Method for producing degradation products of nylon 66

Abstract

This invention provides a method for producing nylon 66 degradation products using enzymes, which increases the degradation rate. It also provides a modified enzyme with superior degradation characteristics, applicable to chemical recycling methods for nylon 66. [Solution] A method for producing a degradation product of nylon 66 is provided, comprising the step of reacting nylon 66 with 6-aminohexanoic acid oligomer end type hydrolase (NylC), wherein NylC is a protein having a specific amino acid sequence.

Description

Technical Field

[0001] The present invention relates to a method for producing nylon 66 decomposition products.

Background Art

[0002] Nylon, which is a type of polyamide, is a material excellent in heat resistance, oil resistance, chemical resistance, fatigue resistance, creep resistance, and toughness. Nylon is widely used in clothing and daily necessities, but 35 - 40% of the total demand is for automobiles and vehicles. It is widely used as parts in the engine room, intake system parts, fuel system parts, airbags, etc. Nylon is also used in fishing gear such as fishing nets, where strong promotion of recycling is desired from the perspective of reducing marine plastic waste. On the other hand, nylon has poor biodegradability, and a general-purpose recycling method has not been established. Therefore, at present, most of the nylon as waste is either incinerated or landfilled.

[0003] A method for producing a diamine compound and / or a dicarboxylic acid compound, which are polymerization raw materials, from a polyamide, specifically, from a polyamide (1) represented by a specific general formula (1), a dicarboxylic acid compound (2) represented by a specific general formula (2) and / or a diamine compound (3) represented by a specific general formula (3), and optionally, a salt compound (4) represented by a specific general formula (4), a polyamide (5) represented by a specific general formula (5) and / or a polyamide (6) represented by a specific general formula (6), the method comprising: (i) a first hydrolysis step of hydrolyzing the polyamide (1) in high-temperature water to obtain a first hydrolyzate, and (ii) subjecting the first hydrolyzate to enzymatic hydrolysis to obtain a second hydrolyzate, wherein the first hydrolyzate contains a water-soluble polyamide that dissolves in water at 20°C, and the second hydrolyzate contains the dicarboxylic acid compound (2) and / or the diamine compound (3), and optionally, the salt compound (4), the polyamide (5) and / or the polyamide (6), has been proposed (Patent Document 1).

[0004] Regarding nylon, three enzymes with different decomposition modes derived from microorganisms are known: NylA (6-aminohexanoate-cyclic dimer hydrolase), NylB (6-aminohexanoate-dimer hydrolase), and NylC (6-aminohexanoate oligomer endo-hydrolase). In particular, NylC, specifically p2-NylC found from the soil bacterium Arthrobacter sp. KI72 (encoded on plasmid pOAD2), has been modified with four amino acid substitutions (122G, 130Y, 36A, and 263Q) to form NylC-GYAQ. This modified form exhibits high activity and converts high molecular weight nylon into oligomers, hence it has been named nylon hydrolase (Non-patent documents 1-4). Furthermore, it has recently been reported that heat resistance has been improved by amino acid substitution at 111 sites (Non-Patent Document 5).

[0005] On the other hand, NylC is expressed as an inactive precursor (36 kDa), which is autocatalytically cleaved at Asn266 / Thr267, separating 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 monomers in a heterodimer structure (Non-Patent Literature 4).

[0006] Furthermore, studies are underway to reduce the molecular weight of nylon 6 and nylon 66 by performing chemical limited decomposition with formic acid, thereby reducing the decomposition rate by NylC-GYAQ. However, NylC-GYAQ cannot decompose the dimer, and nylon 6 dimer or nylon 66 monomer (66MU) remains in the system. Therefore, in order to achieve complete decomposition of nylon 6 to 6-aminohexanoic acid (Ahx) and nylon 66 to hexamethylenediamine (HMD) and adipic acid (AD), studies are underway to use NylB, which can decompose nylon 6 dimer or nylon 66 monomer (66MU), in combination with NylB to perform nylon decomposition (Patent Document 2). [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] International release WO2023 / 149514 [Patent Document 2] International release WO2023 / 167314 [Patent Document 3] Japanese Patent Application No. 2024-143286 (Not published at the time of this application) [Non-patent literature]

[0008] [Non-Patent Document 1] 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. [Non-Patent Document 2] 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.

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Summary of the Invention

[0009] On the other hand, the present inventors have provided modified NylC with excellent degradation characteristics, and a process for further increasing the degradation rate when degrading with NylC. They have also provided modified NylC that does not require a self-cleavage process to activate it (Patent Document 3).

[0010] One method for processing waste plastics is chemical recycling. In chemical recycling, the collected waste is generally sorted, crushed, and washed to remove foreign matter, and then depolymerized to chemically break it down into raw materials or intermediate materials. These intermediate materials are then refined and polymerized to produce new plastic products. For nylon 66, it would be desirable to establish a chemical recycling method that utilizes enzymatic reactions instead of depolymerization.

[0011] In one aspect, the present invention aims to provide a modified NylC with excellent decomposition characteristics that can be applied to chemical recycling methods for nylon 66, and to provide a process for further increasing the decomposition rate (monomerization rate - 66MU decomposition rate) when decomposing with NylC. Considering future application to actual products, the monomerization rate for nylon 66 is preferably 55% or higher, and more preferably 75% or higher.

[0012] In one aspect, the present invention also aims to provide a modified NylC that does not require a process for self-cleavage. [Means for solving the problem]

[0013] The present invention provides the following: [1] A method for producing a degradation product of nylon 66, comprising the step of reacting nylon 66 with 6-aminohexanoic acid oligomer end type hydrolase (NylC), wherein NylC is the protein described in (4), (5), or (6) below: (4) A protein having the amino acid sequence of sequence number 1 to 355, wherein at least one amino acid selected from M50, R52, D99, G111, L139, D304, and L331 is substituted; (5)(4) The amino acid sequence of the protein described in (4) is a sequence in which one or more amino acids are deleted, substituted, or added, 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 acid corresponding to at least one of the substitutions are maintained, and the amino acid corresponding to T267 of SEQ ID NO: 1 is maintained or may be substituted with S or C, and the protein has 6-aminohexanoic acid oligomer endo-type hydrolase (NylC) activity; A protein having NylC activity, comprising an amino acid sequence having 70% or more sequence identity with the protein described in (6)(4), wherein 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 acid corresponding to at least one of these substitutions are maintained, and the amino acid corresponding to T267 may be maintained or substituted with S or C. [2] A method for producing a degradation product of nylon 66 according to 1, wherein NylC is the protein of (4'), (5'), or (6') below: (4') A protein having the amino acid sequence of positions 1-355 of sequence number 1, with at least one substitution selected from M50I, R52Y, D99A, G111A, L139R, D304A, L331D, and L331E; A protein having NylC activity, comprising a sequence in which 1 to 36 amino acids are deleted, substituted, or added in the amino acid sequence of the protein described in (5')(4'); A protein having a sequence identity of 70% or more with the protein described in (6')(4'), and possessing NylC activity. [3] A method for producing a degradation product of nylon 66 according to 1 or 2, wherein NylC is a protein having at least two substitutions selected from M50I, R52Y, D99A, G111A, L139R, D304A, L331D, and L331E. [4] A method for producing a degradation product of nylon 66 according to any one of items 1 to 3, wherein NylC is a protein in which G111A and D304A are substituted. [5] A method for producing a degradation product of nylon 66 according to any one of items 1 to 4, wherein NylC is 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 sequences 19-260 of sequence number 1; (a2) A polypeptide comprising an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the polypeptide described in (a1), provided 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 an α-subunit of NylC; (a3) A polypeptide consisting of an amino acid sequence having 70% or more sequence identity with the polypeptide described in (a1), provided that the amino acids corresponding to F134, L137, Y146, K189, and N219 of SEQ ID NO: 1 are maintained, and which is capable of functioning as the α-subunit of NylC; (b1) A polypeptide having sequences 267-355 of sequence number 1; (b2) A polypeptide comprising an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the polypeptide described in (b1), provided that the 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 which is capable of functioning as a β-subunit of NylC; A polypeptide having a sequence identity of 70% or more with the polypeptide described in (b3)(b1), provided that the 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, consisting of an amino acid sequence, and capable of functioning as the β subunit of NylC. [6] The method for producing a nylon 66 degradation product according to any one of items 1 to 5, wherein NylC is a protein having a structure in which the N-terminus of a polypeptide represented by the following (a1'), (a2'), or (a3') is linked to the C-terminus of a polypeptide represented by 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 consisting of a sequence in which 1 to 24 amino acids are deleted, substituted, or added in the polypeptide described in (a1'), and capable of functioning as the α subunit of NylC; (a3') A polypeptide consisting of a sequence having a sequence identity of 90% or more with the polypeptide described in (a1'), and 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 consisting of 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 the β subunit of NylC; (b3') A polypeptide consisting of a sequence having a sequence identity of 90% or more with the polypeptide described in (b1'), and capable of functioning as the β subunit of NylC. [7] The method for producing a nylon 66 degradation product according to any one of items 1 to 6, wherein NylC is a protein represented by the following (1), (2), or (3): (1) A protein having the sequence of positions 1 to 333 of SEQ ID NO: 2; (2) A protein having NylC activity, comprising an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the polypeptide described in (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; (3) A protein having NylC activity, comprising a sequence having 70% or more sequence identity with the polypeptide described in (1), wherein 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] A method for producing a degradation product of nylon 66 according to any one of items 1 to 7, wherein NylC is the protein (1'), (2'), or (3') below: (1') Proteins having sequences 1-333 of sequence number 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 (2')(1'); A protein having NylC activity, consisting of a sequence with 90% or more sequence identity with the polypeptide described in (3')(1'). [9] A method for producing a degradation product of nylon 66 according to any one of items 1 to 8, wherein NylC is a protein in which at least one substitution is made from amino acids selected from those corresponding to M50I, R52Y, D99A, G111A, L139R, D304A, L331D, and L331E of SEQ ID NO: 1.

[10] A method for producing a degradation product of nylon 66 according to any one of items 1 to 9, wherein NylC is a protein in which amino acids corresponding to G111A and D304A of SEQ ID NO: 1 have been substituted.

[11] A method for producing a decomposition product of nylon 66 according to any one of items 1 to 10, comprising the step of contacting nylon 66 with high-temperature water to obtain low-molecular-weight nylon 66, and reacting the low-molecular-weight nylon 66 with at least one of NylB and NylC.

[12] An enzyme preparation for degrading nylon 66, comprising NylC as defined in any one of items 1 to 10.

[13] A method for chemically recycling nylon 66, comprising all steps of the manufacturing method described in any one of items 1 to 11. [Effects of the Invention]

[0014] One embodiment of the present invention provides a method for producing degradation products of nylon 66 using a modified NylC. Furthermore, one embodiment of the present invention provides a novel application for the modified NylC: the production of degradation products of nylon 66. One embodiment of the present invention provides a method for decomposing nylon 66 using a modified NylC that has a high decomposition rate (monomerization rate) of nylon 66 and is applicable to chemical recycling methods for nylon 66. [Brief explanation of the drawing]

[0015] [Figure 1] Comparison of various properties of the created cpGYAQ protein - 1. The cpGYAQ protein showed a Tm value 2°C higher than p2-GYAQ. [Figure 2] Comparison of various properties of the created cpGYAQ protein - Part 2. The cpGYAQ protein showed substrate recognition equivalent to that of p2-GYAQ. [Figure 3] Comparison of various properties of the created cpGYAQ protein - Part 3. The cpGYAQ protein showed enzymatic activity equivalent to that of p2-GYAQ against Aco (Ahx cyclic oligomer). [Figure 4]The amino acid sequence of NylC-GYAQ (SEQ ID NO: 1), the amino acid sequence of cp-modified NylC obtained in the examples of this specification (SEQ ID NO: 2), and the amino acid sequence of NylB-DNY (SEQ ID NO: 5). In the sequence of SEQ ID NO: 1, □ represents M50, R52, D99, G111, L139, D304, and L331 respectively; underlined represents A36, G122, Y130, and Q263 respectively; and shaded represents F134, L137, Y146, K189, N219, N266, D306, and D308 respectively. Italics represent T267. A wavy underline indicates the β-chain portion. In sequence number 2, the squares represent D38, L65, M121, R123, D170, G182, and L210 respectively; the underlines represent A107, G193, and Y201 respectively; and the shaded areas represent D40, D42, F206, L208, Y217, K260, and N290 respectively. Italics indicate T1. Wavy underlines indicate the β-chain portion. [Modes for carrying out the invention]

[0016] The following abbreviations may be used in this specification, the claims, and the drawings. 66MU: Monomer unit (see below) [ka] AD: Adipic acid HMD: Hexamethylenediamine (1,6-diaminohexane) Ahx: 6-aminohexanoic acid (sometimes called ε-aminocaproic acid). ALD: 6-aminohexanoate linear dimer Aco: 6-aminohexanoic acid cyclic oligomer (Ahx cyclic oligomer) NOM: Nylon oligomer mixture NylB: 6-aminohexanoate dimer hydrolase NylC: 6-aminohexanoate oligomer endo-hydrolase p2-NylC: NylC derived from Arthrobacter sp. KI72. When simply referred to as NylC, this is what is meant unless otherwise specified. Hyb-24: A variant of the NylB and NylB' hybrid enzyme with several amino acid substitutions. Sometimes simply referred to as DNY. NylC-GYAQ: A quadruple mutant in which p2-NylC is substituted with 122G, 130Y, 36A, and 263Q. It is sometimes simply called GYAQ.

[0017] In this specification, claims, and drawings, unless otherwise specified, amino acids are denoted by a single letter of the alphabet. Specifically, A represents alanine, L represents leucine, R represents arginine, K represents lysine, N represents asparagine, M represents methionine, D represents aspartic acid, F represents phenylalanine, C represents cysteine, P represents proline, Q represents glutamine, S represents serine, E represents glutamic acid, T represents threonine, G represents glycine, W represents tryptophan, H represents histidine, Y represents tyrosine, I represents isoleucine, and V represents valine.

[0018] Furthermore, in the case of proteins or enzymes, when an amino acid substitution is represented by a variant consisting of a single letter of the alphabet, a number, and another letter of the alphabet following the number, the leftmost letter indicates the amino acid before the mutation, the middle 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 replaced by the rightmost amino acid. For example, M50I indicates that methionine at position 50 in the amino acid sequence has been replaced with isoleucine. Note that in this invention, the term "amino acid" may sometimes be used to mean "amino acid residue," but this will be clear to those skilled in the art.

[0019] In relation to proteins or enzymes, specific amino acids in their amino acid sequence may be represented by a single letter of the alphabet and a number. For example, M50 refers to the 50th amino acid, M, in the amino acid sequence. Similarly, 50I indicates that the 50th amino acid in the sequence has been substituted with I. The original amino acid before the substitution is irrelevant.

[0020] I. Method for producing degradation products of nylon 66 (Embodiment 1) This embodiment relates to a method for decomposing nylon 66 using enzymes.

[0021] More specifically, this embodiment provides a method for producing a breakdown product of nylon 66, comprising the following steps:

[0022] Nylon 66 is treated with 6-aminohexanoic acid oligomer end hydrolase (NylC).

[0023] (Enzyme reaction) In this process, an enzyme having nylon 66-degrading activity is added to nylon 66 to hydrolyze the nylon. The nylon 66 may optionally include nylon 66 that has been degraded by pretreatment as described later. The nylon hydrolysates may be 66MU, AD, and HMD.

[0024] In this embodiment, NylC is used.

[0025] The temperature of this process can be adjusted as appropriate depending on the enzyme used. For example, the temperature can be 20-80°C, 25-70°C, or 30-60°C.

[0026] The enzyme concentration is not particularly limited as long as the target monomerization rate is achieved. However, when using multiple enzymes, the concentration of each enzyme can be, for example, 0.001 to 10 mg / mL, or 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.

[0027] The time required for this process is not particularly limited, as long as the target monomerization rate is achieved, but 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.

[0028] (enzyme) Regarding nylon, three enzymes of different decomposition modes derived from microorganisms are known: NylA (6-aminohexanoate-cyclic dimer hydrolase, EC 3.5.2.12), NylB (EC 3.5.1.46), and NylC (EC 3.5.1.117). In the method for producing the degradation products of nylon 66 of this embodiment, these can be used individually or in combination. In one embodiment, NylC and NylB are used in combination.

[0029] NylB is generally known to be involved in the degradation of nylon-6 oligomers. It degrades linear oligomers of 6-aminohexanoic acid with a degree of polymerization of 2 to 20 by exo-type cleavage, sequentially removing residues from the N-terminus.

[0030] With respect to nylon 66, NylB can decompose linear nylon-66 oligomers and 1,6-diaminohexyl-adipyl through exo-type cleavage, producing AD and HMD.

[0031] In one embodiment, the enzyme used may be NylB-DNY (SEQ ID NO: 5) or its homolog. Such a mutant enzyme is one of the proteins (10), (11), and (12) below: (10) A protein consisting of the amino acid sequence of Sequence ID No. 5; (11) A protein having a sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 5, provided that the amino acids corresponding to G180, H265, and D369 of SEQ ID NO: 5 are maintained, and which has NylB activity; (12) A protein having a sequence that is 70% or more identical to the amino acid sequence of SEQ ID NO: 5, provided that the amino acids corresponding to G180, H265, and D369 of SEQ ID NO: 5 are maintained, and which also has NylB activity.

[0032] NylC is generally known to be involved in the decomposition of nylon-6 oligomers. It decomposes linear or cyclic oligomers of poly(6-aminohexanoyl) with a degree of polymerization of 3 or higher by endo-type cleavage, usually yielding oligomers with a length of 2 or more.

[0033] With respect to nylon 66, NylC can decompose linear or cyclic oligomers of poly(1,6-diaminohexyl-adipyl) with a degree of polymerization of 2 or more by endo-type cleavage, potentially producing 1,6-diaminohexyl-adipyl and its oligomers, as well as AD and HMD.

[0034] In one embodiment, the enzyme used may be NylC-GYAQ (SEQ ID NO: 1) or its homolog. Such a mutant enzyme is one of the proteins (7), (8), and (9) below: (7) A protein consisting of the amino acid sequence of Sequence ID No. 1; (8) A protein having a sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 1, provided that the amino acids corresponding to G122, Y130, A36, and Q263 of SEQ ID NO: 1 are maintained, and which has NylC activity; (9) A protein having a sequence that is 70% or more identical to the amino acid sequence of SEQ ID NO: 1, provided that the amino acids corresponding to G122, Y130, A36, and Q263 of SEQ ID NO: 1 are maintained, and which also has NylC activity.

[0035] (mutant enzyme) In one embodiment, the enzyme used is more active or has better heat resistance compared to the original NylC-GYAQ (SEQ ID NO: 1). Such a mutant enzyme is one of the proteins (4), (5), and (6) below: (4) A protein having the amino acid sequence of sequence number 1 to 355, wherein at least one amino acid selected from M50, R52, D99, G111, L139, D304, and L331 is substituted; (5)(4) The amino acid sequence of the protein described above, wherein one or more amino acids are deleted, substituted, or added, and the sequence consists of amino acids that satisfy (i) and (ii), and the protein has NylC activity; (i) 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 these substitutions are retained. (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 a sequence identity of 70% or more with the protein described in (4), provided that it consists of an amino acid sequence satisfying (iii) and (iv), and having NylC activity; (iii) 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 these substitutions are retained. (iv) The amino acid corresponding to T267 may be maintained or substituted with S or C.

[0036] 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. Furthermore, it is important that amino acid 267, the nucleophilic residue of this enzyme, is T (although S or C may also be acceptable, albeit with slightly reduced activity) (Non-Patent Literature 4). In SEQ ID NO: 1, G122, Y130, A36, and Q263 are NylC derived from Arthrobacter sp. KI72. p2 This corresponds to four substitutions from D122G, H130Y, D36A, and E263Q.

[0037] The term "corresponding amino acid" refers to the amino acid at the position corresponding to position 122 in the sequence of Sequence ID No. 1, for example, position 122 in the reference Sequence ID No. 1 sequence. However, if one or more amino acids are deleted, substituted, or added to the Sequence ID No. 1 sequence, the position may shift and it may no longer be position 122.

[0038] "The at least one substitution" refers to all of the "at least one mutation selected from M50I, R52Y, D99A, G111A, L139R, D304A, L331D, and L331E" present in the sequence of sequence number 1 of reference (4). For example, if the protein of (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. If the protein of (4) has both 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 "at least two mutations," it means all mutations present in the sequence of sequence number 1 of reference (4), such as "at least two mutations selected from M50I, R52Y, D99A, G111A, L139R, D304A, L331D, and L331E." The same applies to other items such as (6).

[0039] Those skilled in the art can appropriately identify the corresponding amino acids and their corresponding positions by aligning the two sequences in an optimal manner.

[0040] With respect to M50, R52, D99, G111, L139, D304, and L331, the substituted amino acids are not particularly limited as long as they have the desired activity.

[0041] Specifically, M50, that is, the amino acid corresponding to methionine (M) at position 50 in the sequence of Sequence ID No. 1, may be substituted with isoleucine (I), or an amino acid with similar properties to isoleucine, or it may be substituted with an amino acid with different properties from isoleucine.

[0042] In one embodiment, the amino acid corresponding to methionine at position 50 of the sequence of Sequence ID No. 1 is replaced with isoleucine or an amino acid with similar properties to isoleucine. Examples of amino acids with similar properties to isoleucine include the following: Hydrophobic amino acids: alanine, valine, glycine, leucine, phenylalanine, proline, tryptophan, tyrosine; The neutral amino acids are alanine, asparagine, cysteine, glutamine, glycine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Valine and leucine are branched-chain amino acids.

[0043] R52, that is, the amino acid corresponding to arginine (R) at position 52 in the sequence of Sequence ID No. 1, may be substituted with tyrosine (Y), or an amino acid with similar properties to tyrosine, or it may be substituted with an amino acid with different properties from tyrosine.

[0044] In one embodiment, the amino acid corresponding to arginine at position 52 of sequence number 1 is replaced with tyrosine or an amino acid with properties similar to tyrosine. Examples of amino acids with properties similar to tyrosine include the following: Hydrophobic amino acids: alanine, valine, glycine, isoleucine, leucine, phenylalanine, proline, tryptophan; The neutral amino acids are alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, and valine. Tryptophan and phenylalanine are aromatic ring amino acids.

[0045] D99, that is, the amino acid corresponding to aspartic acid (D) at position 99 in the sequence of Sequence ID No. 1, may be substituted with alanine (A), or an amino acid similar in properties to alanine, or it may be substituted with an amino acid different in properties from alanine.

[0046] In one embodiment, the amino acid corresponding to aspartic acid at position 99 of the sequence of Sequence ID No. 1 is replaced with alanine or an amino acid with properties similar to alanine. Examples of amino acids with properties similar to alanine include the following: Hydrophobic amino acids: valine, glycine, isoleucine, leucine, phenylalanine, proline, tryptophan, tyrosine; The neutral amino acids are asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0047] The amino acid G111, that is, the glycine (G) at position 111 in the sequence of Sequence ID No. 1, may be substituted with alanine, an amino acid similar in properties to alanine, or an amino acid different in properties from alanine.

[0048] In one embodiment, the amino acid corresponding to glycine at position 111 of the sequence of Sequence ID No. 1 is replaced with alanine or an amino acid with properties similar to alanine. Examples of amino acids with properties similar to alanine include the following: Hydrophobic amino acids: valine, isoleucine, leucine, phenylalanine, proline, tryptophan, tyrosine; The neutral amino acids are asparagine, cysteine, glutamine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0049] L139, that is, the amino acid corresponding to leucine (L) at position 139 of the sequence of Sequence ID No. 1, may be substituted with arginine (R), or an amino acid with similar properties to arginine, or it may be substituted with an amino acid with different properties from arginine.

[0050] In one embodiment, the amino acid corresponding to leucine at position 139 of the sequence of Sequence ID No. 1 is replaced with arginine or an amino acid with similar properties to arginine. Examples of amino acids with similar properties to arginine include the following: Nonhydrophobic amino acids: asparagine, aspartic acid, glutamic acid, glutamine, lysine, serine, threonine, cysteine, histidine; Hydrophilic amino acids: asparagine, aspartic acid, glutamic acid, glutamine, lysine, serine, threonine; Lysine and histidine are basic amino acids.

[0051] D304, that is, the amino acid corresponding to aspartic acid (D) at position 304 in the sequence of Sequence ID No. 1, may be substituted with alanine (A), or an amino acid similar in properties to alanine, or it may be substituted with an amino acid different in properties from alanine.

[0052] In one embodiment, the amino acid corresponding to aspartic acid at position 304 of the sequence of Sequence ID No. 1 is replaced with alanine or an amino acid with properties similar to alanine. Examples of amino acids with properties similar to alanine include the following: Hydrophobic amino acids: valine, glycine, isoleucine, leucine, phenylalanine, proline, tryptophan, tyrosine; The neutral amino acids are asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0053] L331, that is, the amino acid corresponding to leucine (L) at position 331 in the sequence of Sequence ID No. 1, may be substituted with aspartic acid (D), or an amino acid with similar properties to aspartic acid, or it may be substituted with an amino acid with different properties from aspartic acid.

[0054] In one embodiment, the amino acid corresponding to leucine at position 331 of the sequence of Sequence 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 the following: Nonhydrophobic amino acids include arginine, asparagine, glutamic acid, glutamine, lysine, serine, threonine, cysteine, and histidine; Hydrophilic amino acids: arginine, asparagine, glutamic acid, glutamine, lysine, serine, threonine; Glutamic acid is an acidic amino acid.

[0055] Furthermore, the amino acid corresponding to leucine (L) at position 331 of sequence number 1 may be substituted with glutamic acid (E), or an amino acid with similar properties to glutamic acid, or it may be substituted with an amino acid with different properties from glutamic acid.

[0056] In one embodiment, the amino acid corresponding to leucine at position 331 of the sequence of Sequence ID No. 1 is replaced with glutamic acid or an amino acid with similar properties to glutamic acid. Examples of amino acids with similar properties to glutamic acid include the following: Nonhydrophobic amino acids: arginine, aspartic acid, asparagine, glutamine, lysine, serine, threonine, cysteine, histidine; Hydrophilic amino acids: arginine, aspartic acid, asparagine, glutamine, lysine, serine, threonine; Aspartic acid is an acidic amino acid.

[0057] In one embodiment, the mutant enzyme is the following protein (4'), (5'), or (6'): (4') A protein having the amino acid sequence of positions 1 to 355 of sequence number 1, with at least one of the following substitutions: M50I, R52Y, D99A, G111A, L139R, D304A, L331D, and L331E; A protein having NylC activity, comprising a sequence in which 1 to 36 amino acids are deleted, substituted, or added in the amino acid sequence of the protein described in (5')(4'); A protein having a sequence identity of 70% or more with the protein described in (6')(4'), and possessing NylC activity.

[0058] In one embodiment, the mutations in the mutant enzyme having further mutations in NylC-GYAQ are such that the protein has at least two mutations selected from M50I, R52Y, D99A, G111A, L139R, D304A, L331D, and L331E, or is sequence-identical to such a protein. That is, it is one of the following: A protein consisting of amino acid sequences 1-355 of sequence number (4") of sequence number 1, having at least two mutations selected from M50I, R52Y, D99A, G111A, L139R, D304A, L331D, and L331E; A protein having 6-aminohexanoic acid oligomer endo-type hydrolase (NylC) activity, wherein the amino acid sequence of the protein described in (5) and (4) is characterized by the deletion, substitution, or addition of one or more amino acids, provided that the amino acids corresponding to 122G, 130Y, 36A, and 263Q of SEQ ID NO: 1, and at least two of the amino acids corresponding to these mutations, are maintained; A protein having a sequence identity of 90% or more with the protein described in (6) and (4), wherein the amino acids corresponding to 122G, 130Y, 36A, and 263Q of SEQ ID NO: 1, and the amino acids corresponding to at least two of these mutations, are maintained in the amino acid sequence, and the protein has NylC activity.

[0059] In one embodiment, the mutant enzyme mutations having further mutations in NylC-GYAQ are proteins having at least two mutations selected from R52Y and D304A, R52Y and D99A, D99A and D304A, and G111A and D304A. That is, any of the following: (4') A protein consisting of amino acid sequences 1-355 of sequence number 1, having at least two mutations selected from R52Y and D304A, R52Y and D99A, D99A and D304A, and G111A and D304A; A protein having 6-aminohexanoic acid oligomer endo-type hydrolase (NylC) activity, wherein the amino acid sequence of the protein described in (5')"(4')) is characterized by the deletion, substitution, or addition of one or more amino acids, provided that the amino acids corresponding to 122G, 130Y, 36A, and 263Q of SEQ ID NO: 1, and at least two of the amino acids corresponding to these mutations, are maintained; A protein having a sequence identity of 90% or more with the protein described in (6')"(4'"), wherein the amino acids corresponding to 122G, 130Y, 36A, and 263Q of SEQ ID NO: 1, and the amino acids corresponding to at least two of these mutations, are maintained in the amino acid sequence, and the protein has NylC activity.

[0060] In this embodiment, when referring to NylC activity, unless otherwise specified, it refers to the activity of degrading cyclic or linear oligomers of Ahx with a degree of polymerization of 3 or higher in the endo form.

[0061] This embodiment also provides a polynucleotide encoding a mutant enzyme having further mutations in NylC-GYAQ. That is, it provides a polynucleotide encoding one of the proteins described above: (4), (5), (6), (4'), (5'), (6'), (4"), (5"), (6"), (4'"), (5'"), (6'). Such a polynucleotide can be incorporated into a suitable vector to express the desired enzyme in E. coli or the like.

[0062] In relation to the present invention, when referring to the identity of an amino acid sequence, unless otherwise specified, it refers to the 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 95% or more, even more preferably 97.5% or more, and even more preferably 99% or more of the sequence.

[0063] In relation to this invention, when referring to "identity" of an amino acid sequence, unless otherwise specified, it is defined as the value measured by BLAST from NCBI (http: / / www.ncbi.nlm.nih.gov / ). When comparing amino acid sequences with BLAST, Blastp can be used as the default setting for the Algorithm. The measurement results are quantified as Positives or Identities.

[0064] In the description of the present invention, when referring to a protein or amino acid sequence as "a sequence in which one or more amino acids are deleted, substituted, or added," the number of amino acids that are substituted, etc. is not particularly limited for any protein, as long as the protein consisting of that amino acid sequence has the desired function, unless otherwise specified. However, it is generally around 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, or 1-4, or even more substitutions, etc., are possible if the substitutions are with amino acids of similar properties. Means for preparing proteins relating to such amino acid sequences are well known to those skilled in the art.

[0065] Similar amino acids refer to amino acids with similar physical properties such as hydroxyl, charge, pKa, and solubility. Examples include the following: Hydrophobic amino acids: alanine, valine, glycine, isoleucine, leucine, phenylalanine, proline, tryptophan, tyrosine Nonhydrophobic 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 amino acids: tyrosine, tryptophan, phenylalanine; Branched amino acids: valine, leucine, isoleucine.

[0066] (cp-transferase) In the manufacturing method of this embodiment, NylC modified by circular permutation (cp-modification) may be used. Originally, NylC is expressed as an inactive precursor (36 kDa).

[0067] [ka]

[0068] The precursor is autocatalytically cleaved at Asn266 / Thr267, separating into a 27kDa α-chain and a 9kDa β-chain.

[0069] [ka]

[0070] Furthermore, four heterodimer structures, each composed of α and β chains, assemble to form a donut-shaped three-dimensional structure, which then exhibits enzyme activity.

[0071] [ka]

[0072] Conventional NylC requires a 24-hour incubation process at 37°C after enzyme purification to fully enable autocleavage. Furthermore, some amino acid substitutions may not undergo autocleavage, making it impossible to confirm the amino acid substitution effect.

[0073] In one embodiment, the NylC used is cp-modified, having a structure in which the N-terminus of the α-chain polypeptide and the C-terminus of the β-chain polypeptide are linked. Such a cp-modified enzyme does not require processing to promote self-cleavage and, unexpectedly, may have a higher Tm value than the original enzyme.

[0074] In one embodiment, the NylC used is specifically a protein having a structure in which the N-terminus of an α-chain, i.e., a polypeptide which is (a1), (a2), or (a3) ​​below, is linked to the C-terminus of a β-chain, i.e., a polypeptide which is (b1), (b2), or (b3) below. (a1) A polypeptide having sequences 19-260 of sequence number 1; (a2) A polypeptide comprising an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the polypeptide described in (a1), provided 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 an α-subunit of NylC; (a3) A polypeptide consisting of an amino acid sequence having 70% or more sequence identity with the polypeptide described in (a1), provided that the amino acids corresponding to F134, L137, Y146, K189, and N219 of SEQ ID NO: 1 are maintained, and which is capable of functioning as the α-subunit of NylC; (b1) A polypeptide having sequences 267-355 of sequence number 1; (b2) A polypeptide comprising an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the polypeptide described in (b1), provided that the 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 which is capable of functioning as a β-subunit of NylC; A polypeptide comprising an amino acid sequence having 70% or more sequence identity with the polypeptide described in (b3)(b1), wherein the 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 which is capable of functioning as a β-subunit of NylC.

[0075] When we say that the N-terminus of one polypeptide and the C-terminus of another polypeptide are "linked," this includes cases where the N-terminus is directly linked to the C-terminus, and cases where the C-terminus is linked via 1 to 10 amino acids (spacers).

[0076] To be functional as an α-subunit or a β-subunit means that it can exert the desired enzyme activity when combined with the other subunit.

[0077] 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 sequences 19-260 of sequence number 1; A polypeptide comprising a sequence in which 1 to 24 amino acids are deleted, substituted, or added, as described in (a2')(a1'), and capable of functioning as the α-subunit of NylC; A polypeptide consisting of a sequence with 90% or more sequence identity with the polypeptides described in (a3')(a1'), and capable of functioning as the α-subunit of NylC; (b1') A polypeptide having sequences 267-355 of sequence number 1; A polypeptide comprising a sequence in which 1 to 9 amino acids are deleted, substituted, or added, as described in (b2')(b1'), and capable of functioning as a β-subunit of NylC; A polypeptide comprising a sequence with 90% or more sequence identity with the polypeptides described in (b3')(b1'), and capable of functioning as a β-subunit of NylC.

[0078] In one embodiment, the cp-modified enzyme is the protein (1), (2), or (3) below: (1) Proteins having sequences 1-333 of sequence number 2; (2) A protein having NylC activity, comprising an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the polypeptide described in (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; (3) A protein having NylC activity, comprising a sequence having 70% or more sequence identity with the polypeptide described in (1), wherein 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.

[0079] In one embodiment, the cp-modified enzyme is the protein (1'), (2'), or (3') below: (1') Proteins having sequences 1-333 of sequence number 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 (2')(1'); A protein having NylC activity, consisting of a sequence with 90% or more sequence identity with the polypeptide described in (3')(1').

[0080] The sequence listing shows the amino acid sequence of cp-modified NylC obtained in the examples described herein as Sequence ID No. 2. In Sequence 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 (Sequence ID No. 1). Also, T1 (which may be S or C, although its activity may be slightly reduced) corresponds to amino acid 267, which is a nucleophilic residue in NylC-GYAQ (Sequence ID No. 1). A107, G193, and Y201 in Sequence ID No. 2 correspond to A36, G122, and Y130 in NylC-GYAQ (Sequence ID No. 1). D38, L65, M121, R123, D170, G182, and L210 in Sequence ID No. 2 correspond, in order, to D304, L331, M50, R52, D99, G111, and L139 in NylC-GYAQ (Sequence ID No. 1). Portions 1 through 89 of Sequence ID No. 2 are the β-chain.

[0081] In one embodiment, such cpized NylC may further have mutations corresponding to at least one mutation selected from the amino acids corresponding to M50, R52, D99, G111, L139, D304, L331, and L331 of SEQ ID NO: 1, as shown in Embodiment 1 above.

[0082] In one embodiment, such cpized NylC has at least two mutations selected from amino acids corresponding to M50I, R52Y, D99A, G111A, L139R, D304A, L331D, and L331E of SEQ ID NO: 1.

[0083] (Pre-processing) In one embodiment, nylon 66 is pretreated before being treated with enzymes. Examples include, but are not limited to, grinding, washing, removal of foreign matter, particle formation, and demolecularization. These treatments may also be combined. In one embodiment, the pretreatment involves hydrolyzing nylon 66 in high-temperature water to obtain low-molecular-weight nylon 66 (see Patent Document 1). This pretreatment will be described below.

[0084] In the pretreatment process, hydrolysis reduces the molecular weight of nylon 66, loosening the fixation of oligomers via hydrogen bonds and generating water-soluble nylon 66. Therefore, the reduced molecular weight nylon 66 contains water-soluble nylon 66. Here, "water-soluble nylon 66" refers to nylon 66 that is generated by the reduction of the molecular weight of nylon 66 and dissolves in water at 20°C.

[0085] Hydrolysis in the pretreatment step is also referred to herein as "high-temperature hydrolysis."

[0086] The low-molecular-weight nylon 66 further includes, in addition to the water-soluble nylon 66 described above, nylon 66 that is insoluble in water at 20°C, produced by the low molecular weight reduction of nylon 66. In this specification, nylon 66 that is insoluble in water at 20°C is also referred to as "water-insoluble nylon 66". The number-average molecular weight of the water-insoluble nylon 66 in the low-molecular-weight nylon 66 is not particularly limited, but is, for example, 10,000 or less, preferably 7,000 or less, more preferably 5,000 or less, even more preferably 4,000 or less, and even more preferably 3,000 or less. If the molecular weight of water-insoluble nylon 66 is high and its water solubility is low, it is less susceptible to hydrolysis by enzymes, but by lowering the average molecular weight, the reactivity with enzymes in the subsequent second hydrolysis step is improved.

[0087] The content of water-insoluble nylon 66 in the low-molecular-weight nylon 66 is not particularly limited, but may be 1 to 95% by weight, 10 to 90% by weight, or 50 to 85% by weight. In particular, when the content of water-insoluble nylon 66 is 10% by weight or more, the number-average molecular weight of water-soluble nylon 66 is reduced relative to the raw material nylon 66, and the subsequent second hydrolysis tends to proceed more favorably.

[0088] The method for measuring the molecular weight of water-insoluble nylon 66 is not particularly limited, but it can be measured by GPC, for example. Specifically, the method described in the examples below can be used.

[0089] In this specification, "high-temperature water" means water with a temperature of 200°C or higher. High-temperature water includes subcritical water and supercritical water. The water during the reaction is either liquid or in a supercritical state.

[0090] Supercritical water refers to water in a supercritical state. The supercritical state of water is defined as a state where the critical temperature (374°C) and critical pressure (22.1 MPa) of water have been exceeded. Furthermore, the critical point is also included in the definition of supercritical state in this specification.

[0091] Subcritical water is water in a subcritical state. The subcritical state of water is a state in which the temperature is below the critical temperature of water and the pressure is above the saturated vapor pressure. Therefore, the reaction pressure in the pretreatment process is above the saturated vapor pressure when the reaction temperature is below the critical temperature of water, and above the critical pressure when the reaction temperature is above the critical temperature of water.

[0092] The reaction temperature in the pretreatment step is the temperature of the high-temperature water and is not particularly limited, but is preferably 200°C to 400°C, more preferably 230°C to 350°C, and even more preferably 230°C to 300°C. If the reaction temperature is too low, the hydrolysis reaction of nylon 66 tends to be slow. If the reaction temperature exceeds 400°C, the decomposition reaction proceeds significantly, and the yield of monomer tends to decrease.

[0093] In one preferred embodiment, the high-temperature water in the pretreatment step is subcritical water. That is, in this embodiment, the temperature of the high-temperature water in the pretreatment step is below the critical temperature of water. In this case, the reaction pressure should be equal to or greater than the saturated vapor pressure at each reaction temperature. There is no particular upper limit to the reaction pressure, but it is preferably saturated vapor pressure + 20 MPa or less, more preferably saturated vapor pressure + 15 MPa or less, even more preferably saturated vapor pressure + 5 MPa or less, and most preferably saturated vapor pressure. From the viewpoint of selecting the apparatus for carrying out the reaction, it is preferable that the reaction pressure is not too high compared to the saturated vapor pressure.

[0094] The reaction pressure can be set to the saturated vapor pressure by heating in a sealed container, or it can be pressurized by injecting an inert gas into a sealed container. Pressurization with an inert gas can be done before or after heating.

[0095] If the reaction temperature in the pretreatment step is above the critical temperature of water, the reaction pressure should be above the critical pressure. There is no particular upper limit to the reaction pressure, but it is preferably below the critical pressure + 15 MPa, more preferably below the critical pressure + 5 MPa, and most preferably at the critical pressure. From the viewpoint of selecting the apparatus for carrying out the reaction, it is preferable that the reaction pressure is not too high compared to the critical pressure.

[0096] Furthermore, if the temperature of the high-temperature water in the pretreatment process exceeds the critical temperature, the corrosiveness will increase significantly, potentially leading to metal contamination from the equipment and deterioration of the equipment due to corrosion.

[0097] While there are no particular limitations on stirring during the reaction, stirring is preferred. Stirring renews the surface of the nylon 66 solid, ensuring the reaction proceeds uniformly and improving the reaction rate.

[0098] There are no particular restrictions on the shape of the reactor; any shape, such as a tank type or a circulating type, may be used.

[0099] The reaction time is not particularly limited, but is preferably 5 to 300 minutes, more preferably 5 to 100 minutes, and even more preferably 5 to 30 minutes. The reaction time should be measured from the time the desired reaction conditions are reached until heating is stopped. If the reaction time is too short, many high molecular weight components will not react with the enzyme, and if it is too long, the decomposition reaction will proceed and the yield will decrease significantly.

[0100] The concentration of nylon 66 in water (high-temperature water) is not particularly limited, but it is preferably 1 to 50% by weight, more preferably 5 to 40% by weight, and most preferably 7 to 30% by weight. If the concentration is too low, recovery becomes very difficult, and if the concentration is too high, the fluidity of the reaction solution is low, making it difficult to achieve a uniform reaction.

[0101] To improve the reaction efficiency of the pretreatment process in high-temperature water, nylon 66 can also be pretreated.

[0102] For example, the nylon 66 can be atomized to ensure a uniform reaction in the pretreatment process. The method of atomization is not particularly limited, but examples include methods using methods such as recrystallization, reprecipitation, and spray drying after dissolving nylon 66 in a solvent, as well as atomization by pulverization such as freeze-drying. The method of dissolving the nylon 66 first and then atomizing it is preferred because it allows for uniform atomization regardless of the shape of the nylon 66 used as a raw material.

[0103] To improve the reactivity after the reaction starts, the nylon 66 can be thoroughly impregnated with water beforehand. The impregnation method is not particularly limited and includes methods such as immersing the nylon 66 in water and heating it, exposing the nylon 66 to a humid atmosphere, and dissolving the nylon 66 in a solvent and then adding water to precipitate it.

[0104] To ensure a uniform reaction start at the beginning of the reaction, the catalyst can be pre-mixed. The catalyst to be pre-mixed is not particularly limited, but examples include acidic compounds, basic compounds, and inorganic salts. Inorganic salts are preferred as the catalyst to be pre-mixed.

[0105] The method of mixing the catalysts is not particularly limited, and examples include immersing nylon 66 in a solvent in which the catalysts to be mixed are dissolved, melt-kneading the catalysts to be mixed with nylon 66, and dissolving the catalysts to be mixed with nylon 66 in a solvent, mixing them, and then precipitating the nylon 66.

[0106] While there are no particular limitations on the pH of the hydrolysis reaction solution at the end of the pretreatment step, if the reaction is started with a concentration of nylon 66 (initial nylon 66 concentration) of 10% by weight per 100% by weight of water (high-temperature water), it is preferable to stop the reaction at pH 7.0 to 10.0, more preferably at pH 8.0 to 10.0, and even more preferably at pH 9.0 to 9.5. If the pH is 10.0 or lower, enzyme inactivation can be prevented, and the subsequent second hydrolysis tends to proceed well. The progress of the reaction can be confirmed by measuring the pH of the hydrolysis reaction solution.

[0107] Furthermore, if the concentration of nylon 66 used in the preparation is other than 10% by weight, the progress of the reaction can be similarly confirmed and adjusted by, for example, concentrating or diluting a sample taken from the reaction solution for pH measurement at a concentration or dilution ratio that results in a nylon 66 concentration of 10% by weight, and then measuring the pH.

[0108] The method of concentration and pH measurement are not particularly limited, and general methods can be used. Specifically, the methods described in the examples below are examples.

[0109] The solution after the reaction may be post-treated as needed, and the methods used for post-treatment are not particularly limited.

[0110] The pH after the pretreatment step may be adjusted by adding an acidic or basic compound to a range suitable for the subsequent second enzymatic hydrolysis step, and / or the pH fluctuation may be suppressed by a buffer solution.

[0111] Furthermore, the concentrations of nylon 66, hexamethylenediamine, and water-soluble nylon 66 may be concentrated or diluted with water as necessary to a level suitable for carrying out the subsequent second hydrolysis step.

[0112] The solid component (water-insoluble nylon 66) precipitated by the pretreatment step may be subjected to solid-liquid separation or directly used in the next step. When solid-liquid separation is performed, only the soluble component is hydrolyzed by enzymes, resulting in a faster reaction completion. On the other hand, if separation is not performed, the hydrolysis of the low-molecular-weight solid polymer by enzymes also proceeds, resulting in a higher yield of hexamethylenediamine.

[0113] In one embodiment, the degree of molecular weight reduction of nylon 66 can be appropriate, but for example, it can be carried out so that the monomerization rate (sometimes called the decomposition rate), expressed 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 nearly 100%.

[0114]

number

[0115] If the monomerization rate is 50% or higher, nylon 66 becomes water-soluble, and subsequent enzymatic reactions can achieve monomerization with nearly 100% yield.

[0116] In one embodiment, the pH is adjusted to 6-8, preferably 6.8-7.8, more preferably 7.1-7.5, for example, 7.3.

[0117] Furthermore, the solution containing the low-molecular-weight nylon 66 is adjusted to a concentration that allows the enzyme used in the subsequent enzymatic reaction step to exert sufficient activity. This adjustment is preferably carried out using a phosphate buffer with added glycerol, more specifically, Buffer A (10% Glycerol containing 20 mM phosphate buffer, pH 7.3).

[0118] In one embodiment, the solution containing the low molecular weight nylon 66 is adjusted to 1 to 50 mg / mL, preferably 3 to 25 mg / mL, more preferably 5 to 15 mg / mL, for example, 10 mg / mL.

[0119] The manufacturing method of this embodiment is expected to be applicable not only to nylon 66 but also to various other nylons composed of diamines and dicarboxylic acids. Examples of such nylons include, but are not limited to, nylon 610, nylon 6T, nylon 6I, nylon 9T, and nylon M5T.

[0120] II. Other Embodiments This application also provides, as another embodiment, polynucleotides encoding the above-described mutant enzymes and cp-transferases. Such polynucleotides can be incorporated into a suitable vector to express the target enzyme in E. coli or other organisms.

[0121] Furthermore, other embodiments provide novel applications for the mutant enzymes and cpases described above, specifically for the degradation of nylon 66 and for the production of degradation products of nylon 66.

[0122] Another embodiment provides a chemical recycling method for nylon 66 that incorporates the manufacturing process described above. [Examples]

[0123] [Manufacturing Example 1: Amino Acid Substitutions of GYAQ] (GYAQ, and the creation of its amino acid substitutions) The preparation of GYAQ and its amino acid substitutions followed the procedure described in the paper (Non-Patent Documents 3 and 4).

[0124] The GYAQ used in the experiment had a His-tag sequence and other additions at its N-terminus, resulting in a total length of 370 aa. The sequence is shown below. The underlined portion is the His-tag sequence, and amino acids from number 16 onwards are the original GYAQ amino acid sequence. In the following experiments, GYAQ refers to this sequence unless otherwise specified.

[0125] >Histag-NylC-GYAQ_370aa_38712.37Da MNHKVHHHHHHIEGRMNTTPVHALTDIDGGIAVDPAPRLAGPPVFGGPGNAAFDLAPVRSTGREMLRFDFPGVSIGAAHYEEGPTGATVIHIPAGARTAVDARGGAVGLSGGYDFNHAICLAGGAGYGLEAGAGVSGALLERLEYRTGFAELQLVSSAVIYDFSARSTAVYPDKALGRAALEFAVPGEFPQGRAGAGMSASAGKVDWDRTEITGQGAAFRRLGDVRILAVVVPNPVGVIVDRAGTVVRGNYDAQTGVRRHPVFDYQEAFAEQVPPVTQAGNTTISAIVTNVRMSPVELNQFAKQVHSSMHRGIQPFHTDMDGDTLFAVTTDEIDLPTTPGSSRGRLSVNATALGAIASEVMWDAVLEAGK* (Sequence ID 3)

[0126] Since the prepared enzyme was obtained as a mixture of cleaved and uncleaved enzymes, cleavage was completed by dialyzing in Buffer A (10% Glycerol containing 20 mM phosphate buffer, pH 7.3) at 4°C for 5 days, followed by incubation at 37°C for 24 hours.

[0127] (Heat resistance evaluation) I followed the method described in the paper (Non-Patent Document 2).

[0128] [Table 1]

[0129] The results are shown in the table above. The values ​​in parentheses represent the heat resistance temperature, which is the indicator of enzyme reaction activity inactivation. Due to the characteristics of the CD equipment, accurate values ​​cannot be calculated for Tm values ​​exceeding 90°C; therefore, NOM was used as the substrate for the D304A, G111A-D304A, and R52Y-D304A mutants. The heat resistance temperature was estimated based on the inactivation of enzyme reaction activity.

[0130] [Manufacturing Example 2: cpGYAQ] (Creating cpGYAQ) An expression vector was created by inserting a DNA sequence (Sequence ID 2), designed to ligate amino acids from positions 19 to 262 after positions 267 to 355 of GYAQ (NylC consisting of the sequence of SEQ ID NO: 1), and then attaching six histidine molecules, after the start codon (ATG) of a Takara pColdIV vector. The protein was expressed in E. coli BL21 according to standard procedures, and the cpGYAQ protein was prepared by purification using a His-tag affinity column. The purified cpGYAQ protein was confirmed to exhibit the desired activity without requiring processing for self-cleavage.

[0131] >cpGYAQ-His_339aa_35601.04 TTISAIVTNVRMSPVELNQFAKQVHSSMHRGIQPFHTDMDGDTLFAVTTDEIDLPTTPGSSRGRLSVNATALGAIASEVMWDAVLEAGKDPAPRLAGPPVFGGPGNAAFDLAPVRSTGREMLRFDFPGVSIGAAHYEEGPTGATVIHIPAGARTAVDARGGAVGLSGGYDFNHAICLAGGAGYGLEAGAGVSGALLERLEYRTGFAELQLVSSAVIYDFSARSTAVYPDKALGRAALEFAVPGEFPQGRAGAGMSASAGKVDWDRTEITGQGAAFRRLGDVRILAVVVPNPVGVIVDRAGTVVRGNYDAQTGVRRHPVFDYQEAFAEQVPPVTHHHHHH(Sequence ID 2)

[0132] [ka]

[0133] By deleting amino acid segments 1-18 and 263-266 of p2-GYAQ, we successfully reduced its size by 22 aa. In reality, considering that p2-GYAQ has an additional 15 aa and cpGYAQ has an additional 6 aa of His-tag and other sequences, cpGYAQ is 31 aa smaller than p2-GYAQ.

[0134] Furthermore, crystal structure analysis of purified cpGYAQ revealed that it formed a donut-shaped tetramer, similar to p2-GYAQ.

[0135] (Heat resistance test: Circular dichroism (CD) spectral analysis) The method described in the paper (Non-Patent Literature 2) was followed. A spectrophotometer (JASCO, Model J-820Q4) was used to measure the circular dichroism (CD) spectrum at the far-ultraviolet wavelength (220 nm). A cuvette with a path length of 1 mm was used for the measurement, and the protein concentration was set to 0.1 mg / mL. The temperature was increased from 25°C to 95°C at a rate of 1°C / min using a JASCO PTC-423L Peltier system. The measured values ​​obtained at each temperature were converted to molar ellipticity, and the Tm value, which is an indicator of the heat resistance temperature, was calculated.

[0136] The calculated Tm values ​​were 85.0°C for p2-GYAQ and 86.9°C for cpGYAQ, with cpGYAQ showing a value approximately 2 degrees higher (Figure 1).

[0137] (substrate specificity) A mixture of cyclic nylon oligomers (NOM), Ald (Ahx linear dimer), and nylon-6 were used as reaction substrates. NOM was prepared according to the method described in the paper (Methods Enzymol., 2021, 648, 357-389), and nylon-6 was micronized or treated with formic acid according to the method described in the prior patent (Patent Document 1). The substrates were added to Buffer A (10% Glycerol containing 20 mM phosphate buffer, pH 7.3) to a final concentration of 5 mg / mL, and the enzyme protein to a final concentration of 0.5 mg / mL, and the mixture was allowed to stand at 37°C. The control was prepared by adding Buffer A instead of the enzyme. After the predetermined time had elapsed, the reaction was stopped by heating the reaction mixture at 99°C for 10 minutes, and the mixture was subjected to TLC, TNBS, and LC-MS analysis as needed.

[0138] Regarding the substrates examined (NOM and nylon-6), cpGYAQ showed reactivity equivalent to that of p2-GYAQ (Figure 2).

[0139] (kinetic analysis) Trimeric nylon oligomers (Aco) of three or more mers were used as the reaction substrate. Preparation followed the method described in the paper (Methods Enzymol., 2021, 648, 357-389). The enzyme protein was prepared to a final concentration of 0.0025 mg / mL, and Aco was added to Buffer A to final concentrations of 0, 0.25, 0.5, 0.75, 1, 1.25, and 2.5 mg / mL, and allowed to stand at 37°C. After 1, 2, and 3 hours, the reaction was stopped by heating the reaction mixture at 99°C for 10 minutes, and the amino group concentration in the system was measured by TNBS analysis. The initial reaction rate 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).

[0140] The kcat / Km values, which indicate catalytic efficiency, were 5.6 for p2-GYAQ and 5.2 for cpGYAQ, confirming that their reactivity to Aco is equivalent (Figure 3 and table below).

[0141] [Table 2]

[0142] [Example 1: Evaluation of 66MU degradation activity]

[0143] [ka]

[0144] Degradation was performed by dissolving the HMD 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 by an o-ABA assay. Specifically, the generation of aminoaldehydes produced by amino group transfer was monitored by measuring the 450 nm absorption change due to complex formation with o-ABA (see paragraph 0166 of WO2022-270597).

[0145] [Table 3]

[0146] D99A, G111A, L139R, D304A, L331D, or E, either individually or in combination, were found to have higher degradation activity than GYAQ (reference).

[0147] [Example 2: Evaluation of Nylon 66 Degradation Activity]

[0148] [ka]

[0149] Nylon 66 was micronized according to the method described in the prior patent (Patent Document 2). The amino group concentration was determined by TNBS using the method described below (quantification of amino groups by colorimetric method). In summary, when the enzyme was reacted with nylon 66, two types of spots, 66MU and HMD, appeared in TLC. Each was quantified, and the two values ​​were added together as 66MU + HMD.

[0150] [Table 4]

[0151] Experimental results showed high activity in compounds with substitutions of R52Y and D304A, or D99A and D304A.

[0152] (Detection of enzyme activity by thin-layer chromatography (TLC)) The supernatant (2 μL) was developed on a silica gel plate (1.05748; Merck Co.), and the sample was identified by developing it on a solvent mixture (1-propanol / water / ethyl acetate / ammonia = 24:12:4:1.5) and visualizing it with a 0.2% ninhydrin solution (in 1-butanol saturated with water).

[0153] (Quantitative determination of amino groups by colorimetric method) In a 96-well plate, 6 μL of the sample was added to 75 μL of 0.1 M sodium tetraborate solution (pH 9.5). Then, 30 μL of TNBS solution (0.05% sodium trinitrobenzenesulfonate, TNBS, containing 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 the absorbance of a standard substance of known concentration to estimate the concentration of amino groups produced as a result of polymer hydrolysis. [Industrial applicability]

[0154] This invention can be used to recycle nylon 66 back into monomer molecules. Furthermore, this invention contributes to ensuring sustainable production and consumption patterns.

[0155] [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-modified NylC SEQ ID NO:3 Histag-NylC-GYAQ_370aa_38712.37Da

Claims

1. A method for producing a degradation product of nylon 66, comprising the step of reacting nylon 66 with 6-aminohexanoic acid oligomer end type hydrolase (NylC), wherein NylC is the protein described in (4), (5), or (6) below: (4) A protein having the amino acid sequence of positions 1 to 355 of sequence number 1, wherein at least one amino acid selected from M50, R52, D99, G111, L139, D304, and L331 is substituted; (5)(4) The amino acid sequence of the protein described in (4) is a sequence in which one or more amino acids are deleted, substituted or added, 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 acid corresponding to at least one of the substitutions are maintained, and the amino acid corresponding to T267 of SEQ ID NO: 1 is maintained or may be substituted with S or C, and the protein has 6-aminohexanoic acid oligomer endo-type hydrolase (NylC) activity; A protein having NylC activity, comprising an amino acid sequence having 70% or more sequence identity with the protein described in (6)(4), wherein 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 acid corresponding to at least one of these substitutions are maintained, and the amino acid corresponding to T267 may be maintained or substituted with S or C.

2. A method for producing a degradation product of nylon 66 according to claim 1, wherein NylC is the protein described below as (4'), (5'), or (6'): (4') A protein having the amino acid sequence of positions 1 to 355 of sequence number 1, with at least one substitution selected from M50I, R52Y, D99A, G111A, L139R, D304A, L331D, and L331E; A protein having NylC activity, comprising a sequence in which 1 to 36 amino acids are deleted, substituted, or added in the amino acid sequence of the protein described in (5')(4'); A protein having a sequence identity of 70% or more with the protein described in (6')(4'), and possessing NylC activity.

3. A method for producing a degradation product of nylon 66 according to claim 1, wherein NylC is a protein that has been substituted with at least two substitutions selected from M50I, R52Y, D99A, G111A, L139R, D304A, L331D, and L331E.

4. A method for producing a degradation product of nylon 66 according to claim 1, wherein NylC is a protein in which G111A and D304A are substituted.

5. A method for producing a degradation product of nylon 66 according to claim 1, wherein NylC is 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 an amino acid sequence in which one or more amino acids are deleted, substituted or added in the polypeptide described in (a1), provided 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 an α-subunit of NylC; (a3) A polypeptide consisting of an amino acid sequence having 70% or more sequence identity with the polypeptide described in (a1), provided that the amino acids corresponding to F134, L137, Y146, K189, and N219 of SEQ ID NO: 1 are maintained, and which is capable of functioning as the α-subunit of NylC; (b1) Polypeptides having sequences 267-355 of sequence number 1; (b2) A polypeptide comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in the polypeptide described in (b1), provided that the 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 which is capable of functioning as a β-subunit of NylC; (b3) A polypeptide comprising an amino acid sequence having 70% or more sequence identity with the polypeptide described in (b1), wherein the 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 which is capable of functioning as a β-subunit of NylC.

6. A method for producing a degradation product of nylon 66 according to claim 1, wherein NylC is 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 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.

7. A method for producing a degradation product of nylon 66 according to claim 1, wherein NylC is the protein described in (1), (2), or (3) below: (1) Proteins having sequences 1 to 333 of Sequence ID No. 2; (2) A protein having NylC activity, comprising an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the polypeptide described in (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; (3) A protein having NylC activity, comprising a sequence that is 70% or more identical in sequence to the polypeptide described in (1), wherein 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. A method for producing a degradation product of nylon 66 according to claim 1, wherein NylC is the protein described in (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').

9. A method for producing a degradation product of nylon 66 according to claim 1, wherein NylC is a protein in which at least one substitution is made from amino acids selected from those corresponding to M50I, R52Y, D99A, G111A, L139R, D304A, L331D, and L331E of SEQ ID NO:

1.

10. The method for producing a degradation product of nylon 66 according to claim 1, wherein NylC is a protein in which amino acids corresponding to G111A and D304A of SEQ ID NO: 1 have been substituted.

11. A method for producing a decomposition product of nylon 66 according to any one of claims 1 to 10, further comprising a step of reducing the molecular weight of nylon 66.

12. A method for producing a decomposition product of nylon 66 according to any one of claims 1 to 11, wherein the step of reducing the molecular weight of nylon 66 includes a step of contacting nylon 66 with high-temperature water to obtain nylon 66 with reduced molecular weight.

13. An enzyme for degrading nylon 66, comprising NylC as defined in any one of claims 1 to 10.

14. A method for chemical recycling nylon 66, comprising all steps of the manufacturing method described in any one of claims 1 to 12.

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