 to 10 mg / mL, or alternatively, 0.005 to 7 mg / mL, 0.01 to 5 mg / mL, 0.03 to 3 mg / mL, or 0.05 to 2 mg / mL.

[0128] The time for this step is not particularly limited as long as the target monomerization rate can be achieved, but it can be, for example, 0.5 to 72 hours, 0.6 to 24 hours, 0.7 to 12 hours, 0.8 to 8 hours, or 1 to 4 hours.

[0129] NylB is generally known to be involved in the degradation of nylon-6 oligomers. It degrades linear 6-aminohexanoic acid oligomers with a degree of polymerization of 2 to 20 by exo-cleavage, sequentially removing residues from the N-terminus. Regarding nylon-66, NylB degrades linear nylon-66 oligomers and 1,6-diaminohexyl-adipyl by exo-cleavage, potentially producing HMD and other products.

[0130] In one embodiment, the enzyme used as NylB may be NylB-DNY (SEQ ID NO: 5) or a homolog thereof. Such mutant enzymes are any one of the following proteins (10), (11), and (12): (10) a protein consisting of the amino acid sequence of SEQ ID NO: 5; (11) a protein having one or more amino acids deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 5, with the amino acids corresponding to G180, H265, and D369 of SEQ ID NO: 5 being maintained, and having NylB activity; (12) a protein having a sequence identity of 70% or more with the amino acid sequence of SEQ ID NO: 5, with the amino acids corresponding to G180, H265, and D369 of SEQ ID NO: 5 being maintained, and having NylB activity.

[0131] As mentioned above, NylC is generally known to be involved in the degradation of nylon-6 oligomers, and degrades linear or cyclic poly(6-aminohexanoyl) oligomers with a degree of polymerization of 3 or greater by endo-cleavage, usually to oligomers with a length of 2 or greater. In the case of nylon-66, NylC degrades linear or cyclic poly(1,6-diaminohexyl-adipyl) oligomers with a degree of polymerization of 2 or greater by endo-cleavage, and can produce 1,6-diaminohexyl-adipyl and its oligomers.

[0132] In one embodiment, the enzyme used as NylC may be NylC-GYAQ (SEQ ID NO: 1) or a homolog thereof. Such mutant enzymes are any one of the following proteins (7), (8), and (9): (7) a protein consisting of the amino acid sequence of SEQ ID NO: 1; (8) a protein having one or more amino acid deletions, substitutions, or additions in the amino acid sequence of SEQ ID NO: 1, with the amino acids corresponding to G122, Y130, A36, and Q263 of SEQ ID NO: 1 maintained, and having NylC activity; (9) a protein having a sequence identity of 70% or more with the amino acid sequence of SEQ ID NO: 1, with the amino acids corresponding to G122, Y130, A36, and Q263 of SEQ ID NO: 1 maintained, and having NylC activity.

[0133] In one aspect, NylC is a protein according to any one of embodiments 1 and 2.

[0134] The present embodiment also provides a method for chemically recycling nylon, which includes the steps described above.

[0135] The method of this embodiment is expected to be applicable to, but not limited to, nylon-6, nylon-11, nylon-12, nylon-6T, nylon-6I, nylon-M5T, nylon-6i, and photoswitchable biodegradable nylon (iNylon) containing a carboxylic acid unit (MAAli et al., Macromolecules, 2013, 46, 3719).

[0136] The method of this embodiment is also expected to be applicable to nylon 66 and various other nylons composed of diamines and dicarboxylic acids, including, but not limited to, nylon 610, nylon 6T, nylon 6I, nylon 9T, and nylon M5T.

[0137] IV. Other Embodiments This embodiment relates to the processing of composite materials including nylon and / or polyester (eg, PET).

[0138] More specifically, this embodiment provides a method for dissolving nylon and / or polyester from a composite material containing nylon and / or polyester, the method comprising the following steps: heating the composite material together with an alcohol having a boiling point higher than 150°C and sodium carbonate to a temperature higher than 150°C to dissolve the nylon and / or polyester.

[0139] In one embodiment, the alcohol having a boiling point higher than 150° C. may be selected from ethylene glycol, propylene glycol, glycerol, and any mixture thereof. Glycerol is preferred.

[0140] The temperature at which nylon and / or polyester is dissolved is 150°C or higher, preferably 160°C or higher, more preferably 170°C or higher, and even more preferably 175°C or higher. The temperature can also be, for example, 200°C or lower, or 190°C or lower. The heating is not particularly limited as long as it is sufficient to achieve the desired degree of dissolution, but is typically carried out for 0.5 to 6 hours, for example, 1 to 3 hours. Pressure may be applied during heating.

[0141] After dissolving the nylon and / or polyester, the residue obtained by dissolving the nylon and / or polyester can be washed with water, if necessary.

[0142] In one embodiment, the composite material is in the form of a tire containing nylon and / or polyester and rubber. The rubber may be natural rubber, styrene-butadiene rubber, butadiene rubber, isoprene rubber, or butyl rubber. The method of this embodiment can be used to selectively remove the nylon and / or polyester, preferably polyester, more particularly PET, from the tire by dissolving it.

[0143] [Example 1: Various mutants] (Preparation of various mutants) GYAQ, its mutants, and NylB protein (DNY) were prepared according to the procedures described in papers (Non-Patent Documents 3 and 4).

[0144] The GYAQ used in the experiments had a His-tag sequence and other sequences added to the N-terminus, totaling 370 aa. The sequence is shown below. The underlined portion is the sequence of the His-tag, and the amino acid sequence from position 16 onwards is the original GYAQ amino acid sequence (SEQ ID NO: 1). In the following experiments, GYAQ refers to this sequence unless otherwise specified.

[0145] >Histag-NylC-GYAQ_370aa_38712.37DaMNHKVHHHHHHIEGRMNTTPVHALTDIDGGIAVDPAPRLAGPPVFGGGPGNAAFDLAPVRSTGREMLR FDFPGVSIGAAHYEEGPTGATVIHIPAGARTAVDARGGAVGLSGGYDFNHAICLAGGAGYGLEAGAGVSGALLERLEYRTGFAELQLVSSAVIYDFSARST AVYPDKALGRAALEFAVPGEFPQGRAGAGMSASAGKVDWDRTEITGQGAAFRRLGGDVRILAVVVPNPVGVIVDRAGTVVRGNYDAQTGVRRHPVFDYQEAFAEQVPVVTQAGNTTISAIVTNVRMSPVELNQFAKQVHSSMHRGIQPFHTDMDGDTLFAVTTDEIDLPTTPGSSRGRLSVNATALGAIASEVMWDAVLEAGK* (Sequence number 3)

[0146] The sequence of DNY used in the experiments is shown below. The underlined portion is the sequence of the His-tag, etc. Note that in general NylB-DNY (a NylB-NylB' hybrid enzyme with G181D / H266N / D370Y substitutions), the first amino acid, methionine M, is omitted due to the tag junction; the sequence excluding the tag is D180, N265, Y369. In the following experiments, DNY refers to this sequence unless otherwise specified.

[0147] >Hyb24-DNYMRGSHHHHHHGSACLNARSTGQHPARYPGAAAGEPTLDSWQEPPHNRWAFAHLGEMVPSAAVSRRPVNAPGHALARLGAIAAQLPDLEQRLEQTYTDAFLVLRGTEVVAEYYRAGFAPDDRHLLMSVSKSLCGTVVGALVDEGRIDPAQPVTEYVPELAGSVYDGPSVLQVLDMQISIDYNEDYVDPASEVQTHDRSAGWRTRRHGDPADTYEFLTTLRGDGSTGEFQYCSANTDVLAWIVERVTGLRYVEALSTYLWAKLDADRDATITVDTTGFGFANGGVSCTARDLARVGRMMLDGGVAPGGRVVSEDWVRRVLAGGSHEAMTDKGFTNTFPDGSYTRQWWCTGNERGNVSGIGIHGQNLWLDPLTDSVIVKLSSWPDPYTEHWHRLQNGILLDVSRALDAV* (SEQ ID NO: 4)

[0148] The prepared enzyme was obtained as a mixture of cleaved and uncleaved enzymes, so cleavage was completed by dialysis against Buffer A (10% glycerol containing 20 mM phosphate buffer, pH 7.3) at 4°C for 5 days and then incubation at 37°C for 24 hours.

[0149] (Evaluation of NOM Degradation Activity) NOM (nylon oligomer mixture) was prepared using the method described in the paper (Methods Enzymol., 2021, 648, 357-389). When the enzyme was reacted with NOM, two spots appeared on TLC (performed as described in Example 3 below): ALD (6-aminohexanoate linear dimer) and Ahx (6-aminohexanoic acid) (Figure 1). Each spot was quantified. The two values ​​were then added together to calculate Ahx + ALD.

[0150]

[0151] As a result, high activity was observed in the mutants with G111A and D304A substitutions.

[0152] (ALD decomposition activity evaluation) ALD was prepared using the method described in the paper (Methods Enzymol., 2021, 648, 357-389.) When the enzyme reacted with ALD, two spots appeared on TLC: ALD (substrate) and Ahx (product), and each was quantified.

[0153]

[0154] A strong Ahx spot intensity is considered promising. A weaker ALD spot intensity is better. Experimental results showed that substitutions of R52Y and D304A, or D99A and D304A, showed high activity.

[0155] (Evaluation of Nylon-6 Degradation Activity) Nylon-6 was microparticulated or treated with formic acid according to the method described in a prior patent (Patent Document 1). When the enzyme reacted with Nylon-6, two spots, ALD and Ahx, appeared on TLC, and each was quantified. The two values ​​were then added together to calculate Ahx + ALD.

[0156]

[0157] As a result, when used alone, the mutants with substitutions of R52Y, D99A, and G111A had high activity, but when used in combination, the mutants with substitutions of R52Y and D304A, D99A and D304A, and R52Y and D99 showed high activity.

[0158] (Evaluation of Heat Resistance) The method described in the paper (Non-Patent Document 2) was followed.

[0159]

[0160] The results are shown in the table above. The values ​​in parentheses are the heat-resistant temperature, which is measured by the inactivation of enzyme reaction activity. Due to the characteristics of the CD instrument, accurate values ​​cannot be calculated for Tm values ​​above 90°C. Therefore, NOM was used as the substrate for the values ​​of the D304A, G111A-D304A, and R52Y-D304A mutants. The heat-resistant temperature was estimated using the inactivation of enzyme reaction activity as an index.

[0161] Example 2: Cp synthesis (Preparation of cpGYAQ) The amino acids 19 to 262 of GYAQ (NylC consisting of the sequence of SEQ ID NO: 1) were ligated to the end of positions 267 to 355, and six histidines were further added (SEQ ID NO: 2). The DNA sequence encoding this was inserted after the start codon (ATG) of the pColdIV vector (Takara) to create an expression vector. The protein was expressed in Escherichia coli BL21 according to standard methods and purified using a His-tag affinity column to prepare the cpGYAQ protein. It was confirmed that the purified cpGYAQ protein exerted its intended activity without the need for further processing for self-cleavage.

[0162] >cpGYAQ-His_339aa_35601.04 TTISAIVTNVRMSPVELNQFAKQVHSSMHRGIQPFHTDMDGDTLFAVTTDEIDLPTTPGSSRGRLSVNATALGAIASEVMWDAVLEAGKDPAPRLAGPPVFGGPGNAAFDLAPVRSTGREMLRFDFPGVSIGAAHYEEGPTGATVIHIPAGARTAVDARGGAVGLSGGYDFNH AICLAGGAGYGLEAGAGVSGALLERLEYRTGFAELQLVSSAVIYDFSARSTAVYPDKALGRAALEFAVPGEFPQGRAGAGMSASAGKVDWDRTEITGQGAAFRRLGDVRILAVVVPNPVGVIVDRAGTVVRGNYDAQTGVRRHPVFDYQEAFAEQVPPVTHHHHHH (SEQ ID NO: 2)

[0163]

[0164] By deleting amino acids 1-18 and 263-266 of p2-GYAQ, we succeeded in reducing the size by 22 aa. In fact, considering that p2-GYAQ has +15 aa and cpGYAQ has 6 aa of additional sequences such as a His-tag, cpGYAQ is 31 aa shorter than p2-GYAQ.

[0165] Furthermore, crystal structure analysis of the purified cpGYAQ revealed that it formed a donut-shaped tetramer, similar to p2-GYAQ.

[0166] (Heat resistance test: circular dichroism (CD) spectrum analysis) The method described in the paper (Non-Patent Document 2) was followed. Circular dichroism (CD) spectra at a far-ultraviolet wavelength (220 nm) were measured using a spectrophotometer (JASCO, Model J-820Q4). A cuvette with an optical path length of 1 mm was used for the measurement, and the protein concentration was 0.1 mg / mL. The temperature was raised from 25°C to 95°C at a rate of 1°C / min using a JASCO PTC-423L Peltier system, and the measured values ​​obtained at each temperature were converted to molar ellipticity, and the Tm value, which serves as an index of heat resistance temperature, was calculated.

[0167] The calculated Tm values ​​were 85.0°C for p2-GYAQ and 86.9°C for cpGYAQ, with cpGYAQ showing a value approximately 2°C higher (Figure 2-1).

[0168] (Substrate Specificity) Cyclic nylon oligomer mixture (NOM), Ald (Ahx linear dimer), and nylon-6 were used as reaction substrates. NOM was prepared according to a method described in a paper (Methods Enzymol., 2021, 648, 357-389). Nylon-6 was microparticulated or treated with formic acid according to a method described in a prior patent (Patent Document 1). The substrate and enzyme protein were added to Buffer A (10% glycerol containing 20 mM phosphate buffer, pH 7.3) at a final concentration of 5 mg / mL and 0.5 mg / mL, respectively, and incubated at 37°C. A control was prepared by adding Buffer A instead of the enzyme. After the specified time had elapsed, the reaction was stopped by heating the reaction solution at 99°C for 10 minutes, and the reaction was subjected to TLC, TNBS, and LC-MS analysis as needed.

[0169] For the substrates tested (NOM, nylon-6), cpGYAQ showed reactivity equivalent to that of p2-GYAQ (Figure 2-2).

[0170] (Kinetic Analysis) Trimeric or higher cyclic nylon oligomers (Aco) were used as the reaction substrate. Preparation was performed according to the method described in the paper (Methods Enzymol., 2021, 648, 357-389). The enzyme protein was adjusted to a final concentration of 0.0025 mg / mL, and Aco was added to Buffer A at final concentrations of 0, 0.25, 0.5, 0.75, 1, 1.25, or 2.5 mg / mL and incubated at 37°C. After 1, 2, and 3 hours, the reaction was terminated by heating the reaction solution to 99°C for 10 minutes. The amino group concentration in the system was measured by TNBS analysis. The initial reaction velocity was determined from the change in amino group concentration, and kinetic parameters were calculated according to the method described in the paper (J. Appl. Glycosci., 2023, 70, 33-37).

[0171] The kcat / Km values, which indicate catalytic efficiency, were 5.6 for p2-GYAQ and 5.2 for cpGYAQ, confirming that their reactivities toward Aco were equivalent (FIGS. 2-3 and the table below).

[0172]

[0173] Example 3: Monomerization by Acid Treatment and Enzyme Treatment. Formic acid treatment and microparticulation were performed according to the methods described in a prior patent (Patent Document 1). For hydrochloric acid and sulfuric acid treatments, 2 mL of acid (18% hydrochloric acid or 50% sulfuric acid (commercially available stock solution diluted 2-fold with water)) was added to 1 g of nylon-6 (the same as used in the examples) and heated in an oil bath set at 120°C for 2 hours. After the reaction, the mixture was neutralized with 10 M NaOH and diluted with water to 10 mg / mL. An equal amount of enzyme solution (final concentrations: GYAQ, cpGYAQ 0.1 mg / mL, DNY 1 mg / mL) was added to the mixture and allowed to stand at 37°C for 24 hours. As a control, Buffer A was added instead of the enzyme. After the specified time had elapsed, the reaction was stopped by heating the reaction solution at 99°C for 10 minutes. The amino group concentration in the system was measured by TNBS analysis, and the monomerization rate was calculated.

[0174] It was confirmed that the enzymatic hydrolysis activity of p2-GYAQ against chemically pretreated nylon-6 was equivalent to that of p2-GYAQ (Fig. 3).

[0175] [Example 4: Verification using products] (Nylon) The nylon-6 pellets used in the examples were kindly provided by Toyobo Co., Ltd. (Tsuruga City, Japan). The fishing net was kindly provided by Mr. Muneaki Okamoto of Higashikushira Fisheries Cooperative Association in Shibushi City, Kagoshima Prefecture.

[0176] (Nyl Series Enzymes) In this example, two types of nylon hydrolases called Nyl series enzymes derived from Arthrobacter sp. KI72 were used: NylB-DNY (a NylB-NylB' hybrid enzyme with G181D / H266N / D370Y substitutions) and NylC-GYAQ (a NylC enzyme with D122G / H130Y / D36A / E263Q substitutions) (Non-Patent Documents 1 and 4).

[0177] (Detection of Enzyme Activity by Thin-Layer Chromatography (TLC)) The supernatant (2 μL) was developed on a silica gel plate (1.05748; Merck Co.). The sample was developed on a solvent mixture (1-propanol / water / ethyl acetate / ammonia = 24:12:4:1.5), and degradation products were identified by visualization with 0.2% ninhydrin solution (in 1-butanol saturated with water).

[0178] (Colorimetric determination of amino groups) In a 96-well plate, 6 μL of sample was added to 75 μL of 0.1 M sodium tetraborate solution (pH 9.5). Then, 30 μL of TNBS solution (containing 0.05% trinitrobenzenesulfonic acid sodium salt (TNBS) and 0.065% sodium sulfite) was added, and the solution was rapidly mixed. After incubation at 40°C for 60 minutes, the absorbance at 420 nm was measured and compared with that of a known concentration of 6-aminohexanoic acid (Ahx) standard to estimate the concentration of amino groups generated by polymer hydrolysis.

[0179] (Acid Treatment) 1 g of nylon (150, 200, 500, 1,000 g / L) was added to 6.8, 5, 2, or 1 mL of 18% hydrochloric acid (2x diluted concentrated HCl) or 50% sulfuric acid (2x diluted concentrated H2SO4), and the mixture was stirred in an oil bath at 120 °C. The heating time was 2 hours. After cooling to room temperature, the pH of the reaction mixture was adjusted to 7.3 with 10 M NaOH, and the solution was diluted to 10 mg / mL for the enzyme reaction.

[0180] (Enzyme Reaction) 200 μL of NylC-GYAQ and / or DNY ​​enzyme solution (2.0 mg / mL each in buffer A) was added to 200 μL of the acid-treated nylon solution (10 mg / mL) prepared above. The mixture was incubated at 37°C for 24 or 72 hours. During the reaction, samples were taken at regular intervals (30 μL aliquots were taken and heated at 99°C for 10 minutes to inactivate the enzyme activity). The concentration of amino groups produced by hydrolysis of the polymer was evaluated by TLC analysis and a colorimetric method using TNBS. The monomerization rate is the ratio of the estimated amount of amino groups produced by hydrolysis of the polymer to the total amino group concentration in the system. It was calculated using the following equation: The denominator represents the theoretical maximum amino group concentration when nylon is completely hydrolyzed and all amino groups in the nylon are exposed. The numerator represents the concentration of amino groups produced by hydrolysis of the polymer in the reaction mixture, measured by the TNBS method.

[0181]

[0182] (Results) Nylon-6 pellets were immersed in 50% aqueous hydrochloric acid (effective concentration 18%) or 50% aqueous sulfuric acid (concentration 150 g / L) and heated in an oil bath at 120 °C for 2 hours. Although the reaction mixture did not reflux, the pellets completely dissolved within 1 hour. Nylon-6 did not carbonize, even in 50% aqueous sulfuric acid. Interestingly, the pellets completely dissolved within 2 hours even when the nylon-6 concentration was increased to 200, 400, or 1,000 g / L. However, at higher concentrations (over 1,000 g / L), some insoluble residues were observed (Figure 4-1, a). TLC analysis revealed that oligomers remained in the reaction system even after chemical treatment (Figure 4-1, b). These oligomers were confirmed to be converted to monomers in the hydrolysis step using the Nyl series catalyst, achieving extremely high monomerization rates (>90%). Using cpGYAQ instead of GYAQ also achieved comparable monomerization rates.

[0183] The applicability of the established nylon monomerization method was tested on fishing nets used for bottom trawl fishing in Shibushi Bay, Kagoshima Prefecture, for one year (January 2023 to January 2024). Acid treatment was performed in a 50% hydrochloric acid solution (effective concentration 18%) at 120°C for two hours. The net completely dissolved within 30 minutes (Figure 4-2). Enzymatic hydrolysis using a nylon-based enzyme revealed that the net was made of nylon-6, with a monomerization rate of 80% (Figure 4-3). Using cpGYAQ instead of GYAQ confirmed a similar monomerization rate. Considering that the net was composited with other materials for commercialization and that non-nylon deposits were present on the surface due to environmental exposure, this method was successfully applied to commercial products.

[0184] Example 5: Examination of Enzyme Reaction Conditions Nylon-6 pellets were monomerized in the same manner as in Examples 3 and 4. Specifically, 1 g of nylon was added to 2 mL of 18% hydrochloric acid (2x diluted concentrated hydrochloric acid), and the mixture was stirred in an oil bath at 120°C. The heating time was 2 hours. After cooling to room temperature, the pH of the reaction mixture was adjusted to 7.3 with 10 M NaOH, and the solution was diluted to 10 mg / mL. The enzymatic reaction was carried out at 37°C. NylB-DNY and NylC-GYAQ were used as enzymes, and the enzyme concentrations (final concentrations in the reaction system) were set at 1.0, 0.5, and 0.1 mg / mL.

[0185] The results are shown in Figure 5 and the table below. As shown in Figure 5, TLC showed an oligomer spot in the early stage of the enzyme reaction, but the monomer spot became darker over time, and after 3 hours of enzyme reaction, no spots other than the monomer were observed at any enzyme concentration. The monomerization rate calculated by TNBS showed that monomerization was achieved in as short a time as 3 hours at all three enzyme concentrations.

[0186]

[0187] Nylon-6 was dissolved by acid treatment, and then completely monomerized by treating it with a specific enzyme at a final concentration of 0.1 mg / mL for 3 hours.

[0188] [Example 6: Verification 2 using products] (Nyl series enzymes) In this example, two types of nylon hydrolases called Nyl series enzymes derived from Arthrobacter sp. KI72 were used: NylB-DNY (a NylB-NylB' hybrid enzyme with G181D / H266N / D370Y substitutions) and NylC-GYAQ (a NylC enzyme with D122G / H130Y / D36A / E263Q substitutions) (Non-Patent Documents 1 and 4).

[0189] (Detection of Enzyme Activity by Thin-Layer Chromatography (TLC)) The supernatant (2 μL) was developed on a silica gel plate (1.05748; Merck Co.). The sample was developed on a solvent mixture (1-propanol / water / ethyl acetate / ammonia = 24:12:4:1.5), and degradation products were identified by visualization with 0.2% ninhydrin solution (in 1-butanol saturated with water).

[0190] (Quantification of amino groups by colorimetry) In a 96-well plate, 6 μL of sample was added to 75 μL of 0.1 M sodium tetraborate solution (pH 9.5). Then, 30 μL of TNBS solution (containing 0.05% trinitrobenzenesulfonic acid sodium salt (TNBS) and 0.065% sodium sulfite) was added, and the solution was rapidly mixed. After incubation at 40°C for 60 minutes, the absorbance at 420 nm was measured and compared with that of a standard substance of known concentration to estimate the concentration of amino groups generated by hydrolysis of the polymer.

[0191] (Acid Treatment) The product was added to a 50% aqueous hydrochloric acid solution (effective concentration 18%), and the mixture was stirred in an oil bath at 120°C. The heating time was 2 hours. After cooling to room temperature, the pH of the reaction mixture was adjusted to 7.3 with 10 M NaOH, and the solution was diluted to 10 mg / mL for the enzyme reaction.

[0192] (Enzyme Reaction) 200 μL of NylC-GYAQ and / or DNY ​​enzyme solution (2.0 mg / mL each in buffer A) was added to 200 μL of the acid-treated nylon solution (10 mg / mL) prepared above. The mixture was incubated at 37°C for 24 or 72 hours. During the reaction, samples were taken at regular intervals (30 μL aliquots were taken and heated at 99°C for 10 minutes to inactivate the enzyme activity), and the concentration of amino groups generated as a result of hydrolysis of the polymer was evaluated by TLC analysis and colorimetry using TNBS.

[0193] (Test using airbags (coated)) The airbags were completely dissolved by acid treatment in a 50% aqueous hydrochloric acid solution (effective concentration 18%) at 120°C for 2 hours (Figure 6-1). The monomerization rate was approximately 51% before the addition of the enzyme, and after 24 hours of enzyme reaction, it was approximately 58% when GYAQ alone was used, approximately 77% when DNY alone was used, and approximately 83% when both were used (Figure 6-2).

[0194] The airbag was confirmed to be nylon 66, as it was efficiently decomposed by the enzyme to produce HMD. Furthermore, the same monomerization rate was obtained when cpGYAQ from Production Example 2 was used instead of GYAQ. It was confirmed that this method can be applied to commercially available products without any problems.

[0195] (Test using airbags (without coating agent)) The airbags were completely dissolved by acid treatment in a 50% aqueous hydrochloric acid solution (effective concentration 18%) at 120°C for 2 hours (Figure 6-3). The monomerization rate was approximately 47% before the addition of the enzyme, and after 24 hours of enzyme reaction, it was approximately 61% when GYAQ alone was used, approximately 78% when DNY alone was used, and approximately 79% when both were used (Figure 6-4).

[0196] The airbag was confirmed to be nylon 66, as it was efficiently decomposed by the enzyme to produce HMD. Furthermore, the same monomerization rate was obtained when cpGYAQ from Production Example 2 was used instead of GYAQ. It was confirmed that this method can be applied to commercially available products without any problems.

[0197] (Summary of verification results using products) The verification results using products are shown in Figure 6-5. It was confirmed that this method shows a high monomerization rate for airbags. It was also confirmed that the acid treatment and Nyl series reaction process are effective for actual products.

[0198] Example 7: Pretreatment of polyester-nylon composite fabric and monomerization by enzyme treatment The following four types were used for the study. (1) Outer: 100% nylon, Inner: 100% polyester (Product name: "Stylish◇Nylon", Beige, Product number: 14-0524-01[22C], Matsuyama Woolen Co., Ltd., https: / / item.rakuten.co.jp / matsuke5 / 10006969 / ) (2) 100% nylon (Product name: "Plain Nylon Fabric", 011 Ivory, Product number: 6774, Shimura Co., Ltd., https: / / item.rakuten.co.jp / simuraginga / 6774 / ) (3) 40% nylon, 60% polyester (3 Yellow, Product number: 700041 Sarinene ak 3*20, Kyoto Canary Handicraft Store, https: / / item.rakuten.co.jp / ky-kanariya / 10025115 / ?variantId=77316) (4) Nylon: 53% Polyester: 47% (2 Red product number: 112547600, Naka Shoji Co., Ltd., https: / / item.rakuten.co.jp / fabricbird / 112547600 / )

[0199] The acid treatment and subsequent enzyme treatment of the fabric were carried out as follows. 18% hydrochloric acid (2.5 mL) was added to 1 g of each of fabrics (1) to (4) to a final concentration of 400 g / L, and the mixture was heated at 120°C for 2 hours. After dilution with distilled water, the mixture was neutralized to pH 7.0 with NaOH and made up to 40 mL. 80 μL of the resulting solution was mixed with 320 μL of enzyme (final concentrations: GYAQ 0.1 mg / mL, DNY 1.0 mg / mL) and allowed to stand at 37°C. The monomerization rate and other properties of the enzyme-treated solution were calculated.

[0200] Figure 7-1 shows the reaction system after 24 hours of enzyme reaction. Figure 7-2 shows photographs of the cloth before and after acid treatment. The total weight of the cloth before and after hydrochloric acid treatment is shown in the table below.

[0201]

[0202] Assuming that the difference in weight before and after treatment is the weight of nylon, the percentage of nylon in the fabric was calculated. The nylon percentages of Fabrics 1 to 4 were 48%, 93%, 51%, and 59%, respectively. It was confirmed that the percentages for all products were roughly as indicated.

[0203] Figure 7-3 shows the results of TLC analysis after the enzyme reaction. Based on the appearance of the spots in the TLC analysis after the enzyme reaction, it was predicted that all of the four types of fabrics examined contained nylon-6. Figure 7-4 also shows the monomerization rates before and after the enzyme reaction. After the enzyme reaction, the monomerization rate was 90% or higher in all products.

[0204] It was confirmed that nylon can be selectively extracted and monomerized from polyester-nylon composite materials by hydrochloric acid treatment under the same conditions as previously established.

[0205] Example 8: Monomerization by pretreatment and enzyme treatment of natural material-nylon composite fabric The following four types were used for the study. (1) 90% wool, 10% nylon (product name: "Wool Polyamide Loop Check Tweed", made in Italy by VT, wool polyamide loop check tweed, https: / / enop.jp / product / %E3%82%A4%E3%82%BF%E3%83%AA%E3%82%A2-%E8%BC%B8%E5%85%A5%E7%94%9F%E5%9C%B0%E3%80%90vt%E7%A4%BE%E3%80%91%E3%82%A6%E3%83%BC%E3%83%AB%E3%83%BB%E3%83%9D%E3%83%AA%E3%82%A2%E3%83%9F%E3%83%89%E3%83%AB / ) (2) (1) 84% cotton, 16% nylon (product name: "Cotton Nylon Weather Damage Dyed", OW Off-White, product number: IN50728-OW, https: / / www.kijinomori.com / c / 0000000134 / 0000000152 / gr469 / gr424 / in50728-) (2) 68% cotton, 32% nylon (Cotton Nylon Gaba Shiro 05-3090, https: / / daruma-store.jp / ?pid=159204502) (3) 70% cotton, 30% nylon (Cotton Nylon Herringbone Cloth KW Super White IN50620-KW), https: / / www.kijinomori.com / c / gr446 / gr450 / gr453 / in50620-)

[0206] The acid treatment and subsequent enzyme treatment of the fabric were carried out as in Example 7.

[0207] Figure 8-1 shows the state of the reaction system after 24 hours of enzyme reaction. Figure 8-2 shows photographs of the cloth before and after acid treatment. The cotton part has turned black, indicating a high possibility of decomposition by acid. The total weight of the cloth before and after hydrochloric acid treatment is shown in the table below.

[0208]

[0209] The percentage of nylon in the fabric was calculated by assuming that the difference in weight before and after treatment was the weight of nylon. The percentage was higher than indicated on the product label, and it was thought that wool and cotton were also leached out by the acid treatment.

[0210] The results of TLC analysis after the enzyme reaction are shown in Figure 8-3. Based on the appearance of the spots in the TLC analysis after the enzyme reaction, fabrics 1, 3, and 4 were predicted to be nylon-6, and fabric 2 was predicted to be nylon-66.

[0211] Figure 8-4 shows the monomerization rate before and after the enzyme reaction. Because it is highly likely that components other than nylon were eluted during the acid treatment, the monomerization rate was calculated assuming that all of the nylon was eluted as indicated on the product label. For Fabric 1, the yield exceeded 100%, likely due to the elution of amino acids from the wool. For Fabrics 2, 3, and 4, the sugar components of the cotton may have formed a Schiff base with the amino groups of the nylon, resulting in a reduced yield.

[0212] When the previously established hydrochloric acid treatment is applied to natural material-nylon composites, components other than nylon are also eluted, making it thought that selective monomerization of nylon would be difficult. However, TLC and LC analysis results confirmed that Ahx monomer was produced as the main product, confirming that nylon was preferentially monomerized.

[0213] Example 9: Pretreatment of a tire containing nylon fibers. A tire containing nylon fibers was used for the study. A tire section with the remaining fiber portion was cut into thin strips perpendicular to the fiber (thickness: approximately 0.5 mm) using a cutting machine (Leica, microtome). Acid treatment and subsequent enzyme treatment were performed in the same manner as in Example 7.

[0214] Figure 9-1 shows the reaction system after 24 hours of enzyme reaction. Figure 9-2 shows photographs of the tire fragments before and after acid treatment. It was confirmed that the nylon cords had dissolved and holes had appeared due to the acid treatment.

[0215] The results of TLC analysis before and after the enzyme reaction are shown in Figure 9-3. Based on the appearance of the TLC spots, the tire cord was predicted to be nylon-66. The monomerization rate was calculated assuming that the mass difference before and after treatment (0.029 g) was nylon. The results are shown in Figure 9-4. The monomerization rate was 92%, which was satisfactory.

[0216] It was found that nylon could be selectively extracted and monomerized from tires using the same pretreatment method previously established.

[0217] Example 10: Pretreatment of PET-containing tires. PET-containing tires were used for the study. 4 mL of ethylene glycol was added to 49.2 mg of a fibrous tire sample, followed by 100 mg of Na2CO3, and the mixture was treated at 180°C for 2 hours. After the reaction, the residue was washed with distilled water.

[0218] The state after the reaction is shown in Figure 10-1. It was confirmed that the orange fibrous substance had dissolved.

[0219] The results of LC-MS analysis of the solution after the reaction are shown in FIG. 10-2.

[0220] It was confirmed that the pretreatment allowed the PET components to be extracted into the solution phase, demonstrating that this method can be used to extract only the rubber when recycling tires.

[0221] Example 11: Enzyme activity of GYAQ mutant and cpGYAQ mutant toward various substrates Various mutant enzymes were prepared according to the method described in Example 1, and their activities were measured.

[0222] The results for the self-cleaving GYAQ mutant are shown in the table below, with the positions of the mutations based on the amino acid numbering in SEQ ID NO:1.

[0223]

[0224] (Measurement of Nylon-66 Monomer Unit (66MU) Degradation Activity) Degradation was performed by dissolving the substrate in buffer A to a final substrate concentration of 5 mg / mL and an enzyme concentration of 1 mg / mL, incubating at 37°C for 24 hours, and then quantifying the HMD concentration released into the system using an o-ABA assay. Specifically, the production of aminoaldehydes resulting from transamination was monitored by measuring the change in absorbance at 450 nm due to complex formation with ortho-aminobenzaldehyde (o-ABA) (see paragraph 0166 of WO2022-270597).

[0225] Regarding the self-cleaving GYAQ, the mutations that increase the decomposition activity for each substrate are summarized as follows: (1) Nylon-6 substrates Mutations that increase the decomposition activity of Nom: D99A_G111T, G111A-D304A Mutations that increase the decomposition activity of ALD: G111D, F134W, P27Q_D304A, R52Y_D99A, R52Y_D304A, D99A_D304A, G111D_D304A Mutations that increase the decomposition activity of 6-Nylon: Increased ability to produce Ahx: R52Y, D99A, G111A, F134W, R52Y_D99A, R52Y_D304A, D99A_D304A Increased ability to produce ALD: P27A, P27Q, P28A, F301L

[0226] (2) Mutations that enhance the degradation activity of nylon-66MU, a nylon-66 substrate: D99A, G111A, G111T, G111L, G111H, G111C, L139R, D304A, L331D, L331E, R52Y_D99A, D99A_D304A, G111A_D304A, G111A_L331D, G111A_L331E Mutations that enhance the degradation activity of nylon-66: G111A, G111T, F134W, D304A, R52Y_D99A, R52Y_G111T, G90A_G111T, V92A_G111T, D99A_G111T, D99A_D304A, G111T_D304A, D99A_G111A_D304A

[0227] (3) Mutations that increase thermostability (above 90°C): G111Y, D304A, R52Y_D304A, D99A_G111T, G111A_D304A, G111T_D304A, R52Y_G111A_D304A, D99A_G111A_D304A

[0228] Furthermore, from the viewpoint of improving enzyme activity, preferred mutations include P27A, P27Q, P28A, R52Y, D99A, G111D, G111R, G111A, G111T, G111L, G111H, G111C, F134W, L139R, F301L, D304A, L331D, L331E, P27Q_D304A, R52Y_D99A, R52Y_G111T, R52Y_D304A, G90A_G111T, V92A_G111T, D99A_G111T, D99A_D304A, G111A_D304A, G111A_L331D, G111A_L331E, Examples include G111D_D304A, G111T_D304A, R52Y_G111A_D304A, and D99A_G111A_D304A.

[0229] The results for the cpGYAQ mutant are shown in the table below. The positions of the mutations are based on the amino acid numbers in SEQ ID NO: 2. The corresponding amino acid numbers in the self-cleaving NylC (SEQ ID NO: 1) are shown in parentheses.

[0230]

[0231] The mutations that increase the degradation activity of cpGYAQ for each substrate are summarized below. (1) Nylon-6 substrates. Mutations that increase the degradation activity of Nom: T203(132)A. Mutations that increase the degradation activity of ALD: T203(132)A. Mutations that increase the degradation activity of nylon-6: D170(99)A, F35(301)L, T1(267)S, T1(267)C.

[0232] (2) Mutations that enhance the degradation activity of nylon-66MU, a nylon-66 substrate: D38(304)A, D170(99)A, G182(111)A, M39(305)D, T1(267)C. Mutations that enhance the degradation activity of nylon-66: D38(304)A, R123(52)Y, D170(99)A, G182(111)A, P98(27)Q, F35(301)L, F35L_P98Q.

[0233] (3) Mutation K260(189)A that increases thermostability Although no overall improvement in thermostability was observed, this mutation resulted in a Tm value of nearly 90 degrees.

[0234] Furthermore, preferred mutations from the viewpoint of improving enzyme activity include D38(304)A, R123(52)Y, D170(99)A, G182(111)A, M39(305)D, P98(27)Q, F35(301)L, T203(132)A, T1(267)S, T1(267)C, and F35L_P98Q.

[0235] Example 12: Degradation activity against fishing line The activity of a combination of the self-cleaving GYAQ mutant and the DNY enzyme to degrade a fishing line substrate was compared.

[0236] The following three types of fishing line were used in the study: (1) Ginrin (registered trademark) No. 1 (JAN 4996478 112320, Toray Monofilament Co., Ltd., https: / / www.torayfishing.net / product / 626 / ) Nylon-6 / Nylon-66 = 90 / 10 (2) QUEEN STAR No. 1 (JAN 4968813 805723, Sunline Co., Ltd., https: / / fishing.sunline.co.jp / line / 197 / ) Nylon-6 / Nylon-66 = 84 / 16 (3) VALCAN EXTRA V-500 No. 1 (JAN 4994942 004003, Sanyo Nylon Co., Ltd., https: / / www.sanyo-nylon.co.jp / products / valcan_extra_v / ) Nylon-6 / Nylon-66 = 84 / 16

[0237] The micronization was performed according to the method described in a prior patent (Patent Document 1). The enzyme treatment conditions were fishing line concentration: 5 mg / mL, DNY enzyme concentration: 1 mg / mL (added or not added), and GYAQ mutant concentration: 0.1 or 1 mg / mL.

[0238]

[0239]

[0240]

[0241] In the study of Example 11, mutants with improved degradation activity against nylon-6 or nylon-66 were selected and their degradation of copolymer products was examined. As a result, the R52Y, D99A, and D304A mutations, either singly or in combination, successfully improved the degradation rate several tens of times over GYAQ. Although the substrate used was insoluble in water, the amount of precipitate clearly decreased after enzymatic treatment, confirming the progress of enzymatic degradation.

[0242] [Example 13: Summary of processing conditions] Processing conditions for nylon-6 and nylon-66 were investigated according to the method of Example 4. Commercially available pellets of nylon-6 and nylon-66 were used without being subjected to a micronization treatment.

[0243]

[0244]

[0245] In a system where nylon-6 was treated with 150g / L hydrochloric acid at an actual concentration of 10.8% at 120℃ for 168 hours, the water absorption of the sample after acid treatment was too high, so an enzyme reaction was not carried out. If the acid treatment time is prolonged, it is expected that side reactions such as deamination and polymerization may occur.

[0246] Experimental results show that the following are some of the preferred acid treatment conditions. These conditions allow for sufficient oligomerization. Nylon-6: Charge concentration: 100-1200g / L, Acid: 9-27%, Hydrochloric acid temperature: 100-150°C, Time: 1.5-32 hours Nylon-66: Charge concentration: 50-750g / L, Acid: 9-27%, Hydrochloric acid temperature: 110-150°C, Time: 1.5-32 hours

[0247] Experimental results show that the most favorable acid treatment conditions are as follows. Under these conditions, almost complete water-soluble oligomerization can be achieved. Nylon-6: Charge concentration: 1000 g / L, or twice the amount of nylon charged. Acid: 18% hydrochloric acid. Temperature: 120°C. Time: 2 hours. Nylon-66: Charge concentration: 400 g / L, or twice the amount of nylon charged. Acid: 18% hydrochloric acid. Temperature: 120°C. Time: 2 hours.

[0248] The present invention can be used to recycle nylon composite materials into monomer molecules, contributing to ensuring sustainable production and consumption patterns.

[0249] [Sequences listed in the sequence listing] SEQ ID NO: 1 Amino acid sequence of NylC-GYAQ SEQ ID NO: 2 Amino acid sequence of cp-NylC SEQ ID NO: 3 Histag-NylC-GYAQ_370aa_38712.37Da SEQ ID NO: 4 Hyb24-DNY SEQ ID NO: 5 Amino acid sequence of NylB-DNY

Claims

1. A protein that is (4), (5), or (6) below, wherein at least one amino acid selected from D304, R52, D99, and G111 of the sequence of SEQ ID NO: (4) Proteins consisting of the amino acid sequence of Sequence ID No. 1; (5) A protein having an amino acid sequence in which 1 to 36 amino acids are deleted, substituted, or added in the amino acid sequence of the protein described in (4), and which has endo-type nylon hydrolase (NylC) activity; (6) A protein having a sequence identity of 90% or more with the protein described in (4), and possessing NylC activity, A protein in which the mutation corresponds to at least one selected from D304A, R52Y, D99A, and G111A or G111T.

2. The protein according to claim 1, wherein the mutations correspond to at least two mutations selected from D304A, R52Y, D99A, and G111A.

3. The protein according to claim 1, wherein the mutations correspond to at least two mutations selected from R52Y and D304A, R52Y and D99A, D99A and D304A, and G111A and D304A.

4. Nylon is brought into contact with 10% or more hydrochloric acid or sulfuric acid at a concentration of 300 to 1200 g / L at 50 to 160°C for 0.5 hours to 50 hours to obtain a liquid containing low-molecular-weight nylon; Add an alkali to the resulting solution to adjust the pH to 6-8, obtaining a neutral solution containing low-molecular-weight nylon; The resulting neutral solution is then treated with at least one of exo-type nylon hydrolase (NylB) and NylC. A method for producing nylon decomposition products, including the process.

5. The manufacturing method according to claim 5, wherein the low molecular weight of nylon by contact with hydrochloric acid or sulfuric acid is carried out until the monomerization rate is 50% or more.

6. A protein having a structure in which the N-terminus of a polypeptide (a1), (a2), or (a3) ​​below is linked to the C-terminus of a polypeptide (b1), (b2), or (b3) below: (a1) Polypeptides having sequences 19-260 of sequence number 1; (a2) A polypeptide comprising a sequence in which one or more amino acids are deleted, substituted, or added in the polypeptide described in (a1), and which is capable of functioning as an α-subunit of NylC; (a3) A polypeptide consisting of a sequence with 70% or more sequence identity with the polypeptide described in (a1), and capable of functioning as the α-subunit of NylC; (b1) Polypeptides having sequences 267-355 of sequence number 1; (b2) A polypeptide comprising a sequence in which one or more amino acids are deleted, substituted, or added in the polypeptide described in (b1), and which is capable of functioning as a β-subunit of NylC; (b3) A polypeptide comprising a sequence having 70% or more sequence identity with the polypeptide described in (b1), and capable of functioning as a β-subunit of NylC.

7. The protein according to claim 6, having a structure in which the N-terminus of a polypeptide (a1'), (a2'), or (a3') below is linked to the C-terminus of a polypeptide (b1'), (b2'), or (b3') below: (a1') A polypeptide having sequences 19-260 of sequence number 1; (a2') A polypeptide comprising a sequence in which 1 to 24 amino acids are deleted, substituted, or added in the polypeptide described in (a1'), and which is capable of functioning as the α-subunit of NylC; (a3') A polypeptide consisting of a sequence with 90% or more sequence identity with the polypeptide described in (a1'), and capable of functioning as the α-subunit of NylC; (b1') A polypeptide having sequences 267-355 of sequence number 1; (b2') A polypeptide comprising a sequence in which 1 to 9 amino acids are deleted, substituted, or added in the polypeptide described in (b1'), and which is capable of functioning as a β-subunit of NylC; A polypeptide comprising a sequence with 90% or more sequence identity with the polypeptide described in (b3')(b1'), and capable of functioning as a β-subunit of NylC.

8. The protein according to claim 6, which is (1), (2), or (3) below: (1) Proteins having sequences 1 to 333 of Sequence ID No. 2; (2) A protein having NylC activity, comprising a sequence in which one or more amino acids are deleted, substituted, or added in the polypeptide described in (1); (3) A protein having a sequence that is 70% or more identical to the polypeptide described in (1), and which has NylC activity.

9. The protein according to claim 8, which is (1'), (2'), or (3') below: (1') Proteins having sequences 1-333 of sequence number 2; (2') A protein having NylC activity, consisting of a sequence in which 1 to 34 amino acids are deleted, substituted, or added in the polypeptide described in (1'); (3') A protein having NylC activity, consisting of a sequence with 90% or more sequence identity with the polypeptide described in (1').

10. The protein according to claim 9, wherein the protein has a mutation corresponding to at least one mutation selected from T1S, T1C, F35L, D38A, M39D, P98Q, R123Y, D170A, G182A, T203A, and K260A in SEQ ID NO:

2.

11. The protein according to claim 9, wherein the protein has mutations corresponding to F35L and P98Q in SEQ ID NO:

2.

12. The method for producing the protein according to claim 5, wherein NylC is the protein according to any one of claims 1 to 4 and 7 to 12.

13. A method for producing nylon degradation products from a nylon-containing composite material, wherein the nylon-containing composite material includes any one selected from polyester, wool, linen, and cotton, according to claim 12.

14. A method for chemically recycling nylon, comprising all steps of the manufacturing method described in any one of claims 4, 5, and 13.

15. Furthermore, the protein (according to any one of claims 1 to 3) has a mutation corresponding to at least one mutation selected from P27A, P27Q, P28A, F134W, F301L, L331D, L331E, F134W, L139R in SEQ ID NO: 1.