Polypeptide and method for producing amino acid using same

Abstract

A polypeptide that has a sequence having a sequence identity of 90% or more with respect to a sequence obtained by modifying one amino acid residue in the amino acid sequence represented by SEQ ID NO: 1, and that has a catalytic activity for a reductive amination reaction between one or more of compounds A represented by formula (1) or a salt thereof and one or more of compounds B represented by formula (2) or a salt thereof, or for an intramolecular reductive amination reaction of one or more of the compounds B or a salt thereof, wherein the catalytic activity is higher than that of a polypeptide having the amino acid sequence represented by SEQ ID NO: 1 under at least one reaction condition.

Description

Polypeptide and method for producing amino acids using the same

[0001] The present invention relates to a polypeptide and a method for producing amino acids using the polypeptide.

[0002] Amino acids such as N-alkylamino acids are known to exist as part of the structures of various natural physiologically active substances. Some N-methylamino acids, which are a type of N-alkylamino acid, are also known to exist naturally as amino acids themselves (Non-Patent Document 1).

[0003] There are various organic chemistry methods for synthesizing N-alkylamino acids. From the perspective of green chemistry, methods for synthesizing N-alkylamino acids using enzymes have been investigated and reported.

[0004] Mihara et al. screened various microorganisms based on their activity to synthesize N-methylphenylalanine from phenylpyruvic acid and methylamine (Patent Document 1, Non-Patent Documents 2 and 3). As a result, they discovered N-methylamino acid dehydrogenase (NMAADH) from P. putida ATCC12633, which had the highest activity. They also cloned the gene encoding this enzyme (called dpkA), expressed it in Escherichia coli, and successfully isolated a purified version of the enzyme.

[0005] Furthermore, the purified enzyme had the activity to synthesize the corresponding N-alkylamino acids from various α-keto acids (pyruvic acid, phenylpyruvic acid, hydroxypyruvic acid, etc.) and alkylamines (methylamine, ethylamine, propylamine, etc.).

[0006] The amino acid sequence of the NMAADH derived from the P. putida ATCC12633 strain is in good agreement with that of protein PP3591 encoded in the genome of the P. putida KT2440 strain (Patent Document 2, Non-Patent Documents 2, 3, and 4).

[0007] X-ray crystal structure analysis has been performed on NMAADH derived from P. syringae, which shows 71% amino acid sequence identity with NMAADH derived from P. putida strain ATCC 12633 (Non-Patent Documents 3 and 5). The substrate specificity of purified NMAADH derived from P. syringae for α-keto acids and amines has been shown to be similar to that of NMAADH derived from purified P. putida strain ATCC 12633, and the mechanism of the reaction catalyzed by NMAADH derived from P. syringae has been explained based on the crystal structure.

[0008] Literature has reported the introduction of mutations into NMAADH. In the crystal structure of NMAADH derived from P. syringae (Non-Patent Document 5), residues contained in the substrate binding site are also conserved in NMAADH derived from P. putida KT2440 strain. By introducing mutations into these amino acid residues, a mutant F117L of NMAADH derived from P. putida KT2440 strain was found, which has improved specific activity when glyoxylic acid and methylamine, and glyoxylic acid and ethylamine are used as substrates (Non-Patent Document 6).

[0009] NMAADH derived from P. putida strain KT2440 exhibits higher activity toward pyruvate than toward phenylpyruvate, and the mutant P262A, M141L, which has two mutations, has almost equal activity toward phenylpyruvate and pyruvate (Non-Patent Document 7).

[0010] International Publication No. WO 2003 / 072770 International Publication No. WO 2002 / 077183

[0011] JF Hyslop et al., J.Biotechnol. 2019, 293, 56-65.H. Mihara et al., FEBS Journal2005, 272, 1117-1123. Hisaaki Mihara, Biochemistry 2015, 87, 326-332.KE Nelson et al., Environ.Microbiol. 2002, 4, 799-808.M. Goto et al., J. Biol. Chem.2005, 280, 40875-40884.M. Mindet et al., Front.Bioeng. Biotechnol. 2019, 7.A. Kerbs et al.,Microorganisms 2021, 9, 824.

[0012] To the best of the inventor's knowledge, there are only three publications, including the publication mentioned above, that report on the synthesis of N-alkylamino acids using modified NMAADH. In other words, there are very few reports on modified NMAADH.

[0013] Non-Patent Documents 2 and 3 show that NMAADH derived from the P. putida ATCC12633 strain exhibits absolutely no activity toward ammonia, nor toward α-ketoisocaproic acid or α-keto-β-methylvaleric acid, both of which have a branch at the β-position.

[0014] As far as the present inventors know, only sequence information has been made public for protein PP3591 encoded in the genome of the P. putida KT2440 strain described in Non-Patent Document 4, and there is no information examining its catalytic activity, etc. To the present inventors' knowledge, there have been no reports of improving the synthetic activity of a specific amino acid by, for example, modifying the amino acid sequence of PP3591.

[0015] An object of the present invention is to provide a novel polypeptide that can be used as an enzyme that catalyzes reductive amination reactions, and a method for producing amino acids using the polypeptide.

[0016] The present invention relates to, for example, the following inventions: [1] A polypeptide comprising a sequence having 90% or more sequence identity with a sequence in which one amino acid residue in the amino acid sequence represented by SEQ ID NO: 1 has been altered, and which has a catalytic activity for a reductive amination reaction between one or more compounds A represented by the following formula (1) or a salt thereof and one or more compounds B represented by the following formula (2) or a salt thereof, or for an intramolecular reductive amination reaction between one or more compounds B or a salt thereof, which is higher than the catalytic activity of a polypeptide having the amino acid sequence represented by SEQ ID NO: 1 under at least one reaction condition: [In formula (1), R １ and R ２ each independently represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, a heterocyclyl group, or a heteroaryl group, and these groups may be substituted; R １ or R ２ and at least one of the groups is a hydrogen atom. [In the formula (2), X represents a carbon atom, Y represents a hydrogen atom, a group represented by the formula (1′) or a group represented by the formula (3), n represents an integer of 0 or more and 2 or less, and R ６ represents a hydrogen atom, an aliphatic hydrocarbon group having 1 to 6 carbon atoms which may be substituted, an aryl group having 5 to 12 carbon atoms which may be substituted, a heteroaryl group having 5 to 12 ring-constituting atoms which may be substituted, a group containing a nitrogen atom, or a group containing an oxygen atom, indicates the point of attachment to X, and R １ａ is R in formula (1) １ is a group obtained by removing a hydrogen atom from a group represented by the formula (3): represents a bonding point with X, m represents an integer of 0 to 6, p is 0 or 1, q is 0 or 1, r is 0 or 1, Z １represents an alkylene group which may be substituted, or an ether bond-containing group having 1 to 6 carbon atoms; when m is 2 or more, a plurality of Z １ may be the same or different, Z ２ represents a carbon atom, R ３ , R ４ and R ５ each independently represents a hydrogen atom, an optionally substituted aliphatic hydrocarbon group having from 1 to 6 carbon atoms, an optionally substituted aryl group having from 5 to 12 carbon atoms, an optionally substituted heteroaryl group having from 5 to 12 ring-constituting atoms, a group containing a nitrogen atom, or a group containing an oxygen atom; R ３ , R ４ and R ５ Any two or more of the following may be bonded to each other to form Z ２ and R may form a ring structure together, which may be a cycloalkyl group, an aryl group, a heterocyclyl group, or a heteroaryl group, which may be substituted; ３ , R ４ , and R ５ Is Z ２ may form a double bond or a triple bond with R ３ , R ４ , and R ５ is connected to Z by a double bond or a triple bond. ２ When R is bonded to R, any one or more of p, q, and r is 0. １ and R ２ is a methyl group and the other is a hydrogen atom, in formula (2), Y is a group represented by formula (3), m is 0, and R ３ ~R ５two or more of which are not hydrogen atoms.] [2] A polypeptide comprising a sequence having 90% or more sequence identity with a sequence in which two amino acid residues in the amino acid sequence represented by SEQ ID NO: 1 have been altered, and which has a catalytic activity for a reductive amination reaction between one or more of compounds A represented by the following formula (1) or a salt thereof and one or more of compounds B represented by the following formula (2) or a salt thereof, or for an intramolecular reductive amination reaction between one or more of compounds B or a salt thereof, which is higher than the catalytic activity of a polypeptide having the amino acid sequence represented by SEQ ID NO: 1 under at least one reaction condition. [In formula (1), R １ and R ２ each independently represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, a heterocyclyl group, or a heteroaryl group, and these groups may be substituted; R １ or R ２ and at least one of the groups is a hydrogen atom. [In the formula (2), X represents a carbon atom, Y represents a hydrogen atom, a group represented by the formula (1′) or a group represented by the formula (3), n represents an integer of 0 or more and 2 or less, and R ６ represents a hydrogen atom, an aliphatic hydrocarbon group having 1 to 6 carbon atoms which may be substituted, an aryl group having 5 to 12 carbon atoms which may be substituted, a heteroaryl group having 5 to 12 ring-constituting atoms which may be substituted, a group containing a nitrogen atom, or a group containing an oxygen atom, indicates the point of attachment to X, and R １ａ is R in formula (1) １ is a group obtained by removing a hydrogen atom from a group represented by the formula (3): represents a bonding point with X, m represents an integer of 0 to 6, p is 0 or 1, q is 0 or 1, r is 0 or 1, Z １represents an alkylene group which may be substituted, or an ether bond-containing group having 1 to 6 carbon atoms; when m is 2 or more, a plurality of Z １ may be the same or different, Z ２ represents a carbon atom, R ３ , R ４ and R ５ each independently represents a hydrogen atom, an optionally substituted aliphatic hydrocarbon group having from 1 to 6 carbon atoms, an optionally substituted aryl group having from 5 to 12 carbon atoms, an optionally substituted heteroaryl group having from 5 to 12 ring-constituting atoms, a group containing a nitrogen atom, or a group containing an oxygen atom; R ３ , R ４ and R ５ Any two or more of the following may be bonded to each other to form Z ２ and R may form a ring structure together, which may be a cycloalkyl group, an aryl group, a heterocyclyl group, or a heteroaryl group, which may be substituted; ３ , R ４ , and R ５ Is Z ２ may form a double bond or a triple bond with R ３ , R ４ , and R ５ is connected to Z by a double bond or a triple bond. ２ When R is bonded to R, any one or more of p, q, and r is 0. １ and R ２ is a methyl group and the other is a hydrogen atom, in formula (2), Y is a group represented by formula (3), m is 0, and R ３ ~R ５and two or more of the amino acid residues are not hydrogen atoms.] [3] The polypeptide according to [1], which comprises a sequence having 90% or more sequence identity with a sequence in which two amino acid residues in the amino acid sequence represented by SEQ ID NO: 1 have been altered, and which has a catalytic activity for a reductive amination reaction between one or more of compound A represented by the following formula (1) or a salt thereof and one or more of compound B represented by the following formula (2) or a salt thereof, or an intramolecular reductive amination reaction between one or more of compound B or a salt thereof, which is higher than the catalytic activity of a polypeptide having the amino acid sequence represented by SEQ ID NO: 1 under at least one reaction condition. [In formula (1), R １ and R ２ each independently represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, a heterocyclyl group, or a heteroaryl group, and these groups may be substituted; R １ or R ２ and at least one of the groups is a hydrogen atom. [In the formula (2), X represents a carbon atom, Y represents a hydrogen atom, a group represented by the formula (1′) or a group represented by the formula (3), n represents an integer of 0 or more and 2 or less, and R ６ represents a hydrogen atom, an aliphatic hydrocarbon group having 1 to 6 carbon atoms which may be substituted, an aryl group having 5 to 12 carbon atoms which may be substituted, a heteroaryl group having 5 to 12 ring-constituting atoms which may be substituted, a group containing a nitrogen atom, or a group containing an oxygen atom, indicates the point of attachment to X, and R １ａ is R in formula (1) １ is a group obtained by removing a hydrogen atom from a group represented by the formula (3): represents a bonding point with X, m represents an integer of 0 to 6, p is 0 or 1, q is 0 or 1, r is 0 or 1, Z １represents an alkylene group which may be substituted, or an ether bond-containing group having 1 to 6 carbon atoms; when m is 2 or more, a plurality of Z １ may be the same or different, Z ２ represents a carbon atom, R ３ , R ４ and R ５ each independently represents a hydrogen atom, an optionally substituted aliphatic hydrocarbon group having from 1 to 6 carbon atoms, an optionally substituted aryl group having from 5 to 12 carbon atoms, an optionally substituted heteroaryl group having from 5 to 12 ring-constituting atoms, a group containing a nitrogen atom, or a group containing an oxygen atom; R ３ , R ４ and R ５ Any two or more of the following may be bonded to each other to form Z ２ and R may form a ring structure together, which may be a cycloalkyl group, an aryl group, a heterocyclyl group, or a heteroaryl group, which may be substituted; ３ , R ４ , and R ５ Is Z ２ may form a double bond or a triple bond with R ３ , R ４ , and R ５ is connected to Z by a double bond or a triple bond. ２ When R is bonded to R, any one or more of p, q, and r is 0. １ and R ２ is a methyl group and the other is a hydrogen atom, in formula (2), Y is a group represented by formula (3), m is 0, and R ３ ~R ５two or more of which are not hydrogen atoms.] [4] A polypeptide comprising a sequence having 90% or more sequence identity with a sequence in which three amino acid residues in the amino acid sequence represented by SEQ ID NO: 1 have been altered, and which has a catalytic activity for a reductive amination reaction between one or more of compounds A represented by the following formula (1) or a salt thereof and one or more of compounds B represented by the following formula (2) or a salt thereof, or for an intramolecular reductive amination reaction between one or more of compounds B or a salt thereof, which is higher than the catalytic activity of a polypeptide having the amino acid sequence represented by SEQ ID NO: 1 under at least one reaction condition. [In formula (1), R １ and R ２ each independently represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, a heterocyclyl group, or a heteroaryl group, and these groups may be substituted; R １ or R ２ and at least one of the groups is a hydrogen atom. [In the formula (2), X represents a carbon atom, Y represents a hydrogen atom, a group represented by the formula (1′) or a group represented by the formula (3), n represents an integer of 0 or more and 2 or less, and R ６ represents a hydrogen atom, an aliphatic hydrocarbon group having 1 to 6 carbon atoms which may be substituted, an aryl group having 5 to 12 carbon atoms which may be substituted, a heteroaryl group having 5 to 12 ring-constituting atoms which may be substituted, a group containing a nitrogen atom, or a group containing an oxygen atom, indicates the point of attachment to X, and R １ａ is R in formula (1) １ is a group obtained by removing a hydrogen atom from a group represented by the formula (3): represents a bonding point with X, m represents an integer of 0 to 6, p is 0 or 1, q is 0 or 1, r is 0 or 1, Z １represents an alkylene group which may be substituted, or an ether bond-containing group having 1 to 6 carbon atoms; when m is 2 or more, a plurality of Z １ may be the same or different, Z ２ represents a carbon atom, R ３ , R ４ and R ５ each independently represents a hydrogen atom, an optionally substituted aliphatic hydrocarbon group having from 1 to 6 carbon atoms, an optionally substituted aryl group having from 5 to 12 carbon atoms, an optionally substituted heteroaryl group having from 5 to 12 ring-constituting atoms, a group containing a nitrogen atom, or a group containing an oxygen atom; R ３ , R ４ and R ５ Any two or more of the following may be bonded to each other to form Z ２ and R may form a ring structure together, which may be a cycloalkyl group, an aryl group, a heterocyclyl group, or a heteroaryl group, which may be substituted; ３ , R ４ , and R ５ Is Z ２ may form a double bond or a triple bond with R ３ , R ４ , and R ５ is connected to Z by a double bond or a triple bond. ２ When R is bonded to R, any one or more of p, q, and r is 0. １ and R ２ is a methyl group and the other is a hydrogen atom, in formula (2), Y is a group represented by formula (3), m is 0, and R ３ ~R ５and two or more of the amino acid residues are not hydrogen atoms.] [5] The polypeptide according to [1], which comprises a sequence having 90% or more sequence identity with a sequence in which three amino acid residues in the amino acid sequence represented by SEQ ID NO: 1 have been altered, and which has a catalytic activity for a reductive amination reaction between one or more of compound A represented by the following formula (1) or a salt thereof and one or more of compound B represented by the following formula (2) or a salt thereof, or an intramolecular reductive amination reaction between one or more of compound B or a salt thereof, which is higher than the catalytic activity of a polypeptide having the amino acid sequence represented by SEQ ID NO: 1 under at least one reaction condition. [In formula (1), R １ and R ２ each independently represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, a heterocyclyl group, or a heteroaryl group, and these groups may be substituted; R １ or R ２ and at least one of the groups is a hydrogen atom. [In the formula (2), X represents a carbon atom, Y represents a hydrogen atom, a group represented by the formula (1′) or a group represented by the formula (3), n represents an integer of 0 or more and 2 or less, and R ６ represents a hydrogen atom, an aliphatic hydrocarbon group having 1 to 6 carbon atoms which may be substituted, an aryl group having 5 to 12 carbon atoms which may be substituted, a heteroaryl group having 5 to 12 ring-constituting atoms which may be substituted, a group containing a nitrogen atom, or a group containing an oxygen atom, indicates the point of attachment to X, and R １ａ is R in formula (1) １ is a group obtained by removing a hydrogen atom from a group represented by the formula (3): represents a bonding point with X, m represents an integer of 0 to 6, p is 0 or 1, q is 0 or 1, r is 0 or 1, Z １represents an alkylene group which may be substituted, or an ether bond-containing group having 1 to 6 carbon atoms; when m is 2 or more, a plurality of Z １ may be the same or different, Z ２ represents a carbon atom, R ３ , R ４ and R ５ each independently represents a hydrogen atom, an optionally substituted aliphatic hydrocarbon group having from 1 to 6 carbon atoms, an optionally substituted aryl group having from 5 to 12 carbon atoms, an optionally substituted heteroaryl group having from 5 to 12 ring-constituting atoms, a group containing a nitrogen atom, or a group containing an oxygen atom; R ３ , R ４ and R ５ Any two or more of the following may be bonded to each other to form Z ２ and R may form a ring structure together, which may be a cycloalkyl group, an aryl group, a heterocyclyl group, or a heteroaryl group, which may be substituted; ３ , R ４ , and R ５ Is Z ２ may form a double bond or a triple bond with R ３ , R ４ , and R ５ is connected to Z by a double bond or a triple bond. ２ When R is bonded to R, any one or more of p, q, and r is 0. １ and R ２ is a methyl group and the other is a hydrogen atom, in formula (2), Y is a group represented by formula (3), m is 0, and R ３ ~R ５two or more of which are not hydrogen atoms.] [6] A polypeptide comprising a sequence having 90% or more sequence identity with a sequence in which one amino acid residue in the amino acid sequence represented by SEQ ID NO: 1 has been altered, and which has a catalytic activity for a reductive amination reaction between an alkylamine or a salt thereof and one or more compounds represented by the following formula (2') or salts thereof that is higher than the catalytic activity of a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 under at least one reaction condition: wherein Y′ is C ３ ~C ８ a cycloalkyl group, or C ６ ~C ９ represents an aralkyl group, and the aralkyl group is C １ ~C ３ and may be substituted with an alkyl group or a halogen atom.] [7] A polypeptide comprising a sequence having 90% or more sequence identity with a sequence in which two amino acid residues in the amino acid sequence represented by SEQ ID NO: 1 have been altered, and which has a catalytic activity for a reductive amination reaction between an alkylamine or a salt thereof and one or more compounds represented by the following formula (2') or salts thereof that is higher than the catalytic activity of a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 under at least one reaction condition: wherein Y′ is C ３ ~C ８ a cycloalkyl group, or C ６ ~C ９ represents an aralkyl group, and the aralkyl group is C １ ~C ３ The polypeptide according to [6], which comprises a sequence having 90% or more sequence identity with a sequence in which two amino acid residues in the amino acid sequence represented by SEQ ID NO: 1 have been altered, and which has a catalytic activity for a reductive amination reaction between an alkylamine or a salt thereof and one or more compounds represented by the following formula (2') or salts thereof that is higher than the catalytic activity of a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 under at least one reaction condition: wherein Y′ is C ３ ~C ８ a cycloalkyl group, or C６ ~C ９ represents an aralkyl group, and the aralkyl group is C １ ~C ３ and may be substituted with an alkyl group or a halogen atom.] [9] A polypeptide comprising a sequence having 90% or more sequence identity with a sequence in which three amino acid residues in the amino acid sequence represented by SEQ ID NO: 1 have been altered, and which has a catalytic activity for a reductive amination reaction between an alkylamine or a salt thereof and one or more compounds represented by the following formula (2') or salts thereof that is higher than the catalytic activity of a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 under at least one reaction condition: wherein Y′ is C ３ ~C ８ a cycloalkyl group, or C ６ ~C ９ represents an aralkyl group, and the aralkyl group is C １ ~C ３ The polypeptide according to [6], which comprises a sequence having 90% or more sequence identity with a sequence in which three amino acid residues in the amino acid sequence represented by SEQ ID NO: 1 have been altered, and which has a catalytic activity for a reductive amination reaction between an alkylamine or a salt thereof and one or more compounds represented by the following formula (2') or salts thereof that is higher than the catalytic activity of a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 under at least one reaction condition: wherein Y′ is C ３ ~C ８ a cycloalkyl group, or C ６ ~C ９ represents an aralkyl group, and the aralkyl group is C １ ~C ３

[11] The polypeptide according to any one of [1] to

[10] , comprising a sequence having 90% or more sequence identity with a sequence obtained by altering one amino acid residue in the amino acid sequence represented by SEQ ID NO: 17.

[12] The polypeptide according to any one of [1] to

[11] , comprising a sequence having 90% or more sequence identity with a sequence obtained by altering two amino acid residues in the amino acid sequence represented by SEQ ID NO: 17.

[13] The polypeptide according to any one of [1] to

[12] , comprising a sequence having 90% or more sequence identity with a sequence obtained by altering three amino acid residues in the amino acid sequence represented by SEQ ID NO: 17.

[14] A polypeptide comprising a sequence in which amino acid residues located at positions corresponding to one or more amino acid residues selected from the group consisting of the histidine residue at position 44, the phenylalanine residue at position 117, the methionine residue at position 141, the threonine residue at position 156, the histidine residue at position 182, the glutamine residue at position 186, the tryptophan residue at position 253, and the lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1 have been modified.

[15] The polypeptide according to any of [1] to

[13] , comprising a sequence in which amino acid residues located at positions corresponding to one or more amino acid residues selected from the group consisting of the histidine residue at position 44, the phenylalanine residue at position 117, the methionine residue at position 141, the threonine residue at position 156, the histidine residue at position 182, the glutamine residue at position 186, the tryptophan residue at position 253, and the lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1 have been modified.

[16] The polypeptide according to any one of [1] to

[15] , comprising a sequence in which an amino acid residue located at a site corresponding to one or more amino acid residues selected from the group consisting of a methionine residue at position 141, a histidine residue at position 182, a tryptophan residue at position 253, and a lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1 has been modified.

[17] The amino acid residue located at a site corresponding to one amino acid residue selected from the group consisting of a methionine residue at position 141, a histidine residue at position 182, and a tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 has been modified as a first modification site,

[18] A polypeptide comprising a sequence in which an amino acid residue located at a site corresponding to one amino acid residue selected from the group consisting of a histidine residue at position 182, a tryptophan residue at position 253, and a lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1, and which is different from the amino acid residue modified as the first modification site, has been modified as the second modification site.

[19] The polypeptide according to any of [1] to

[20] , comprising a sequence in which an amino acid residue located at a site corresponding to one amino acid residue selected from the group consisting of a methionine residue at position 141, a histidine residue at position 182, and a tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1, and which is different from the amino acid residue modified as the first modification site, has been modified as the second modification site.

[19] A polypeptide comprising a sequence in which an amino acid residue located at a position corresponding to one amino acid residue selected from the group consisting of the methionine residue at position 141 and the histidine residue at position 182 in the amino acid sequence represented by SEQ ID NO: 1 has been modified as a first modification site, an amino acid residue located at a position corresponding to one amino acid residue selected from the group consisting of the histidine residue at position 182 and the tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 and different from the amino acid residue modified as the first modification site has been modified as a second modification site, and an amino acid residue located at a position corresponding to one amino acid residue selected from the group consisting of the tryptophan residue at position 253 and the lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1 and different from the amino acid residues modified as the first and second modification sites has been modified as a third modification site.

[20] A polypeptide comprising a sequence in which an amino acid residue located at a position corresponding to one amino acid residue selected from the group consisting of the methionine residue at position 141 and the histidine residue at position 182 in the amino acid sequence represented by SEQ ID NO: 1 has been modified as a first modification site,

[21] The polypeptide according to any one of [1] to

[16] , comprising a sequence in which an amino acid residue at a site corresponding to one amino acid residue selected from the group consisting of the histidine residue at position 182 and the tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 and different from the amino acid residue modified as the first modification site is modified as the second modification site, and an amino acid residue at a site corresponding to one amino acid residue selected from the group consisting of the tryptophan residue at position 253 and the lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1 and different from the amino acid residues modified as the first modification site and the second modification site is modified as the third modification site.

[22] The polypeptide according to any one of [1], [6],

[11] , and

[23] to

[24] , comprising a sequence in which an amino acid residue at a site corresponding to one or more amino acid residues selected from the group consisting of the methionine residue at position 141, the tryptophan residue at position 253, and the lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1 is modified.

[22] The polypeptide according to any one of [1], [6],

[11] ,

[14] to

[16] and

[21] , wherein the amino acid sequence represented by SEQ ID NO: 1 is modified at one position.

[23] The polypeptide according to any one of [1], [6],

[11] ,

[14] to

[16] and

[21] to

[22] , wherein the polypeptide comprises a sequence in which the amino acid residue at a position corresponding to the histidine residue at position 44 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a methionine residue.

[24] The polypeptide according to any one of [1], [6],

[11] ,

[14] to

[16] and

[21] to

[23] , wherein the polypeptide comprises a sequence in which the amino acid residue at a position corresponding to the phenylalanine residue at position 117 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a leucine residue.

[25] The polypeptide according to any one of [1], [6],

[11] ,

[14] to

[16] and

[21] to

[24] , comprising a sequence in which the amino acid residue at a position corresponding to the methionine residue at position 141 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with one or more amino acid residues selected from the group consisting of tyrosine residues, tryptophan residues, valine residues, threonine residues, serine residues, arginine residues, leucine residues, lysine residues, isoleucine residues, histidine residues, phenylalanine residues and alanine residues.

[26] The polypeptide according to any one of [1], [6],

[11] ,

[14] to

[16] and

[21] to

[25] , comprising a sequence in which the amino acid residue at a position corresponding to the methionine residue at position 141 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with one or more amino acid residues selected from the group consisting of tyrosine residues, tryptophan residues, valine residues, lysine residues, isoleucine residues, phenylalanine residues and alanine residues.

[27] The polypeptide according to any one of [1], [6],

[11] ,

[14] to

[16] , and

[21] to

[26] , which comprises a sequence in which an amino acid residue located at a position corresponding to the threonine residue at position 156 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a serine residue.

[28] The polypeptide according to any one of [1], [6],

[11] ,

[14] to

[16] , and

[21] to

[27] , which comprises a sequence in which an amino acid residue located at a position corresponding to the histidine residue at position 182 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with one or more amino acid residues selected from the group consisting of tyrosine residues, glutamine residues, methionine residues, leucine residues, glycine residues, phenylalanine residues, and alanine residues.

[29] The polypeptide according to any one of [1], [6],

[11] ,

[14] to

[16] , and

[21] to

[28] , comprising a sequence in which the amino acid residue located at a position corresponding to the histidine residue at position 182 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with one or more amino acid residues selected from the group consisting of methionine residues, leucine residues, and phenylalanine residues.

[30]

[31] The polypeptide according to any one of [1], [6],

[11] ,

[14] to

[16] and

[21] to

[29] , comprising a sequence in which an amino acid residue located at a position corresponding to the glutamine residue at position 186 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with one or more amino acid residues selected from the group consisting of methionine residues and glutamic acid residues.

[32] The polypeptide according to any one of [1], [6],

[11] ,

[14] to

[16] and

[21] to

[32] , comprising a sequence in which an amino acid residue located at a position corresponding to the tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with one or more amino acid residues selected from the group consisting of tyrosine residues, valine residues, threonine residues, serine residues, arginine residues, glutamine residues, proline residues, asparagine residues, methionine residues, leucine residues, lysine residues, isoleucine residues, histidine residues, phenylalanine residues and alanine residues.

[32] The polypeptide according to any one of [1], [6],

[11] ,

[14] to

[16] , and

[21] to

[31] , comprising a sequence in which the amino acid residue at a position corresponding to the tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with one or more amino acid residues selected from the group consisting of leucine residues, isoleucine residues, and histidine residues.

[33] The polypeptide according to any one of [1], [6],

[11] ,

[14] to

[16] , and

[21] to

[32] , comprising a sequence in which the amino acid residue at a position corresponding to the tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a histidine residue.

[34] The polypeptide according to any one of [1], [6],

[11] ,

[14] to

[16] , and

[21] to

[33] , comprising a sequence in which the amino acid residue located at a site corresponding to the lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with one or more amino acid residues selected from the group consisting of tyrosine residues, tryptophan residues, threonine residues, serine residues, arginine residues, glutamine residues, asparagine residues, methionine residues, leucine residues, histidine residues, glycine residues, phenylalanine residues, glutamic acid residues, and alanine residues.

[35] The polypeptide according to any one of [1], [6],

[11] ,

[14] to

[16] , and

[21] to

[34] , comprising a sequence in which an amino acid residue located at a site corresponding to the lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with one or more amino acid residues selected from the group consisting of glutamine residues, methionine residues, glutamic acid residues, and asparagine residues.

[36] The polypeptide according to any one of [1], [6],

[11] ,

[14] to

[16] , and

[21] to

[35] , wherein the sequence in which one amino acid residue in the amino acid sequence represented by SEQ ID NO: 1 has been modified is the amino acid sequence represented by SEQ ID NO: 6 in which X is a valine residue (M141V), the amino acid sequence represented by SEQ ID NO: 6 in which X is a tyrosine residue (M141Y), the amino acid sequence represented by SEQ ID NO: 8 in which X is a leucine residue (H182L), the amino acid sequence represented by SEQ ID NO: 11 in which X is a histidine residue (W253H), or the amino acid sequence represented by SEQ ID NO: 12 in which X is a glutamic acid residue (K260E).

[37] The polypeptide according to any one of [1], [6],

[11] ,

[14] to

[16] , and

[21] to

[36] , comprising a sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6, in which X is a valine residue (M141V), the amino acid sequence represented by SEQ ID NO: 6, in which X is a tyrosine residue (M141Y), the amino acid sequence represented by SEQ ID NO: 8, in which X is a leucine residue (H182Y), the amino acid sequence represented by SEQ ID NO: 11, in which X is a histidine residue (W253H), or the amino acid sequence represented by SEQ ID NO: 12, in which X is a glutamic acid residue (K260E).

[38] The polypeptide according to any one of [1], [6],

[11] ,

[14] to

[16] , and

[21] to

[37] , comprising the amino acid sequence represented by SEQ ID NO: 6, in which X is a valine residue (M141V), the amino acid sequence represented by SEQ ID NO: 6, in which X is a tyrosine residue (M141Y), the amino acid sequence represented by SEQ ID NO: 8, in which X is a leucine residue (H182Y), the amino acid sequence represented by SEQ ID NO: 11, in which X is a histidine residue (W253H), or the amino acid sequence represented by SEQ ID NO: 12, in which X is a glutamic acid residue (K260E).

[39]

[40] The polypeptide according to any one of [2], [3], [7], [8],

[12] ,

[17] and

[18] , which has an amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22.

[41] The polypeptide according to any one of [2], [3], [7], [8],

[12] ,

[17] and

[40] , which has two or more alterations in the amino acid sequence represented by SEQ ID NO: 1.

[42] The polypeptide according to any one of [2], [3], [7], [8],

[12] ,

[17] ,

[18] and

[40] , which has two alterations in the amino acid sequence represented by SEQ ID NO: 1.

[42] The polypeptide according to [2], [3], [7], [8],

[12] ,

[17] ,

[18] ,

[40] and

[41] , comprising: a sequence in which, in the amino acid sequence represented by SEQ ID NO: 1, amino acid residues located at positions corresponding to the methionine residue at position 141 and the histidine residue at position 182 are modified; a sequence in which amino acid residues located at positions corresponding to the methionine residue at position 141 and the tryptophan residue at position 253 are modified; a sequence in which amino acid residues located at positions corresponding to the methionine residue at position 141 and the lysine residue at position 260 are modified; a sequence in which amino acid residues located at positions corresponding to the histidine residue at position 182 and the tryptophan residue at position 253 are modified; a sequence in which amino acid residues located at positions corresponding to the histidine residue at position 182 and the lysine residue at position 260 are modified; or a sequence in which amino acid residues located at positions corresponding to the tryptophan residue at position 253 and the lysine residue at position 260 are modified.

[43] The polypeptide according to [2], [3], [7], [8],

[12] ,

[17] ,

[18] , and

[40] to

[42] , which comprises an amino acid sequence represented by SEQ ID NO: 1 in which amino acid residues located at positions corresponding to the methionine residue at position 141 and the lysine residue at position 260 have been modified, an amino acid sequence in which amino acid residues located at positions corresponding to the histidine residue at position 182 and the lysine residue at position 260 have been modified, or an amino acid sequence in which amino acid residues located at positions corresponding to the tryptophan residue at position 253 and the lysine residue at position 260 have been modified.

[44] The polypeptide according to any one of [2], [3], [7], [8],

[12] ,

[17] ,

[18] , and

[40] to

[43] , comprising a sequence in which the amino acid residue at a position corresponding to the methionine residue at position 141 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with one amino acid residue selected from the group consisting of tyrosine residue, tryptophan residue, valine residue, threonine residue, arginine residue, lysine residue, isoleucine residue, and alanine residue.

[45] The polypeptide according to any one of [2], [3], [7], [8],

[12] ,

[17] ,

[18] , and

[40] to

[44] , comprising a sequence in which the amino acid residue at a position corresponding to the methionine residue at position 141 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with one amino acid residue selected from the group consisting of tyrosine residue, valine residue, and alanine residue.

[46] The polypeptide according to any one of [2], [3], [7], [8],

[12] ,

[17] ,

[18] , and

[40] to

[45] , comprising a sequence in which the amino acid residue at a site corresponding to the histidine residue at position 182 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with one amino acid residue selected from the group consisting of tyrosine residues, methionine residues, leucine residues, and phenylalanine residues.

[47] The polypeptide according to any one of [2], [3], [7], [8],

[12] ,

[17] ,

[18] , and

[40] to

[46] , comprising a sequence in which the amino acid residue at a site corresponding to the histidine residue at position 182 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with one amino acid residue selected from the group consisting of methionine residues and leucine residues.

[48] ​​The polypeptide according to any one of [2], [3], [7], [8],

[12] ,

[17] ,

[18] , and

[40] to

[47] , which comprises a sequence in which the amino acid residue located at a position corresponding to the tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with one amino acid residue selected from the group consisting of tyrosine residues, threonine residues, serine residues, asparagine residues, methionine residues, histidine residues, and alanine residues.

[49] The polypeptide according to any one of [2], [3], [7], [8],

[12] ,

[17] ,

[18] , and

[40] to

[48] , comprising a sequence in which an amino acid residue located at a position corresponding to the tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a histidine residue.

[50] The polypeptide according to any one of [2], [3], [7], [8],

[12] ,

[17] ,

[18] , and

[40] to

[49] , comprising a sequence in which an amino acid residue located at a position corresponding to the lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with one amino acid residue selected from the group consisting of serine residues, glutamine residues, asparagine residues, leucine residues, isoleucine residues, histidine residues, glycine residues, phenylalanine residues, glutamic acid residues, and alanine residues.

[51] The polypeptide according to any one of [2], [3], [7], [8],

[12] ,

[17] ,

[18] , and

[40] to

[50] , comprising a sequence in which the amino acid residue located at a site corresponding to the lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with one amino acid residue selected from the group consisting of glutamine residues and glutamic acid residues.

[52] The amino acid sequence of SEQ ID NO: 1 in which two amino acid residues are modified is: an amino acid sequence of SEQ ID NO: 25 in which X at the position corresponding to the amino acid residue at position 141 is an alanine residue and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (M141A_K260E); an amino acid sequence of SEQ ID NO: 25 in which X at the position corresponding to the amino acid residue at position 141 is an alanine residue and X at the position corresponding to the amino acid residue at position 260 is a glutamine residue (M141A_K260Q); an amino acid sequence of SEQ ID NO: 25 in which X at the position corresponding to the amino acid residue at position 141 is a tyrosine residue and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (M141Y_K260E); an amino acid sequence (M141V_K260E) represented by SEQ ID NO:25, in which X at the position corresponding to the amino acid residue at position 141 is a valine residue and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue;an amino acid sequence represented by SEQ ID NO:27 in which X at the position corresponding to the amino acid residue at position 182 is a leucine residue and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (H182L_K260E); an amino acid sequence represented by SEQ ID NO:27 in which X at the position corresponding to the amino acid residue at position 182 is a leucine residue and X at the position corresponding to the amino acid residue at position 260 is a glutamine residue (H182L_K260Q); an amino acid sequence represented by SEQ ID NO:27 in which X at the position corresponding to the amino acid residue at position 182 is a methionine residue and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (H182M_K260E); an amino acid sequence represented by SEQ ID NO:27 in which X at the position corresponding to the amino acid residue at position 182 is a methionine residue and X at the position corresponding to the amino acid residue at position 260 is a glutamine residue (H182M_K260Q); or The polypeptide according to any one of [2], [3], [7], [8],

[12] ,

[17] ,

[18] , and

[40] to

[51] , which is the amino acid sequence (W253H_K260E) represented by SEQ ID NO: 28, in which X at the site corresponding to the amino acid residue at position 253 is a histidine residue and X at the site corresponding to the amino acid residue at position 260 is a glutamic acid residue.

[53] an amino acid sequence represented by SEQ ID NO:25, in which X at the position corresponding to the amino acid residue at position 141 is an alanine residue and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (M141A_K260E); an amino acid sequence represented by SEQ ID NO:25, in which X at the position corresponding to the amino acid residue at position 141 is an alanine residue and X at the position corresponding to the amino acid residue at position 260 is a glutamine residue (M141A_K260Q); an amino acid sequence represented by SEQ ID NO:25, in which X at the position corresponding to the amino acid residue at position 141 is a tyrosine residue and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (M141Y_K260E); an amino acid sequence represented by SEQ ID NO:25, in which X at the position corresponding to the amino acid residue at position 141 is a valine residue and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (M141V_K260E);an amino acid sequence represented by SEQ ID NO:27 in which X at the position corresponding to the amino acid residue at position 182 is a leucine residue and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (H182L_K260E); an amino acid sequence represented by SEQ ID NO:27 in which X at the position corresponding to the amino acid residue at position 182 is a leucine residue and X at the position corresponding to the amino acid residue at position 260 is a glutamine residue (H182L_K260Q); an amino acid sequence represented by SEQ ID NO:27 in which X at the position corresponding to the amino acid residue at position 182 is a methionine residue and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (H182M_K260E); an amino acid sequence represented by SEQ ID NO:27 in which X at the position corresponding to the amino acid residue at position 182 is a methionine residue and X at the position corresponding to the amino acid residue at position 260 is a glutamine residue (H182M_K260Q); or The polypeptide according to any one of [2], [3], [7], [8],

[12] ,

[17] ,

[18] , and

[40] to

[52] , comprising a sequence having 90% or more sequence identity with the amino acid sequence (W253H_K260E) represented by SEQ ID NO: 28, in which X at the site corresponding to the amino acid residue at position 253 is a histidine residue and X at the site corresponding to the amino acid residue at position 260 is a glutamic acid residue.

[54] an amino acid sequence represented by SEQ ID NO:25, in which X at the position corresponding to the amino acid residue at position 141 is an alanine residue and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (M141A_K260E); an amino acid sequence represented by SEQ ID NO:25, in which X at the position corresponding to the amino acid residue at position 141 is an alanine residue and X at the position corresponding to the amino acid residue at position 260 is a glutamine residue (M141A_K260Q); an amino acid sequence represented by SEQ ID NO:25, in which X at the position corresponding to the amino acid residue at position 141 is a tyrosine residue and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (M141Y_K260E);an amino acid sequence represented by SEQ ID NO:25 in which X at the position corresponding to the amino acid residue at position 141 is a valine residue and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (M141V_K260E); an amino acid sequence represented by SEQ ID NO:27 in which X at the position corresponding to the amino acid residue at position 182 is a leucine residue and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (H182L_K260E); an amino acid sequence represented by SEQ ID NO:27 in which X at the position corresponding to the amino acid residue at position 182 is a leucine residue and X at the position corresponding to the amino acid residue at position 260 is a glutamine residue (H182L_K260Q); an amino acid sequence represented by SEQ ID NO:27 in which X at the position corresponding to the amino acid residue at position 182 is a methionine residue and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (H182M_K260E); The polypeptide according to any one of [2], [3], [7], [8],

[12] ,

[17] ,

[18] , and

[40] to

[53] , comprising the amino acid sequence of SEQ ID NO: 27, in which X at the position corresponding to amino acid residue 182 is a methionine residue and X at the position corresponding to amino acid residue 260 is a glutamine residue (H182M_K260Q), or the amino acid sequence of SEQ ID NO: 28, in which X at the position corresponding to amino acid residue 253 is a histidine residue and X at the position corresponding to amino acid residue 260 is a glutamic acid residue (W253H_K260E).

[55] The polypeptide according to any one of [2], [3], [7], [8],

[12] ,

[17] ,

[18] , and

[40] to

[54] , having the amino acid sequence of any one of SEQ ID NOs: 36 to 44.

[56] The polypeptide according to any one of [4], [5], [9],

[10] ,

[13] ,

[19] and

[20] , wherein the amino acid sequence represented by SEQ ID NO: 1 has three or more alterations.

[57] The polypeptide according to any one of [4], [5], [9],

[10] ,

[13] ,

[19] ,

[20] and

[56] , wherein the amino acid sequence represented by SEQ ID NO: 1 has three alterations.

[58] In the amino acid sequence represented by SEQ ID NO: 1,The polypeptide according to any one of [4], [5], [9],

[10] ,

[13] ,

[19] ,

[20] ,

[56] and

[57] , comprising: a sequence in which amino acid residues corresponding to the methionine residue at position 141, the histidine residue at position 182 and the tryptophan residue at position 253 have been modified; a sequence in which amino acid residues corresponding to the methionine residue at position 141, the histidine residue at position 182 and the lysine residue at position 260 have been modified; a sequence in which amino acid residues corresponding to the methionine residue at position 141, the tryptophan residue at position 253 and the lysine residue at position 260 have been modified; a sequence in which amino acid residues corresponding to the histidine residue at position 182, the tryptophan residue at position 253 and the lysine residue at position 260 have been modified.

[59] The polypeptide according to any one of [4], [5], [9],

[10] ,

[13] ,

[19] ,

[20] , and

[56] to

[58] , comprising: a sequence in which amino acid residues corresponding to the methionine residue at position 141, the histidine residue at position 182, and the lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1 have been modified; a sequence in which amino acid residues corresponding to the methionine residue at position 141, the tryptophan residue at position 253, and the lysine residue at position 260 have been modified; and a sequence in which amino acid residues corresponding to the histidine residue at position 182, the tryptophan residue at position 253, and the lysine residue at position 260 have been modified.

[60] The polypeptide according to any one of [4], [5], [9],

[10] ,

[13] ,

[19] ,

[20] , and

[56] to

[59] , which has three alterations in the amino acid sequence represented by SEQ ID NO: 1, and which comprises a sequence in which the amino acid residue at the site corresponding to the methionine residue at position 141 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with one amino acid residue selected from the group consisting of tyrosine residue, valine residue, threonine residue, serine residue, leucine residue, lysine residue, isoleucine residue, and alanine residue.

[61] The polypeptide according to any one of [4], [5], [9],

[10] ,

[13] ,

[19] ,

[20] , and

[56] to

[59] , which has three alterations in the amino acid sequence represented by SEQ ID NO: 1, andThe polypeptide according to any one of [4], [5], [9],

[10] ,

[13] ,

[19] ,

[20] , and

[56] to

[60] , which comprises a sequence in which the amino acid residue at a position corresponding to the methionine residue at position 141 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with one amino acid residue selected from the group consisting of tyrosine residues, valine residues, and isoleucine residues.

[62] The polypeptide according to any one of [4], [5], [9],

[10] ,

[13] ,

[19] ,

[20] , and

[56] to

[61] , which comprises three modifications in the amino acid sequence represented by SEQ ID NO: 1, and which comprises a sequence in which the amino acid residue at a position corresponding to the histidine residue at position 182 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with one amino acid residue selected from the group consisting of methionine residues, leucine residues, and phenylalanine residues.

[63] The polypeptide according to any one of [4], [5], [9],

[10] ,

[13] ,

[19] ,

[20] , and

[56] to

[62] , which has three alterations in the amino acid sequence represented by SEQ ID NO: 1, and which comprises a sequence in which the amino acid residue at a position corresponding to the histidine residue at position 182 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with one amino acid residue selected from the group consisting of methionine residues and leucine residues.

[64] The polypeptide according to any one of [4], [5], [9],

[10] ,

[13] ,

[19] ,

[20] , and

[56] to

[63] , which has three alterations in the amino acid sequence represented by SEQ ID NO: 1, and which comprises a sequence in which the amino acid residue at a position corresponding to the tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with one amino acid residue selected from the group consisting of valine residues, threonine residues, serine residues, glutamine residues, methionine residues, leucine residues, histidine residues, phenylalanine residues, and glutamic acid.

[65] The amino acid sequence represented by SEQ ID NO: 1 has three alterations, andThe polypeptide according to any one of [4], [5], [9],

[10] ,

[13] ,

[19] ,

[20] , and

[56] to

[64] , which comprises a sequence in which the amino acid residue at a position corresponding to the tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with one amino acid residue selected from the group consisting of glutamine residues, methionine residues, leucine residues, and histidine residues.

[66] The polypeptide according to any one of [4], [5], [9],

[10] ,

[13] ,

[19] ,

[20] , and

[56] to

[65] , which comprises three alterations in the amino acid sequence represented by SEQ ID NO: 1, and which comprises a sequence in which the amino acid residue at a position corresponding to the lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with one amino acid residue selected from the group consisting of glutamine residues and glutamic acid residues.

[67] The amino acid sequence of SEQ ID NO: 1 in which three amino acid residues have been modified is: an amino acid sequence of SEQ ID NO: 33 in which X at the position corresponding to the amino acid residue at position 141 is a tyrosine residue, X at the position corresponding to the amino acid residue at position 182 is a methionine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (M141Y_H182M_K260E); an amino acid sequence of SEQ ID NO: 31 in which X at the position corresponding to the amino acid residue at position 141 is a valine residue, X at the position corresponding to the amino acid residue at position 253 is a methionine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (M141V_W253M_K260E); an amino acid sequence (H182L_W253H_K260E) represented by SEQ ID NO: 32, in which X at the position corresponding to the amino acid residue at position 182 is a leucine residue, X at the position corresponding to the amino acid residue at position 253 is a histidine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue;an amino acid sequence represented by SEQ ID NO:33 in which X at the position corresponding to the amino acid residue at position 141 is a tyrosine residue, X at the position corresponding to the amino acid residue at position 182 is a methionine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamine residue (M141Y_H182M_K260Q); an amino acid sequence represented by SEQ ID NO:33 in which X at the position corresponding to the amino acid residue at position 141 is a valine residue, X at the position corresponding to the amino acid residue at position 182 is a methionine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (M141V_H182M_K260E); an amino acid sequence represented by SEQ ID NO:33 in which X at the position corresponding to the amino acid residue at position 141 is a valine residue, X at the position corresponding to the amino acid residue at position 182 is a leucine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamine residue (M141V_H182L_K260Q); an amino acid sequence represented by SEQ ID NO:33 in which X at the position corresponding to the amino acid residue at position 141 is a tyrosine residue, X at the position corresponding to the amino acid residue at position 182 is a leucine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamine residue (M141Y_H182L_K260Q); an amino acid sequence represented by SEQ ID NO:31 in which X at the position corresponding to the amino acid residue at position 141 is a leucine residue, X at the position corresponding to the amino acid residue at position 253 is a glutamine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (M141L_W253Q_K260E); an amino acid sequence represented by SEQ ID NO:32 in which X at the position corresponding to the amino acid residue at position 182 is a leucine residue, X at the position corresponding to the amino acid residue at position 253 is a glutamine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (H182L_W253Q_K260E);an amino acid sequence represented by SEQ ID NO: 31, in which X at the position corresponding to the amino acid residue at position 141 is an isoleucine residue, X at the position corresponding to the amino acid residue at position 253 is a histidine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (M141I_W253H_K260E); an amino acid sequence represented by SEQ ID NO: 33, in which X at the position corresponding to the amino acid residue at position 141 is a valine residue, X at the position corresponding to the amino acid residue at position 182 is a leucine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (M141V_H182L_K260E); an amino acid sequence represented by SEQ ID NO:33, in which X at the position corresponding to the amino acid residue at position 141 is a tyrosine residue, X at the position corresponding to the amino acid residue at position 182 is a leucine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (M141Y_H182L_K260E); an amino acid sequence represented by SEQ ID NO:32, in which X at the position corresponding to the amino acid residue at position 182 is a leucine residue, X at the position corresponding to the amino acid residue at position 253 is a methionine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (H182L_W253M_K260E); an amino acid sequence represented by SEQ ID NO:31, in which X at the position corresponding to the amino acid residue at position 141 is a valine residue, X at the position corresponding to the amino acid residue at position 253 is a glutamine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamine residue (M141V_W253Q_K260Q); an amino acid sequence (M141V_W253H_K260Q) represented by SEQ ID NO: 31, in which X at the position corresponding to the amino acid residue at position 141 is a valine residue, X at the position corresponding to the amino acid residue at position 253 is a histidine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamine residue;an amino acid sequence represented by SEQ ID NO: 31, in which X at the position corresponding to the amino acid residue at position 141 is a valine residue, X at the position corresponding to the amino acid residue at position 253 is a leucine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamine residue (M141V_W253L_K260Q); an amino acid sequence represented by SEQ ID NO: 31, in which X at the position corresponding to the amino acid residue at position 141 is an isoleucine residue, X at the position corresponding to the amino acid residue at position 253 is a histidine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamine residue (M141I_W253H_K260Q); or The polypeptide according to any one of [4], [5], [9],

[10] ,

[13] ,

[19] ,

[20] , and

[56] to

[66] , which is represented by SEQ ID NO: 31 and has the amino acid sequence (M141V_W253M_K260Q) in which X at the position corresponding to the amino acid residue of position 141 is a valine residue, X at the position corresponding to the amino acid residue of position 253 is a methionine residue, and X at the position corresponding to the amino acid residue of position 260 is a glutamine residue.

[68] an amino acid sequence represented by SEQ ID NO: 33, in which X at the position corresponding to the amino acid residue at position 141 is a tyrosine residue, X at the position corresponding to the amino acid residue at position 182 is a methionine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (M141Y_H182M_K260E); an amino acid sequence represented by SEQ ID NO: 31, in which X at the position corresponding to the amino acid residue at position 141 is a valine residue, X at the position corresponding to the amino acid residue at position 253 is a methionine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (M141V_W253M_K260E); an amino acid sequence (H182L_W253H_K260E) represented by SEQ ID NO: 32, in which X at the position corresponding to the amino acid residue at position 182 is a leucine residue, X at the position corresponding to the amino acid residue at position 253 is a histidine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue;an amino acid sequence represented by SEQ ID NO:33 in which X at the position corresponding to the amino acid residue at position 141 is a tyrosine residue, X at the position corresponding to the amino acid residue at position 182 is a methionine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamine residue (M141Y_H182M_K260Q); an amino acid sequence represented by SEQ ID NO:33 in which X at the position corresponding to the amino acid residue at position 141 is a valine residue, X at the position corresponding to the amino acid residue at position 182 is a methionine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (M141V_H182M_K260E); an amino acid sequence represented by SEQ ID NO:33 in which X at the position corresponding to the amino acid residue at position 141 is a valine residue, X at the position corresponding to the amino acid residue at position 182 is a leucine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamine residue (M141V_H182L_K260Q); an amino acid sequence represented by SEQ ID NO:33 in which X at the position corresponding to the amino acid residue at position 141 is a tyrosine residue, X at the position corresponding to the amino acid residue at position 182 is a leucine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamine residue (M141Y_H182L_K260Q); an amino acid sequence represented by SEQ ID NO:31 in which X at the position corresponding to the amino acid residue at position 141 is a leucine residue, X at the position corresponding to the amino acid residue at position 253 is a glutamine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (M141L_W253Q_K260E); an amino acid sequence represented by SEQ ID NO:32 in which X at the position corresponding to the amino acid residue at position 182 is a leucine residue, X at the position corresponding to the amino acid residue at position 253 is a glutamine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (H182L_W253Q_K260E);an amino acid sequence represented by SEQ ID NO: 31, in which X at the position corresponding to the amino acid residue at position 141 is an isoleucine residue, X at the position corresponding to the amino acid residue at position 253 is a histidine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (M141I_W253H_K260E); an amino acid sequence represented by SEQ ID NO: 33, in which X at the position corresponding to the amino acid residue at position 141 is a valine residue, X at the position corresponding to the amino acid residue at position 182 is a leucine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (M141V_H182L_K260E); an amino acid sequence represented by SEQ ID NO:33, in which X at the position corresponding to the amino acid residue at position 141 is a tyrosine residue, X at the position corresponding to the amino acid residue at position 182 is a leucine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (M141Y_H182L_K260E); an amino acid sequence represented by SEQ ID NO:32, in which X at the position corresponding to the amino acid residue at position 182 is a leucine residue, X at the position corresponding to the amino acid residue at position 253 is a methionine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (H182L_W253M_K260E); an amino acid sequence represented by SEQ ID NO:31, in which X at the position corresponding to the amino acid residue at position 141 is a valine residue, X at the position corresponding to the amino acid residue at position 253 is a glutamine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamine residue (M141V_W253Q_K260Q); an amino acid sequence (M141V_W253H_K260Q) represented by SEQ ID NO: 31, in which X at the position corresponding to the amino acid residue at position 141 is a valine residue, X at the position corresponding to the amino acid residue at position 253 is a histidine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamine residue;an amino acid sequence represented by SEQ ID NO: 31, in which X at the position corresponding to the amino acid residue at position 141 is a valine residue, X at the position corresponding to the amino acid residue at position 253 is a leucine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamine residue (M141V_W253L_K260Q); an amino acid sequence represented by SEQ ID NO: 31, in which X at the position corresponding to the amino acid residue at position 141 is an isoleucine residue, X at the position corresponding to the amino acid residue at position 253 is a histidine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamine residue (M141I_W253H_K260Q); or The polypeptide according to any one of [4], [5], [9],

[10] ,

[13] ,

[19] ,

[20] , and

[56] to

[67] , comprising a sequence having 90% or more sequence identity with the amino acid sequence (M141V_W253M_K260Q) represented by SEQ ID NO: 31, in which X at the site corresponding to the amino acid residue of position 141 is a valine residue, X at the site corresponding to the amino acid residue of position 253 is a methionine residue, and X at the site corresponding to the amino acid residue of position 260 is a glutamine residue.

[69] an amino acid sequence represented by SEQ ID NO: 33, in which X at the position corresponding to the amino acid residue at position 141 is a tyrosine residue, X at the position corresponding to the amino acid residue at position 182 is a methionine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (M141Y_H182M_K260E); an amino acid sequence represented by SEQ ID NO: 31, in which X at the position corresponding to the amino acid residue at position 141 is a valine residue, X at the position corresponding to the amino acid residue at position 253 is a methionine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (M141V_W253M_K260E); an amino acid sequence (H182L_W253H_K260E) represented by SEQ ID NO: 32, in which X at the position corresponding to the amino acid residue at position 182 is a leucine residue, X at the position corresponding to the amino acid residue at position 253 is a histidine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue;an amino acid sequence represented by SEQ ID NO:33 in which X at the position corresponding to the amino acid residue at position 141 is a tyrosine residue, X at the position corresponding to the amino acid residue at position 182 is a methionine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamine residue (M141Y_H182M_K260Q); an amino acid sequence represented by SEQ ID NO:33 in which X at the position corresponding to the amino acid residue at position 141 is a valine residue, X at the position corresponding to the amino acid residue at position 182 is a methionine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (M141V_H182M_K260E); an amino acid sequence represented by SEQ ID NO:33 in which X at the position corresponding to the amino acid residue at position 141 is a valine residue, X at the position corresponding to the amino acid residue at position 182 is a leucine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamine residue (M141V_H182L_K260Q); an amino acid sequence represented by SEQ ID NO:33 in which X at the position corresponding to the amino acid residue at position 141 is a tyrosine residue, X at the position corresponding to the amino acid residue at position 182 is a leucine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamine residue (M141Y_H182L_K260Q); an amino acid sequence represented by SEQ ID NO:31 in which X at the position corresponding to the amino acid residue at position 141 is a leucine residue, X at the position corresponding to the amino acid residue at position 253 is a glutamine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (M141L_W253Q_K260E); an amino acid sequence represented by SEQ ID NO:32 in which X at the position corresponding to the amino acid residue at position 182 is a leucine residue, X at the position corresponding to the amino acid residue at position 253 is a glutamine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (H182L_W253Q_K260E);an amino acid sequence represented by SEQ ID NO: 31, in which X at the position corresponding to the amino acid residue at position 141 is an isoleucine residue, X at the position corresponding to the amino acid residue at position 253 is a histidine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (M141I_W253H_K260E); an amino acid sequence represented by SEQ ID NO: 33, in which X at the position corresponding to the amino acid residue at position 141 is a valine residue, X at the position corresponding to the amino acid residue at position 182 is a leucine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (M141V_H182L_K260E); an amino acid sequence represented by SEQ ID NO:33, in which X at the position corresponding to the amino acid residue at position 141 is a tyrosine residue, X at the position corresponding to the amino acid residue at position 182 is a leucine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (M141Y_H182L_K260E); an amino acid sequence represented by SEQ ID NO:32, in which X at the position corresponding to the amino acid residue at position 182 is a leucine residue, X at the position corresponding to the amino acid residue at position 253 is a methionine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue (H182L_W253M_K260E); an amino acid sequence represented by SEQ ID NO:31, in which X at the position corresponding to the amino acid residue at position 141 is a valine residue, X at the position corresponding to the amino acid residue at position 253 is a glutamine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamine residue (M141V_W253Q_K260Q); an amino acid sequence (M141V_W253H_K260Q) represented by SEQ ID NO: 31, in which X at the position corresponding to the amino acid residue at position 141 is a valine residue, X at the position corresponding to the amino acid residue at position 253 is a histidine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamine residue;an amino acid sequence represented by SEQ ID NO: 31, in which X at the position corresponding to the amino acid residue at position 141 is a valine residue, X at the position corresponding to the amino acid residue at position 253 is a leucine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamine residue (M141V_W253L_K260Q); an amino acid sequence represented by SEQ ID NO: 31, in which X at the position corresponding to the amino acid residue at position 141 is an isoleucine residue, X at the position corresponding to the amino acid residue at position 253 is a histidine residue, and X at the position corresponding to the amino acid residue at position 260 is a glutamine residue (M141I_W253H_K260Q); or The polypeptide according to any one of [4], [5], [9],

[10] ,

[13] ,

[19] ,

[20] , and

[56] to

[68] , comprising the amino acid sequence (M141V_W253M_K260Q) represented by SEQ ID NO: 31, in which X at the position corresponding to the amino acid residue of position 141 is a valine residue, X at the position corresponding to the amino acid residue of position 253 is a methionine residue, and X at the position corresponding to the amino acid residue of position 260 is a glutamine residue.

[70] The polypeptide according to any one of [4], [5], [9],

[10] ,

[13] ,

[19] ,

[20] , and

[56] to

[69] , having the amino acid sequence represented by any one of SEQ ID NOs: 45 to 62.

[71] The polypeptide according to any one of [6] to

[70] , which has a catalytic activity for the reductive amination reaction between any one or more of compound A represented by formula (1) or a salt thereof and any one or more of compound B represented by formula (2) or a salt thereof, or for the intramolecular reductive amination reaction between any one or more of compound B or a salt thereof, higher than the catalytic activity of a polypeptide having the amino acid sequence represented by SEQ ID NO: 1 under at least one reaction condition.

[72] The polypeptide according to any of [1] to

[71] , wherein the catalytic activity for the reductive amination reaction between any one or more of compound A represented by formula (1) or a salt thereof and any one or more of compound B represented by formula (2) or a salt thereof, or for the intramolecular reductive amination reaction of any one or more of compound B or a salt thereof, under at least one reaction condition, is higher than the catalytic activity of a polypeptide having the amino acid sequence represented by SEQ ID NO: 17.

[73] The polypeptide according to any of [6] to

[10] , wherein the alkylamine or a salt thereof is one or more selected from the group consisting of methylamine, ethylamine, and salts thereof.

[74] The polypeptide according to any of [6] to

[10] and

[73] , wherein the compound represented by formula (2') or a salt thereof is one or more selected from the group consisting of phenylpyruvic acid, 2-oxo-3-(p-tolyl)propanoic acid, 2-cyclopentyl-2-oxo-acetic acid, and salts thereof.

[75] The polypeptide according to any of [1] to

[74] , which has a catalytic activity for a reductive amination reaction between ethylamine or a salt thereof and phenylpyruvic acid or a salt thereof higher than the catalytic activity of a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 17, under at least one reaction condition.

[76] The polypeptide according to any of [1] to

[75] , which has a catalytic activity for a reductive amination reaction between ethylamine or a salt thereof and 2-oxo-3-(p-tolyl)propanoic acid or a salt thereof higher than the catalytic activity of a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 17, under at least one reaction condition.

[77] The polypeptide according to any of [1] to

[76] , which has a catalytic activity for a reductive amination reaction between methylamine or a salt thereof and 2-cyclopentyl-2-oxo-acetic acid or a salt thereof higher than the catalytic activity of a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 17, under at least one reaction condition.

[78] The polypeptide according to any one of [1] to

[77] , which has catalytic activity for the reductive amination reaction of ammonia or a salt thereof with phenylpyruvic acid or a salt thereof under at least one reaction condition.

[79]

[80] The polypeptide according to any one of [1] to

[79] , wherein the modification is one or more selected from the group consisting of substitution, deletion, and insertion.

[81] The polypeptide according to any one of [1] to

[80] , wherein the modification is a conservative modification.

[82] The polypeptide according to any one of [1] to

[81] , wherein the modification is a substitution with a natural amino acid different from the amino acid residue before the modification.

[83] The polypeptide according to

[82] , wherein the natural amino acid is one or more selected from the group consisting of glycine, alanine, serine, threonine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, histidine, glutamine, asparagine, glutamic acid, aspartic acid, methionine, lysine, arginine, and proline.

[84] The polypeptide according to any one of [1] to

[83] , which comprises one or more sequences selected from the group consisting of a streptavidin-binding peptide tag sequence and a His tag sequence at either or both of the N-terminus and the C-terminus.

[85] The polypeptide according to any one of [1] to

[84] , which has 300 to 400 amino acid residues when present as a monomer.

[86] The polypeptide according to any one of [1] to

[85] , wherein the temperature at which the catalytic activity is higher than that of the polypeptide having the amino acid sequence represented by SEQ ID NO: 1 is in the range of 0°C to 50°C.

[87] The polypeptide according to any one of [1] to

[86] , wherein the pH at which the catalytic activity is higher than that of the polypeptide having the amino acid sequence represented by SEQ ID NO: 1 is in the range of 7 to 11.

[88] The polypeptide according to any one of [1] to [5], wherein the catalytic activity is higher than that of the polypeptide having the amino acid sequence represented by SEQ ID NO: 1 at least at one point in the range of 100 mM to 3000 mM in the total amount of reaction solution during reductive amination.

[89] The polypeptide according to any one of [1] to [5] and

[88] , wherein the catalytic activity is higher than that of the polypeptide having the amino acid sequence represented by SEQ ID NO: 1 at least at one point when the total concentration of compound B and its salt in the total amount of reaction solution at the start of the reductive amination reaction is in the range of 0.001 mM to 1000 mM.

[90] The polypeptide according to any one of [1] to [5] and

[88] and

[89] , wherein the catalytic activity is higher than that of the polypeptide having the amino acid sequence represented by SEQ ID NO: 1 at least at one point when the ratio of the total number of moles of compound A and its salt to the total number of moles of compound B and its salt in the total amount of reaction solution at the start of the reductive amination reaction is 1 or more.

[91] The polypeptide according to any one of [1] to

[90] , wherein the catalytic activity is higher than that of the polypeptide having the amino acid sequence represented by SEQ ID NO: 1 under reaction conditions of 37°C and pH 8.

[92] The polypeptide according to any one of [1] to

[91] , which has a catalytic activity higher than that of a polypeptide having the amino acid sequence represented by SEQ ID NO: 1 under reaction conditions of 25°C and pH 9.

[93] The polypeptide according to any one of [1] to

[92] , wherein the catalytic activity is evaluated under the following reaction conditions: 50 mM phenylpyruvic acid, 2-oxo-3-(p-tolyl)propanoic acid, or 2-cyclopentyl-2-oxo-acetic acid, 100 mM D(+)-glucose, 500 mM methylamine or ethylamine, 100 mM phosphate buffer, 0.89 mM β-NADPH, 0.002 unit / µL GDH solution, and 2.5 µM of the polypeptide to be evaluated. The reaction is started under conditions of 37°C and pH 8, and the yield of amino acid produced by the reductive amination reaction is determined after 19 hours.

[94] The polypeptide according to any one of [1] to

[93] , which is an enzyme that catalyzes a reductive amination reaction.

[95] The polypeptide according to any one of [1] to

[94] , wherein the catalytic activity is 1.2 times or more that of a polypeptide having the amino acid sequence represented by SEQ ID NO: 1.

[96] In the formula (1), R １ is a hydrogen atom, and R２ is C １ ~C ６

[97] The polypeptide according to any one of [1] to [5] and

[88] to

[90] , wherein R is an alkyl group. １ is a hydrogen atom, and R ２

[98] The polypeptide according to any one of [1] to [5],

[88] to

[90] , and

[96] , wherein R is an ethyl group. １ is a hydrogen atom, and R ２

[99] The polypeptide according to any one of [1] to [5],

[88] to

[90] , and

[96] , wherein R is a methyl group. １ and R ２

[100] The polypeptide according to any one of [1] to [5] and

[88] to

[90] , wherein in formula (2), Y is C ３ ~C ８ Cycloalkyl group or C ６ ~C ９ an aralkyl group, the aralkyl group being C １ ~C ３ The polypeptide according to any one of [1] to [5],

[88] to

[90] , and

[96] to

[99] , which may be substituted with an alkyl group or a halogen atom.

[101] In formula (2), Y is C ３ ~C ８

[102] The polypeptide according to any one of [1] to [5],

[88] to

[90] , and

[96] to

[100] , wherein Y is a cycloalkyl group. ６ ~C ９ an aralkyl group, the aralkyl group being C １ ~C ３

[103] The polypeptide according to any one of [1] to [5],

[88] to

[90] , and

[96] to

[100] , wherein in the formula (1), R １ is a hydrogen atom, and R ２ is an ethyl group, in the formula (2), n and m are both 0, Y is a group represented by the formula (3), and R ３ is a phenyl group, and R ４ , R５ and R ６

[104] The polypeptide according to any one of [1] to [5] and

[88] to

[90] , wherein R are hydrogen atoms, and p, q, and r are all 1. １ is a hydrogen atom, and R ２ is an ethyl group, in the formula (2), n and m are both 0, Y is a group represented by the formula (3), and R ３ is a p-tolyl group, and R ４ , R ５ and R ６

[105] The polypeptide according to any one of [1] to [5] and

[88] to

[90] , wherein each of R is a hydrogen atom, and p, q, and r are each 1. １ is a hydrogen atom, and R ２ is a methyl group, in the formula (2), n and m are both 0, Y is a group represented by the formula (3), and R ３ and R ４ are linked together to form a cyclopentane ring, and R ５ and R ６are all hydrogen atoms, and p, q, and r are all 1.

[106] An isolated nucleic acid encoding the polypeptide according to any of [1] to

[105] .

[107] A vector comprising the nucleic acid according to

[106] .

[108] A transformant comprising the nucleic acid according to

[106] or the vector according to

[107] .

[109] A method for producing a polypeptide, the method comprising a step of culturing the transformant according to

[108] so as to produce the polypeptide.

[110] The method according to

[109] , further comprising a step of recovering the polypeptide.

[111] A reductive amination agent comprising the polypeptide according to any of [1] to

[105] .

[112] A method for producing an amino acid, comprising the step of reacting one or more compounds selected from the group consisting of amines, amine analogs, and salts thereof with one or more compounds selected from the group consisting of keto acids, keto acid analogs, and salts thereof, or reacting a compound selected from the group consisting of keto acids, keto acid analogs, and salts thereof intramolecularly in the presence of the polypeptide according to any one of [1] to

[105] and a reducing agent.

[113] The production method according to

[112] , wherein the reaction is carried out under appropriate conditions.

[114] The production method according to

[112] or

[113] , wherein the reaction involves a reductive amination reaction between an amine, an amine analog, or a salt thereof and a keto acid, a keto acid analog, or a salt thereof.

[115] The amine or amine analog is represented by the above formula (1), and R in formula (1) １ and R ２ is R according to formula (1) of [1] １ and R ２

[116] The method according to any one of

[112] to

[114] , wherein the keto acid or keto acid analog is represented by the above formula (2), and in the formula (2), the formula (1'), the formula (3), X, Y, Z １ , Z ２ , R １ａ , R ３ , R ４ , R ５ , R ６, m, n, p, q and r are the formula (1') or formula (3) described in formula (2) of [1], X, Y, Z １ , Z ２ , R １ａ , R ３ , R ４ , R ５ , R ６ , m, n, p, q, and r are respectively defined as Y' in [6].

[117] The method according to any of

[112] to

[116] , wherein the keto acid or keto acid analog is represented by formula (2'), and Y' in formula (2') is defined as Y' in [6].

[118] The method according to any of

[112] to

[117] , wherein the reducing agent is one or more selected from the group consisting of nicotinamide adenine dinucleotide phosphate (NADPH), NADP+, nicotinamide adenine dinucleotide (NADH), and NAD+.

[119] The method according to any of

[112] to

[118] , wherein the reaction is carried out in the presence of compound C represented by the following formula (4): In formula (4), v and w each independently represent 0 or 1, at least one of v and w represents 1, T represents a carbon atom, a phosphorus atom, or a sulfur atom, and a compound represented by the following formula (4a): The functional group represented by the formula (4a) represents ═O, —ORd, or a hydroxy group; when v and w are both 1, two functional groups represented by the formula (4a) may be the same or different; Ra, Rb, and Rc each independently represent a hydrogen atom, C １ ~C ３ Alkyl group, alkylamino group or -CH ２ any two or more of Ra, Rb, and Rc may be bonded to each other together with T to form a ring structure, and Rd represents C １ ~C ３represents an alkyl group, d, e, and f each independently represent 0 or 1, any one or more of d, e, and f represent 1, when v and w are both 1, any one or more of Ra, Rb, and Rc are methyl groups, and Ra, Rb, and Rc are not bonded to each other to form a ring structure together with T, when any one or more of Ra, Rb, and Rc are methylamino groups, Ra, Rb, and Rc are not bonded to each other to form a ring structure together with T, when the functional group represented by formula (4a) is a hydroxy group, and T is a carbon atom, v is 1, w is 0, d, e, and f are all 1, and Ra, Rb, and Rc are all hydrogen atoms.]

[120] The production method according to

[119] , wherein in formula (4), T is a phosphorus atom or a sulfur atom, the functional group represented by formula (4a) is ═O, and Ra, Rb, and Rc are all methyl groups.

[121] The manufacturing method according to

[119] or

[120] , wherein the compound C is one or more compounds selected from the group consisting of dimethyl sulfoxide, dimethyl sulfone, dimethoxyethane, trimethylphosphine oxide, N,N-dimethylformamide, N,N-dimethylacetamide, tetramethylene sulfoxide, diethyl sulfoxide, methanol, and methylformamide.

[122] The manufacturing method according to any of

[119] to

[121] , wherein the compound C is dimethyl sulfoxide.

[123] The manufacturing method according to any of

[112] to

[122] , comprising the following steps (A) and (B): Step (A): A step of contacting a target product to be purified, which is a mixture of the following (i) as a target product to be purified and the following (ii) as an impurity, obtained by the method according to any of

[112] to

[122] , with a lithium-containing substance: (i) the amino acid having a protecting group at the N-terminus; and (ii) a compound other than the target product to be purified. Step (B): A step of precipitating a lithium salt of the target product to be purified.

[124] A method for producing a peptide compound, comprising the following steps: (1) producing an amino acid by the method according to any one of

[112] to

[122] ; and (2) linking the amino acid to one or more selected from the group consisting of other amino acids and peptides to produce a peptide compound.

[0017] According to the present invention, a novel polypeptide that can be used as an enzyme that catalyzes a reductive amination reaction can be provided, and a method for producing an amino acid using the polypeptide can be provided.

[0018] FIG. 1 shows the compound (2S,3S)-2-amino-3-phenyl-butanoic acid (I) obtained in the reaction in the examples, the purchased standard compound (2S,3R)-2-amino-3-phenyl-butanoic acid hydrochloride (II), and (2S,3S)-2-amino-3-phenyl-butanoic acid hydrochloride (III). １ 2 shows the chiral HPLC analysis data of the compound (2S,3S)-2-amino-3-phenyl-butanoic acid (I) obtained in the reaction in the examples, and a mixture (V) prepared by mixing purchased authentic compounds (III) and (IV) in a ratio of (III):(IV)=7:3.

[0019] Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments.

[0020] As used herein, "one or more" means one or more than one. When "one or more" is used in the context of substituents on a group, the term means a number from one to the maximum number of substituents permitted by that group. Specific examples of "one or more" include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and / or more.

[0021] In this specification, the term "to" indicating a range includes both ends of the range. For example, "A to B" means a range equal to or greater than A and equal to or less than B.

[0022] As used herein, the term "about" when used in conjunction with a numerical value means a range of values ​​of plus and minus 10% of that numerical value.

[0023] In this specification, the meaning of the term "and / or" includes any combination of "and" and "or" appropriately combined. Specifically, for example, "A, B and / or C" includes the following seven variations: (i) A, (ii) B, (iii) C, (iv) A and B, (v) A and C, (vi) B and C, and (vii) A, B and C.

[0024] In this specification, the term "alkyl group" refers to a monovalent group derived from an aliphatic hydrocarbon by removing any one hydrogen atom, and does not contain heteroatoms (atoms other than carbon atoms and hydrogen atoms) or unsaturated carbon-carbon bonds in the skeleton, but has a hydrocarbyl or hydrocarbon group structure subset containing hydrogen atoms and carbon atoms. The alkyl group includes not only linear ones but also branched ones. Specific examples of alkyl groups include those having 1 to 20 carbon atoms (C １ ~C ２０ , hereinafter referred to as “C ｐ ~C ｑ " means that the number of carbon atoms is p to q), and preferably C １ ~C １０ alkyl group, more preferably C １ ~C ６ Specific examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an i-propyl group, an n-butyl group, an s-butyl group, a t-butyl group, an isobutyl (2-methylpropyl) group, an n-pentyl group, an s-pentyl (1-methylbutyl) group, a t-pentyl (1,1-dimethylpropyl) group, a neopentyl (2,2-dimethylpropyl) group, an isopentyl (3-methylbutyl) group, a 3-pentyl (1-ethylpropyl) group, a 1,2-dimethylpropyl group, a 2-methylbutyl group, an n-hexyl group, a 1,1,2-trimethylpropyl group, a 1,2,2-trimethylpropyl group, a 1,1,2,2-tetramethylpropyl group, a 1,1-dimethylbutyl group, a 1,2-dimethylbutyl group, a 1,3-dimethylbutyl group, a 2,2-dimethylbutyl group, a 2,3-dimethylbutyl group, a 3,3-dimethylbutyl group, a 1-ethylbutyl group, and a 2-ethylbutyl group.

[0025] As used herein, the term "alkenyl group" refers to an alkenyl group having at least one double bond (two adjacent SP ２ Depending on the configuration of the double bond and the substituents (if any), the geometry of the double bond can be Entgegen (E) or Zusammen (Z), cis or trans. Alkenyl groups include not only straight chain but also branched chain. The alkenyl group is preferably C ２ ~C １０ Alkenyl groups, more preferably C ２ ~C ６ Examples of alkenyl groups include a vinyl group, an allyl group, a 1-propenyl group, a 2-propenyl group, a 1-butenyl group, a 2-butenyl group (including cis and trans), a 3-butenyl group, a pentenyl group, a 3-methyl-2-butenyl group, and a hexenyl group.

[0026] As used herein, the term "alkynyl group" refers to a monovalent group having at least one triple bond (two adjacent SP carbon atoms). The alkynyl group includes not only linear but also branched groups. The preferred alkynyl group is C ２ ~C １０ Alkynyl groups, more preferably C ２ ~C ６ Specific examples thereof include an alkynyl group, such as an ethynyl group, a 1-propynyl group, a propargyl group, a 3-butynyl group, a pentynyl group, a hexynyl group, a 3-phenyl-2-propynyl group, a 3-(2'-fluorophenyl)-2-propynyl group, a 2-hydroxy-2-propynyl group, a 3-(3-fluorophenyl)-2-propynyl group, and a 3-methyl-(5-phenyl)-4-pentynyl group.

[0027] In the present specification, the term "cycloalkyl group" refers to a saturated or partially saturated cyclic monovalent aliphatic hydrocarbon group, including a monocyclic, bicyclic, and spirocyclic ring. ３ ~C ８Examples thereof include cycloalkyl groups, and specific examples thereof include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, a cyclooctyl group, a bicyclo[2.2.1]heptyl group, and a spiro[3.3]heptyl group.

[0028] As used herein, the term "aryl group" refers to a monovalent aromatic hydrocarbon ring, preferably C ６ ~C １０ Examples of the aryl group include a phenyl group and a naphthyl group (for example, 1-naphthyl, 2-naphthyl).

[0029] As used herein, the term "heterocyclyl group" refers to a non-aromatic, cyclic, monovalent group containing, in addition to carbon atoms, 1 to 5 heteroatoms. The heterocyclyl group may have a double and / or triple bond in the ring, and a carbon atom in the ring may be oxidized to form a carbonyl, and may be a monocyclic or fused ring. The number of atoms constituting the ring is preferably 4 to 10 (4- to 10-membered heterocyclyl group), more preferably 4 to 7 (4- to 7-membered heterocyclyl group). Specific examples of the heterocyclyl group include an azetidinyl group, an oxiranyl group, an oxetanyl group, a dihydrofuryl group, a tetrahydrofuryl group, a dihydropyranyl group, a tetrahydropyranyl group, a tetrahydropyridyl group, a tetrahydropyrimidyl group, a morpholinyl group, a thiomorpholinyl group, a pyrrolidinyl group, a piperidinyl group, a piperazinyl group, a pyrazolidinyl group, an imidazolinyl group, an imidazolidinyl group, an oxazolidinyl group, an isoxazolidinyl group, a thiazolidinyl group, an isothiazolidinyl group, and an isothiazolidinyl group. azolidinyl group, 1,2-thiazinane group, thiadiazolidinyl group, oxazolidone group, benzodioxanyl group, benzoxazolyl group, dioxolanyl group, dioxanyl group, tetrahydropyrrolo[1,2-c]imidazole group, thietanyl group, 3,6-diazabicyclo[3.1.1]heptanyl group, 2,5-diazabicyclo[2.2.1]heptanyl group, 3-oxa-8-azabicyclo[3.2.1]octanyl group, sultam group, 2-oxaspiro[3.3]heptyl group, and the like.

[0030] As used herein, the term "protected heterocyclyl group" refers to a group in which one or more functional groups included in the above-defined "heterocyclyl group," such as an amino group, are protected with any protecting group, and preferably includes a protected 4- to 7-membered heterocyclyl group. Specific examples of the protecting group include Boc, Fmoc, Cbz, Troc, and Alloc, and specific examples of the protected heterocyclyl group include a Boc-protected azetidine group.

[0031] As used herein, the term "heterocycloalkylidene group" refers to a divalent group resulting from the removal of two hydrogen atoms from one carbon atom of a "heterocyclyl group" as defined above, in which the free valence becomes part of a double bond. Preferred examples of heterocycloalkylidene groups include 4- to 7-membered heterocycloalkylidene groups, and specific examples include tetrahydropyran-4-ylidene and azetidin-3-ylidene groups.

[0032] As used herein, the term "protected heterocycloalkylidene group" refers to a group in which one or more functional groups included in the above-defined "heterocycloalkylidene group," such as an amino group, are protected with any protecting group, and preferably includes a protected 4- to 7-membered heterocycloalkylidene group. Specific examples of the protecting group include Boc, Fmoc, Cbz, Troc, and Alloc, and specific examples of the protected heterocycloalkylidene group include a Boc-protected azetidin-3-ylidene group.

[0033] As used herein, the term "heteroaryl group" refers to an aromatic, cyclic, monovalent group containing 1 to 5 heteroatoms in addition to carbon atoms. The ring may be a monocyclic ring or a condensed ring with another ring, and may be partially saturated. The number of atoms constituting the ring may be 5 to 12 (5- to 12-membered heteroaryl), 6 to 10 (6- to 10-membered heteroaryl), or 6 to 7 (6- to 7-membered heteroaryl). Specific examples of heteroaryl groups include furyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, triazinyl, benzofuranyl, benzothienyl, benzothiadiazolyl, benzothiazolyl, benzoxazolyl, benzoxadiazolyl, benzimidazolyl, indolyl, isoindolyl, indazolyl, quinolyl, isoquinolyl, cinnolinyl, quinazolinyl, quinoxalinyl, benzodioxolyl, indolizinyl, and imidazopyridyl groups.

[0034] In the present specification, the term "alkoxy group" refers to an oxy group to which the above-defined "alkyl group" is bonded, and preferably C １ ~C ６ Specific examples of the alkoxy group include a methoxy group, an ethoxy group, a 1-propoxy group, a 2-propoxy group, an n-butoxy group, an i-butoxy group, an s-butoxy group, a t-butoxy group, a pentyloxy group, and a 3-methylbutoxy group.

[0035] As used herein, the term "alkylthio group" refers to a thiol group to which the above-defined "alkyl group" is bonded, and preferably C １ ~C ６ Specific examples of the alkylthio group include a methylthio group, an ethylthio group, a 1-propylthio group, a 2-propylthio group, an n-butylthio group, an i-butylthio group, an s-butylthio group, and a t-butylthio group.

[0036] As used herein, the term "alkenyloxy group" refers to an oxy group to which the above-defined "alkenyl group" is bonded, and preferably C ２ ~C ６ Specific examples of the alkenyloxy group include a vinyloxy group, an allyloxy group, a 1-propenyloxy group, a 2-propenyloxy group, a 1-butenyloxy group, a 2-butenyloxy group (including cis and trans), a 3-butenyloxy group, a pentenyloxy group, and a hexenyloxy group.

[0037] As used herein, the term "cycloalkoxy group" refers to an oxy group to which the above-defined "cycloalkyl group" is bonded, and preferably C ３ ~C ８ Examples of the cycloalkoxy group include a cyclopropoxy group, a cyclobutoxy group, and a cyclopentyloxy group.

[0038] As used herein, the term "aryloxy group" refers to an oxy group to which the above-defined "aryl group" is bonded, and preferably C ６ ~C １０ Examples of the aryloxy group include a phenoxy group, a 1-naphthyloxy group, and a 2-naphthyloxy group.

[0039] In the present specification, the term "amino group" means, in a narrow sense, -NH ２ and in a broader sense, -NRR', where R and R' are independently selected from a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a heterocyclyl group, an aryl group, or a heteroaryl group, or R and R' together with the nitrogen atom to which they are attached form a ring. ２ , Mono C １ ~C ６ Alkylamino group, diC １ ~C ６ Examples include alkylamino groups and 4- to 8-membered cyclic amino groups.

[0040] In the present specification, the term "monoalkylamino group" refers to an "amino group" as defined above, in which R is a hydrogen atom and R' is an "alkyl group" as defined above, and preferably a mono-C １ ~C ６ Specific examples of the monoalkylamino group include a methylamino group, an ethylamino group, an n-propylamino group, an i-propylamino group, an n-butylamino group, an s-butylamino group, and a t-butylamino group.

[0041] As used herein, the term "dialkylamino group" refers to an "amino group" as defined above, in which R and R' are independently an "alkyl group" as defined above, and preferably a diC １ ~C ６ Specific examples of the dialkylamino group include a dimethylamino group and a diethylamino group.

[0042] As used herein, the term "cyclic amino group" refers to the above-defined "amino group" in which R and R' form a ring together with the nitrogen atom to which they are bonded, and preferably includes 4- to 8-membered cyclic amino groups. Specific examples of the cyclic amino group include a 1-azetidyl group, a 1-pyrrolidyl group, a 1-piperidyl group, a 1-piperazyl group, a 4-morpholinyl group, a 3-oxazolidyl group, a 1,1-dioxidethiomorpholinyl-4-yl group, and a 3-oxa-8-azabicyclo[3.2.1]octan-8-yl group.

[0043] As used herein, the term "protected amino group" refers to an amino group protected with any protecting group. Specific examples of the protected amino include amino groups protected with protecting groups such as Boc, Fmoc, Cbz, Troc, and Alloc.

[0044] As used herein, the term "aminocarbonyl group" refers to a carbonyl group to which the above-defined "amino group" is bonded, and preferably -CONH ２ , Mono C １ ~C ６ Alkylaminocarbonyl group, diC １ ~C ６Examples of the aminocarbonyl group include an alkylaminocarbonyl group and a 4- to 8-membered cyclic aminocarbonyl group. ２ , dimethylaminocarbonyl group, 1-azetidinylcarbonyl group, 1-pyrrolidinylcarbonyl group, 1-piperidinylcarbonyl group, 1-piperazinylcarbonyl group, 4-morpholinylcarbonyl group, 3-oxazolidinylcarbonyl group, 1,1-dioxidethiomorpholinyl-4-ylcarbonyl group, 3-oxa-8-azabicyclo[3.2.1]octan-8-ylcarbonyl group, and the like.

[0045] As used herein, the term "alkenyloxycarbonyl group" refers to a carbonyl group to which the above-defined "alkenyloxy group" is bonded, and preferably C ２ ~C ６ Specific examples of the alkenyloxycarbonyl group include a vinyloxycarbonyl group, an allyloxycarbonyl group, a 1-propenyloxycarbonyl group, a 2-propenyloxycarbonyl group, a 1-butenyloxycarbonyl group, a 2-butenyloxycarbonyl group (including cis and trans), a 3-butenyloxycarbonyl group, a pentenyloxycarbonyl group, and a hexenyloxycarbonyl group.

[0046] As used herein, the term "alkylsulfonyl group" refers to a sulfonyl group having an "alkyl group" as defined above bonded thereto, and preferably C １ ~C ６ Examples of the alkylsulfonyl group include a methylsulfonyl group.

[0047] The term "hydroxyalkyl group" as used herein means a group in which one or more hydrogen atoms of the "alkyl group" defined above have been substituted with hydroxyl groups, and C １ ~C ６ Hydroxyalkyl groups are preferred, and specific examples of the hydroxyalkyl group include a hydroxymethyl group, a 1-hydroxyethyl group, a 2-hydroxyethyl group, a 2-hydroxy-2-methylpropyl group, and a 5-hydroxypentyl group.

[0048] The term "haloalkyl group" as used herein means a group in which one or more hydrogen atoms of the "alkyl group" defined above are substituted with halogen atoms, and C １ ~C ６ Haloalkyl groups are preferred, C １ ~C ６ Specific examples of haloalkyl groups include a difluoromethyl group, a trifluoromethyl group, a 2,2-difluoroethyl group, a 2,2,2-trifluoroethyl group, a 3,3-difluoropropyl group, a 4,4-difluorobutyl group, and a 5,5-difluoropentyl group.

[0049] The term "cyanoalkyl group" as used herein means a group in which one or more hydrogen atoms of the above-defined "alkyl group" are substituted with a cyano group, and C １ ~C ６ Cyanoalkyl groups are preferred, and specific examples of cyanoalkyl groups include cyanomethyl and 2-cyanoethyl groups.

[0050] The term "aminoalkyl group" as used herein means a group in which one or more hydrogen atoms of an "alkyl group" as defined above are substituted with an "amino group" as defined above, and C １ ~C ６ An aminoalkyl group is preferred. Specific examples of the aminoalkyl group include a 1-pyridylmethyl group, a 2-(1-piperidyl)ethyl group, a 3-(1-piperidyl)propyl group, and a 4-aminobutyl group.

[0051] The term "carboxyalkyl group" as used herein means a group in which one or more hydrogen atoms of the "alkyl group" defined above are substituted with a carboxy group, and C ２ ~C ６ Carboxyalkyl groups are preferred. Specific examples of carboxyalkyl groups include carboxymethyl groups.

[0052] The term "alkenyloxycarbonylalkyl group" as used herein means an "alkyl group" defined above in which one or more hydrogen atoms are substituted with an "alkenyloxycarbonyl group" defined above, and C ２ ~C ６ Alkenyloxycarbonyl C １ ~C ６ Alkyl groups are preferred, and C ２ ~C ６ Alkenyloxycarbonyl C １ ~C ２ Alkyl groups are more preferred. Specific examples of alkenyloxycarbonylalkyl groups include an allyloxycarbonylmethyl group and a 2-(allyloxycarbonyl)ethyl group.

[0053] The term "alkoxyalkyl group" as used herein means a group in which one or more hydrogen atoms of an "alkyl group" as defined above are substituted with an "alkoxy group" as defined above, and C １ ~C ６ Alkoxy C １ ~C ６ Alkyl groups are preferred, and C １ ~C ６ Alkoxy C １ ~C ２ Specific examples of the alkoxyalkyl group include a methoxymethyl group, an ethoxymethyl group, a 1-propoxymethyl group, a 2-propoxymethyl group, an n-butoxymethyl group, an i-butoxymethyl group, an s-butoxymethyl group, a t-butoxymethyl group, a pentyloxymethyl group, a 3-methylbutoxymethyl group, a 1-methoxyethyl group, a 2-methoxyethyl group, and a 2-ethoxyethyl group.

[0054] The term "alkylthioalkyl group" as used herein means a group in which one or more hydrogen atoms of an "alkyl group" as defined above are substituted with an "alkylthio group" as defined above, and C １ ~C ６ Alkylthio C １ ~C ６ Alkyl groups are preferred, and C １ ~C ６ Alkylthio C １ ~C ２Specific examples of the alkylthioalkyl group include a methylthiomethyl group, an ethylthiomethyl group, a 1-propylthiomethyl group, a 2-propylthiomethyl group, an n-butylthiomethyl group, an i-butylthiomethyl group, an s-butylthiomethyl group, and a t-butylthiomethyl group.

[0055] The term "alkenyloxyalkyl group" as used herein means a group in which one or more hydrogen atoms of an "alkyl group" as defined above are substituted with an "alkenyloxy group" as defined above, and C ２ ~C ６ Alkenyloxy C １ ~C ６ Alkyl groups are preferred, and C １ ~C ６ Alkenyloxy C １ ~C ２ Alkyl groups are more preferred. Specific examples of alkenyloxyalkyl groups include vinyloxymethyl and allyloxymethyl groups.

[0056] The term "cycloalkylalkyl group" as used herein means a group in which one or more hydrogen atoms of an "alkyl group" as defined above are substituted with a "cycloalkyl group" as defined above, and C ３ ~C ８ Cycloalkyl C １ ~C ６ Alkyl groups are preferred, and C ３ ~C ６ Cycloalkyl C １ ~C ２ An alkyl group is more preferred. Specific examples of the cycloalkylalkyl group include a cyclopropylmethyl group, a cyclobutylmethyl group, a cyclopentylmethyl group, and a cyclohexylmethyl group.

[0057] The term "cycloalkoxyalkyl group" as used herein means a group in which one or more hydrogen atoms of an "alkyl group" as defined above are substituted with a "cycloalkoxy group" as defined above, and C ３ ~C ８ Cycloalkoxy C １ ~C ６ Alkyl groups are preferred, and C ３ ~C６ Cycloalkoxy C １ ~C ２ An alkyl group is more preferred. Specific examples of the cycloalkoxyalkyl group include a cyclopropoxymethyl group and a cyclobutoxymethyl group.

[0058] As used herein, the term "heterocyclylalkyl group" refers to a group in which one or more hydrogen atoms of an "alkyl group" defined above are substituted with a "heterocyclyl group" defined above, and includes 4- to 7-membered heterocyclyl C １ ~C ６ Alkyl groups are preferred, and 4- to 7-membered heterocyclyl C １ ~C ２ An alkyl group is more preferred. Specific examples of heterocyclylalkyl groups include a 2-(tetrahydro-2H-pyran-4-yl)ethyl group and a 2-(azetidin-3-yl)ethyl group.

[0059] The term "alkylsulfonylalkyl group" as used herein means a group in which one or more hydrogen atoms of an "alkyl group" as defined above are substituted with an "alkylsulfonyl group" as defined above, and C １ ~C ６ Alkylsulfonyl C １ ~C ６ Alkyl groups are preferred, and C １ ~C ６ Alkylsulfonyl C １ ~C ２ An alkyl group is more preferred. Specific examples of the alkylsulfonylalkyl group include a methylsulfonylmethyl group and a 2-(methylsulfonyl)ethyl group.

[0060] The term "aminocarbonylalkyl group" as used herein means a group in which one or more hydrogen atoms of an "alkyl group" as defined above are substituted with an "aminocarbonyl group" as defined above, and an aminocarbonyl C １ ~C ６ Alkyl groups are preferred, and aminocarbonyl C １ ~C ４Specific examples of the aminocarbonylalkyl group include a methylaminocarbonylmethyl group, a dimethylaminocarbonylmethyl group, a t-butylaminocarbonylmethyl group, a 1-azetidinylcarbonylmethyl group, a 1-pyrrolidinylcarbonylmethyl group, a 1-piperidinylcarbonylmethyl group, a 4-morpholinylcarbonylmethyl group, a 2-(methylaminocarbonyl)ethyl group, a 2-(dimethylaminocarbonyl)ethyl group, a 2-(1-azetidinylcarbonyl)ethyl group, a 2-(1-pyrrolidinylcarbonyl)ethyl group, a 2-(4-morpholinylcarbonyl)ethyl group, a 3-(dimethylaminocarbonyl)propyl group, and a 4-(dimethylaminocarbonyl)butyl group.

[0061] The term "aryloxyalkyl group" as used herein means a group in which one or more hydrogen atoms of an "alkyl group" as defined above are substituted with an "aryloxy group" as defined above, and C ６ ~C １０ Aryloxy C １ ~C ６ Alkyl groups are preferred, and C ６ ~C １０ Aryloxy C １ ~C ２ An alkyl group is more preferred. Specific examples of the aryloxyalkyl group include a phenoxymethyl group and a 2-phenoxyethyl group.

[0062] As used herein, the term "aralkyl (arylalkyl) group" refers to a group in which at least one hydrogen atom of an "alkyl group" as defined above is substituted with an "aryl group" as defined above, and C ７ ~C １４ An aralkyl group is preferred, and C ７ ~C １０ An aralkyl group is more preferred. Specific examples of the aralkyl group include a benzyl group, a phenethyl group, and a 3-phenylpropyl group.

[0063] In the present specification, the term "aralkoxy group" refers to an oxy group to which the above-defined "aralkyl group" is bonded, and ７ ~C １４ An aralkoxy group is preferred, and C７ ~C １０ An aralkoxy group is more preferred. Specific examples of the aralkoxy group include a benzyloxy group, a phenethyloxy group, and a 3-phenylpropoxy group.

[0064] The term "aralkoxyalkyl group" as used herein means a group in which one or more hydrogen atoms of an "alkyl group" as defined above are substituted with an "aralkoxy group" as defined above, and C ７ ~C １４ Aralkoxy C １ ~C ６ Alkyl groups are preferred, and C ７ ~C １４ Aralkoxy C １ ~C ２ An alkyl group is more preferred. Specific examples of the aralkoxyalkyl group include a benzyloxymethyl group and a 1-(benzyloxy)ethyl group.

[0065] As used herein, the term "heteroarylalkyl group" refers to a group in which at least one hydrogen atom of an "alkyl group" as defined above is substituted with a "heteroaryl group" as defined above, and includes 5- to 10-membered heteroaryl C １ ~C ６ Alkyl groups are preferred, and 5- to 10-membered heteroaryl C １ ~C ２ Specific examples of heteroarylalkyl groups include a 3-thienylmethyl group, a 4-thiazolylmethyl group, a 2-pyridylmethyl group, a 3-pyridylmethyl group, a 4-pyridylmethyl group, a 2-(2-pyridyl)ethyl group, a 2-(3-pyridyl)ethyl group, a 2-(4-pyridyl)ethyl group, a 2-(6-quinolyl)ethyl group, a 2-(7-quinolyl)ethyl group, a 2-(6-indolyl)ethyl group, a 2-(5-indolyl)ethyl group, and a 2-(5-benzofuranyl)ethyl group.

[0066] As used herein, the term "heteroarylalkoxy group" refers to an oxy group to which the above-defined "heteroarylalkyl group" is bonded, and is a 5- to 10-membered heteroaryl C １ ~C ６ Alkoxy groups are preferred, and 5- to 10-membered heteroaryl C１ ~C ２ An alkoxy group is more preferred. Specific examples of the heteroarylalkoxy group include a 3-thienylmethoxy group and a 3-pyridylmethoxy group.

[0067] The term "heteroarylalkoxyalkyl group" as used herein means a group in which one or more hydrogen atoms of an "alkyl group" as defined above are substituted with a "heteroarylalkoxy group" as defined above, and is a 5- to 10-membered heteroaryl C １ ~C ６ Alkoxy C １ ~C ６ Alkyl groups are preferred, and 5- to 10-membered heteroaryl C １ ~C ２ Alkoxy C １ ~C ２ An alkyl group is more preferred. Specific examples of heteroarylalkoxyalkyl groups include a 3-pyridylmethoxymethyl group.

[0068] As used herein, the term "heterocycloalkylidenealkyl group" refers to a group in which one or more hydrogen atoms of an "alkyl group" defined above are substituted with a "heterocycloalkylidene group" defined above, and is a 4- to 7-membered heterocycloalkylidene C １ ~C ６ Alkyl groups are preferred, and 4- to 7-membered heterocycloalkylidene C １ ~C ２ An alkyl group is more preferred. Specific examples of heterocycloalkylidenealkyl groups include a tetrahydro-4H-pyran-4-ylidenemethyl group and an azetidin-3-ylidenemethyl group.

[0069] As used herein, the term "alkoxyalkenyl group" refers to an "alkenyl group" defined above in which one or more hydrogen atoms are substituted with an "alkoxy group" defined above, and C １ ~C ６ Alkoxy C ２ ~C ６ An alkenyl group is preferred. Specific examples of the alkoxyalkenyl group include an (E)-4-methoxybut-2-en-1-yl group.

[0070] The term "aminocarbonylalkenyl group" as used herein means an "alkenyl group" defined above in which one or more hydrogen atoms are substituted with an "aminocarbonyl group" defined above, and ２ ~C ６ An alkenyl group is preferred. Specific examples of aminocarbonylalkenyl groups include (E)-3-(dimethylaminocarbonylcarbonyl)-prop-2-en-1-yl groups.

[0071] As used herein, the term "haloalkoxy group" refers to an "alkoxy group" defined above in which one or more hydrogen atoms have been substituted with halogen atoms, and C １ ~C ６ A haloalkoxy group is preferred. Specific examples of the haloalkoxy group include a difluoromethoxy group, a trifluoromethoxy group, a 2,2-difluoroethoxy group, and a 2,2,2-trifluoroethoxy group.

[0072] In the present specification, the term "alkylene group" refers to a divalent group derived by further removing one arbitrary hydrogen atom from the "alkyl group", and C ４ ~C ８ An alkylene group is preferred. Specific examples of the alkylene group include —CH ２ -, -(CH ２ ) ２ -, -(CH ２ ) ３ -, -CH(CH ３ ) CH ２ -, -C(CH ３ ) ２ -, -(CH ２ ) ４ -, -CH(CH ３ ) CH ２ CH ２ -, -C(CH ３ ) ２ CH ２ -, -CH ２ CH (CH ３ ) CH ２ -, -CH ２ C(CH ３ ) ２ -, -CH ２ CH２ CH (CH ３ ) -, -(CH ２ ) ５ -, -(CH ２ ) ６ -, -(CH ２ ) ７ -, -(CH ２ ) ８ - and others.

[0073] In the present specification, the term "cycloalkylene group" refers to a divalent group derived by further removing one arbitrary hydrogen atom from the "cycloalkyl group", and C ３ ~C ８ A cycloalkylene group is preferred, and specific examples of the cycloalkylene group include a cyclopropane-1,2-diyl group, a cyclobutane-1,2-diyl group, a cyclopentane-1,2-diyl group, and a cyclohexane-1,2-diyl group.

[0074] As used herein, the term "alkenylene group" refers to a divalent group derived from the aforementioned "alkenyl group" by further removing any one hydrogen atom. Depending on the configuration of the double bond and substituents (if any), the geometry of the double bond can be entgegen (E) or zusammen (Z), cis or trans. The alkenylene group includes linear and branched groups, and can be C ２ ~C １０ Alkenylene groups are preferred, C ２ ~C ６ An alkenylene group is more preferred.

[0075] As used herein, the term "alkynylene group" refers to a divalent group derived from the above-mentioned "alkynyl group" by further removing one arbitrary hydrogen atom. The alkynylene group includes linear and branched groups, and is C ２ ~C １０ Alkynylene groups are preferred, C ２ ~C ６ An alkynylene group is more preferred.

[0076] In this specification, the term "arylene group" refers to a divalent group derived from the aforementioned "aryl group" by further removing any one hydrogen atom. The arylene group may be a single ring or a condensed ring. The number of atoms constituting the ring is not particularly limited, but is preferably 6 to 10 (C ６ ~C １０ Arylene group. Specific examples of the arylene group include a 1,2-phenylene group, a 1,3-phenylene group, a 1,4-phenylene group, a 1,2-naphthylene group, a 1,3-naphthylene group, and a 1,4-naphthylene group.

[0077] As used herein, the term "spirocycloalkyl group" refers to a group formed by sharing one carbon atom constituting a cycloalkane ring with a carbon atom in a group to which it is bound. Preferred spirocycloalkyl groups include C ３ ~C ８ Examples include spirocycloalkyl groups, and specific examples include a spirocyclopropyl group, a spirocyclobutyl group, a spirocyclopentyl group, a spirocyclohexyl group, a spirocycloheptyl group, and a spirocyclooctyl group.

[0078] As used herein, the term "spiroheterocyclyl group" refers to a group in which one or more carbon atoms in the above-mentioned "spirocycloalkyl group" are replaced by heteroatoms. Preferred examples of the spiroheterocyclyl group include 4- to 10-membered spiroheterocyclyl groups.

[0079] As used herein, "alicyclic ring" refers to a non-aromatic hydrocarbon ring. The alicyclic ring may have an unsaturated bond within the ring, or may be a polycyclic ring having two or more rings. Furthermore, the carbon atoms constituting the ring may be oxidized to form a carbonyl group. Preferred examples of the alicyclic ring include 3- to 8-membered alicyclic rings, and specific examples include a cyclopropane ring, a cyclobutane ring, a cyclopentane ring, a cyclohexane ring, a cycloheptane ring, a cyclooctane ring, and a bicyclo[2.2.1]heptane ring.

[0080] As used herein, the term "heterocycle" refers to a non-aromatic heterocycle containing preferably 1 to 5, more preferably 1 to 3, heteroatoms among the atoms constituting the ring. The heterocycle may have a double and / or triple bond in the ring, and a carbon atom in the ring may be oxidized to form a carbonyl, and may be a monocyclic, fused, or spirocyclic ring. The number of atoms constituting the ring is preferably 3 to 12 (3- to 12-membered heterocycle), more preferably 4 to 8 (4- to 8-membered heterocycle). Specific examples of the heterocyclic ring include an azetidine ring, an oxetane ring, a tetrahydrofuran ring, a tetrahydropyran ring, a morpholine ring, a thiomorpholine ring, a pyrrolidine ring, a 4-oxopyrrolidine ring, a piperidine ring, a 4-oxopiperidine ring, a piperazine ring, a pyrazolidine ring, an imidazolidine ring, an oxazolidine ring, an isoxazolidine ring, a thiazolidine ring, an isothiazolidine ring, a thiadiazolidine ring, an oxazolidone ring, a dioxolane ring, a dioxane ring, a thietane ring, an octahydroindole ring, or an azocane ring, as well as rings in which one or more single bonds in these saturated heterocyclic rings have been replaced with double bonds or triple bonds.

[0081] As used herein, the term "saturated heterocycle" refers to a non-aromatic heterocycle containing 1 to 5 heteroatoms in addition to carbon atoms and containing no double and / or triple bonds within the ring. The saturated heterocycle may be a monocycle or may form a condensed ring with another ring, for example, an aromatic ring such as a benzene ring. When a saturated heterocycle forms a fused ring, the saturated heterocycle is preferably a 4- to 7-membered saturated heterocycle, and specific examples thereof include an azetidine ring, an oxetane ring, a tetrahydrofuran ring, a tetrahydropyran ring, a morpholine ring, a thiomorpholine ring, a pyrrolidine ring, a 4-oxopyrrolidine ring, a piperidine ring, a 4-oxopiperidine ring, a piperazine ring, a pyrazolidine ring, an imidazolidine ring, an oxazolidine ring, an isoxazolidine ring, a thiazolidine ring, an isothiazolidine ring, a thiadiazolidine ring, an oxazolidone ring, a dioxolane ring, a dioxane ring, a thietane ring, an octahydroindole ring, an indoline ring, and an azepane ring.

[0082] As used herein, "amino acid" includes natural amino acids and unnatural amino acids. As used herein, "natural amino acids" refers to Gly, Ala, Ser, Thr, Val, Leu, Ile, Phe, Tyr, Trp, His, Glu, Asp, Gln, Asn, Cys, Met, Lys, Arg, and Pro. Unnatural amino acids are not particularly limited, and examples include β-amino acids, γ-amino acids, D-amino acids, N-substituted amino acids, α,α-disubstituted amino acids, amino acids with side chains different from natural amino acids, and hydroxycarboxylic acids. As used herein, unnatural N-substituted amino acids refer to N-substituted amino acids other than Pro. As used herein, amino acids may have any configuration. The side chain of the amino acid is not particularly limited, and may be freely selected from, in addition to a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group, and a cycloalkyl group, and one or two non-adjacent methylene groups in these groups may be replaced by an oxygen atom, a carbonyl group (—CO—), or a sulfonyl group (—SO ２ -). Each of these may be substituted with a substituent, and the substituents are not limited, and may be independently selected from any substituents containing a halogen atom, an O atom, a S atom, a N atom, a B atom, a Si atom, or a P atom. That is, examples include optionally substituted alkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, aralkyl groups, and cycloalkyl groups. In a non-limiting embodiment, the amino acid herein may be a compound having a carboxy group and an amino group in the same molecule (even in this case, imino acids such as proline and hydroxyproline are also included in the amino acid).

[0083] The main chain amino group of the amino acid is unsubstituted (NH ２ group), and may be substituted (i.e., -NHR group: R represents an alkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, aralkyl group, or cycloalkyl group which may have a substituent, and one or two non-adjacent methylene groups in these groups are replaced by an oxygen atom, a carbonyl group (-CO-), or a sulfonyl group (-SO ２-), or the carbon chain bonded to the N atom and the carbon atom at the α-position may form a ring, as in proline. The substituent of R is selected in the same manner as the substituent in the amino acid side chain described above. When the main chain amino group is substituted, R is included in the "amino acid side chain" in this specification. An amino acid in which such a main chain amino group is substituted is referred to as an "N-substituted amino acid" in this specification. As used herein, the "N-substituted amino acid" is preferably an N-alkyl amino acid, an N-C １ ~C ６ Alkyl amino acids, N-C １ ~C ４ Examples include, but are not limited to, alkyl amino acids and N-methyl amino acids.

[0084] As used herein, "amino acids" include all corresponding isotopes. An isotope of an "amino acid" is one in which at least one atom has been replaced with an atom having the same atomic number (number of protons) but a different mass number (sum of the number of protons and neutrons). Examples of isotopes included in "amino acids" as used herein include hydrogen atoms, carbon atoms, nitrogen atoms, oxygen atoms, phosphorus atoms, sulfur atoms, fluorine atoms, and chlorine atoms, each of which is ２ H. ３ H. １３ C. １４ C. １５ N. １７ O. １８ O. ３１ P. ３２ P. ３５ S. １８ F. ３６ Cl and the like are included.

[0085] In this specification, examples of the substituent containing a halogen atom include an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group, and the like, each of which has a halogen atom as a substituent, and more specific examples thereof include a fluoroalkyl group, a difluoroalkyl group, a trifluoroalkyl group, and the like.

[0086] In this specification, examples of the substituent containing an oxygen atom include a hydroxy group (—OH), an oxy group (—OR), a carbonyl group (—C(═O)—R), a carboxy group (—CO ２ H), oxycarbonyl group (-C(=O)-OR), carbonyloxy group (-O-C(=O)-R), thiocarbonyl group (-C(=O)-SR), carbonylthio group (-S-C(=O)-R), aminocarbonyl group (-C(=O)-NHR), carbonylamino group (-NH-C(=O)-R), oxycarbonylamino group (-NH-C(=O)-OR), sulfonylamino group (-NH-SO ２ -R), aminosulfonyl group (-SO ２ -NHR), sulfamoylamino group (-NH-SO ２ -NHR), thiocarboxy group (-C(=O)-SH), carboxycarbonyl group (-C(=O)-CO ２ H).

[0087] Examples of the oxy group (—OR) include an alkoxy group, a cycloalkoxy group, an alkenyloxy group, an alkynyloxy group, an aryloxy group, a heteroaryloxy group, and an aralkyloxy group. １ ~C ４ Alkoxy group, C １ ~C ２ An alkoxy group is preferred, and among these, a methoxy group or an ethoxy group is more preferred.

[0088] Examples of the carbonyl group (-C(=O)-R) include a formyl group (-C(=O)-H), an alkylcarbonyl group, a cycloalkylcarbonyl group, an alkenylcarbonyl group, an alkynylcarbonyl group, an arylcarbonyl group, a heteroarylcarbonyl group, and an aralkylcarbonyl group.

[0089] Examples of the oxycarbonyl group (—C(═O)—OR) include an alkyloxycarbonyl group, a cycloalkyloxycarbonyl group, an alkenyloxycarbonyl group, an alkynyloxycarbonyl group, an aryloxycarbonyl group, a heteroaryloxycarbonyl group, and an aralkyloxycarbonyl group.

[0090] Examples of the carbonyloxy group (—O—C(═O)—R) include an alkylcarbonyloxy group, a cycloalkylcarbonyloxy group, an alkenylcarbonyloxy group, an alkynylcarbonyloxy group, an arylcarbonyloxy group, a heteroarylcarbonyloxy group, and an aralkylcarbonyloxy group.

[0091] Examples of the thiocarbonyl group (-C(=O)-SR) include an alkylthiocarbonyl group, a cycloalkylthiocarbonyl group, an alkenylthiocarbonyl group, an alkynylthiocarbonyl group, an arylthiocarbonyl group, a heteroarylthiocarbonyl group, and an aralkylthiocarbonyl group.

[0092] Examples of the carbonylthio group (—S—C(═O)—R) include an alkylcarbonylthio group, a cycloalkylcarbonylthio group, an alkenylcarbonylthio group, an alkynylcarbonylthio group, an arylcarbonylthio group, a heteroarylcarbonylthio group, and an aralkylcarbonylthio group.

[0093] Examples of aminocarbonyl groups (—C(═O)—NHR) include alkylaminocarbonyl groups (e.g., C １ ~C ６ or C １ ~C ４ Examples include alkylaminocarbonyl groups, particularly ethylaminocarbonyl groups and methylaminocarbonyl groups. Also included are cycloalkylaminocarbonyl groups, alkenylaminocarbonyl groups, alkynylaminocarbonyl groups, arylaminocarbonyl groups, heteroarylaminocarbonyl groups, and aralkylaminocarbonyl groups. In addition to these, examples include compounds in which the H atom bonded to the N atom in —C(═O)—NHR is further substituted with an alkyl group, cycloalkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, or aralkyl group.

[0094] Examples of carbonylamino groups (-NH-C(=O)-R) include alkylcarbonylamino groups, cycloalkylcarbonylamino groups, alkenylcarbonylamino groups, alkynylcarbonylamino groups, arylcarbonylamino groups, heteroarylcarbonylamino groups, and aralkylcarbonylamino groups. In addition to these, compounds in which the H atom bonded to the N atom in -NH-C(=O)-R is further substituted with an alkyl group, cycloalkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, or aralkyl group are also included.

[0095] Examples of oxycarbonylamino groups (-NH-C(=O)-OR) include alkoxycarbonylamino groups, cycloalkoxycarbonylamino groups, alkenyloxycarbonylamino groups, alkynyloxycarbonylamino groups, aryloxycarbonylamino groups, heteroaryloxycarbonylamino groups, and aralkyloxycarbonylamino groups. In addition to these, examples include compounds in which the H atom bonded to the N atom in -NH-C(=O)-OR is further substituted with an alkyl group, cycloalkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, or aralkyl group.

[0096] Sulfonylamino group (-NH-SO ２ Examples of —R) include alkylsulfonylamino groups, cycloalkylsulfonylamino groups, alkenylsulfonylamino groups, alkynylsulfonylamino groups, arylsulfonylamino groups, heteroarylsulfonylamino groups, and aralkylsulfonylamino groups. ２ Examples include compounds in which the H atom bonded to the N atom in —R is further substituted with an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or an aralkyl group.

[0097] Aminosulfonyl group (-SO ２Examples of —NHR) include alkylaminosulfonyl groups, cycloalkylaminosulfonyl groups, alkenylaminosulfonyl groups, alkynylaminosulfonyl groups, arylaminosulfonyl groups, heteroarylaminosulfonyl groups, and aralkylaminosulfonyl groups. ２ Examples include compounds in which the H atom bonded to the N atom in —NHR is further substituted with an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or an aralkyl group.

[0098] Sulfamoylamino group (-NH-SO ２ Examples of —NHR) include alkylsulfamoylamino groups, cycloalkylsulfamoylamino groups, alkenylsulfamoylamino groups, alkynylsulfamoylamino groups, arylsulfamoylamino groups, heteroarylsulfamoylamino groups, and aralkylsulfamoylamino groups. ２ The two H atoms bonded to the N atom in -NHR may be substituted with substituents independently selected from the group consisting of an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, and an aralkyl group, and these two substituents may form a ring.

[0099] Examples of the substituent containing an S atom include a thiol group (-SH), a thio group (-S-R), a sulfinyl group (-S(=O)-R), and a sulfonyl group (-SO ２ -R), sulfo group (-SO ３ H).

[0100] Examples of the thio group (-S-R) are selected from alkylthio groups, cycloalkylthio groups, alkenylthio groups, alkynylthio groups, arylthio groups, heteroarylthio groups, aralkylthio groups, and the like.

[0101] Sulfonyl group (-SO ２Examples of —R) include alkylsulfonyl groups, cycloalkylsulfonyl groups, alkenylsulfonyl groups, alkynylsulfonyl groups, arylsulfonyl groups, heteroarylsulfonyl groups, and aralkylsulfonyl groups.

[0102] As used herein, examples of the nitrogen-containing substituent include an azide group (—N ３ ), cyano group (-CN), primary amino group (-NH ２ ), secondary amino group (-NH-R; also called monosubstituted amino), tertiary amino group (-NR(R'); also called disubstituted amino group), amidino group (-C(=NH)-NH ２ ), a substituted amidino group (—C(═NR)—NR′R″), a guanidino group (—NH—C(═NH)—NH ２ ), substituted guanidino group (—NR—C(═NR′″)—NR′R″), aminocarbonylamino group (—NR—CO—NR′R″), pyridyl group, piperidino group, morpholino group, azetidinyl group, and the like.

[0103] Examples of secondary amino groups (—NH—R; monosubstituted amino) include alkylamino groups, cycloalkylamino groups, alkenylamino groups, alkynylamino groups, arylamino groups, heteroarylamino groups, and aralkylamino groups.

[0104] Examples of tertiary amino groups (-NR(R'); disubstituted amino groups) include alkyl(aralkyl)amino groups and other amino groups having any two substituents independently selected from alkyl groups, cycloalkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, aralkyl groups, and the like, and these two substituents may form a ring. Specifically, dialkylamino groups, particularly C １ -C ６ Dialkylamino group, C １ -C ４ Examples include a dialkylamino group, a dimethylamino group, and a diethylamino group. ｐ -C ｑ "Dialkylamino group" means an amino group having C ｐ -C ｑIt means a group substituted with two alkyl groups, and both C ｐ -C ｑ The alkyl groups may be the same or different.

[0105] Examples of substituted amidino groups (-C(=NR)-NR'R") include groups in which the three substituents R, R', and R" on the N atom are each independently selected from an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, and an aralkyl group, such as an alkyl(aralkyl)(aryl)amidino group.

[0106] Examples of the substituted guanidino group (-NR-C(=NR'")-NR'R") include groups in which R, R', R", and R'" are each independently selected from an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, and an aralkyl group, or groups in which these groups form a ring.

[0107] Examples of aminocarbonylamino groups (—NR—CO—NR′R″) include groups in which R, R′, and R″ are each independently selected from a hydrogen atom, an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, and an aralkyl group, or groups in which these groups form a ring.

[0108] In this specification, "peptide residue" and "amino acid residue" may be simply referred to as "peptide" and "amino acid", respectively.

[0109] As used herein, the term "corresponding site" can be used to characterize amino acid residues in the amino acid sequence of the polypeptide according to this embodiment by reference to the amino acid sequence represented by SEQ ID NO: 1. Alignment to determine corresponding sites can be achieved by various methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX (registered trademark) (Genetyx Corporation). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared.

[0110] A polypeptide according to one embodiment comprises a sequence having 90% or more sequence identity with a sequence in which one amino acid residue in the amino acid sequence represented by SEQ ID NO: 1 has been modified, and has a catalytic activity for a reductive amination reaction between one or more compounds A represented by formula (1) described below or salts thereof and one or more compounds B represented by formula (2) described below or salts thereof, or an intramolecular reductive amination reaction between one or more compounds B or salts thereof, which are also represented by formula (1) described below, and a reductive amination reaction between one or more compounds B or salts thereof, which are also represented by formula (2) described below, and a catalytic activity for an intramolecular reductive amination reaction between one or more compounds B or salts thereof, which are also represented by formula (1) described below, and a reductive amination reaction between one or more compounds B or salts thereof, which are also represented by formula (2) below, and a reductive amination reaction between one or more compounds B or salts thereof, which are also represented by formula (1)

[0111] A polypeptide according to one embodiment comprises a sequence having 90% or more sequence identity with a sequence in which two amino acid residues in the amino acid sequence represented by SEQ ID NO: 1 have been modified, and has catalytic activity for a reductive amination reaction between one or more compounds A represented by formula (1) described below or salts thereof and one or more compounds B represented by formula (2) described below or salts thereof, or for an intramolecular reductive amination reaction between one or more compounds B or salts thereof, which are also represented by formula (1) described below, and a reductive amination reaction between one or more compounds B or salts thereof, which are also represented by formula (2) described below, and a reductive amination reaction between one or more compounds B or salts thereof, which are also represented by formula (1) described below, and a reductive amination reaction between one or more compounds B or salts thereof, which are also represented by formula (2) below, and a reductive amination reaction between one or more compounds B or salts thereof, which are also represented by formula (1)

[0112] A polypeptide according to one embodiment comprises a sequence having 90% or more sequence identity with a sequence in which three amino acid residues in the amino acid sequence represented by SEQ ID NO: 1 have been modified, and has a catalytic activity for a reductive amination reaction between one or more compounds A represented by formula (1) described below or salts thereof and one or more compounds B represented by formula (2) described below or salts thereof, or an intramolecular reductive amination reaction between one or more compounds B or salts thereof, which are also represented by formula (1) described below, and a reductive amination reaction between one or more compounds B or salts thereof, which are also represented by formula (2) described below, and a reductive amination reaction between one or more compounds B or salts thereof, which are also represented by formula (1) described below, and a reductive amination reaction between one or more compounds B or salts thereof, which are also represented by formula (2) below, and a reductive amination reaction between one or more compounds B or salts thereof, which are also represented by formula (1)

[0113] The polypeptide of one embodiment may be an enzyme that catalyzes a reductive amination reaction. The polypeptide of this embodiment may have catalytic activity for the reductive amination reaction of compound A or a salt thereof with compound B or a salt thereof, or for the intramolecular reductive amination reaction of compound B or a salt thereof, under at least one reaction condition. In this specification, compound A or a salt thereof may be collectively referred to as compound A, and compound B or a salt thereof may be collectively referred to as compound B.

[0114] The polypeptide according to one embodiment has catalytic activity for reductive amination reactions and is therefore suitable for producing amino acids.

[0115] A polypeptide according to one embodiment has high synthetic activity even in the production of amino acids for which conventional enzymes that catalyze reductive amination reactions have low synthetic activity. Specifically, the protein PP3591 (hereinafter referred to as the wild-type enzyme) encoded by the genome of the P. putida KT2440 strain described in Non-Patent Document 4 (September 2004) showed low synthetic activity for amino acids in which an alkyl group with a carbon number larger than that of a methyl group (e.g., an ethyl group) is bound to the nitrogen atom (hereinafter also referred to as "N-Et or higher N-substituted amino acids") and amino acids having a branched structure at the β-position relative to the amino group (hereinafter also referred to as "β-branch amino acids"). A polypeptide according to one embodiment has higher activity for N-Et or higher N-substituted amino acids and / or β-branch amino acids than the wild-type enzyme. Furthermore, the polypeptide according to this embodiment allows the production of target amino acids with high stereoselectivity.

[0116] The amino acid sequence represented by SEQ ID NO: 1 is a known amino acid sequence described in Non-Patent Document 4.

[0117] The sequence identity of the above polypeptide to a sequence in which one amino acid residue in the amino acid sequence represented by SEQ ID NO: 1 has been modified may be 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0118] The sequence identity of the above polypeptide to a sequence in which two amino acid residues in the amino acid sequence represented by SEQ ID NO: 1 have been modified may be 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0119] The sequence identity of the above polypeptide to a sequence in which three amino acid residues in the amino acid sequence represented by SEQ ID NO: 1 have been modified may be 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0120] The sequence identity of an amino acid sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in a reference polypeptide sequence, after aligning the sequences to achieve the maximum percent sequence identity and introducing gaps, if necessary, and excluding any conservative substitutions from the sequence identity. Alignment for the purpose of determining sequence identity of amino acid sequences can be achieved by various methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX® (Genetyx Corporation). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment across the entire length of the sequences being compared.

[0121] The sequence identity of amino acid sequences can be determined by the BLAST algorithm by Karlin and Altschul (Proc. Natl. Acad. Sci. USA (1993) 90:5873-7). Based on this algorithm, a program called BLASTN or BLASTX has been developed (Altschul et al., J. Mol. Biol. (1990) 215:403-10). When analyzing an amino acid sequence using BLASTX based on BLAST, the parameters are, for example, score = 50 and wordlength = 3. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. For specific techniques for these analysis methods, information on the BLAST (Basic Local Alignment Search Tool) website of the National Center for Biotechnology Information (NCBI) is available.

[0122] The ALIGN-2 sequence comparison computer program is the copyright of Genentech, Inc., and its source code, together with user documentation, has been filed with the U.S. Copyright Office, Washington, DC 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code. The ALIGN-2 program is compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0123] In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (alternatively, one can say that a given amino acid sequence A has or contains a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored by the sequence alignment program ALIGN-2 as identical matches in that program's alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0124] As used herein, "having a catalytic activity higher than that of a polypeptide having the amino acid sequence represented by SEQ ID NO: 1" means that the catalytic activity is higher than that of the polypeptide under at least one reaction condition. "Having a catalytic activity higher than that of a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 17" means that the catalytic activity is higher than that of the polypeptide under at least one reaction condition. In one aspect, the catalytic activity of a polypeptide can be evaluated using the reaction conditions described in the Examples. For example, the concentrations of each compound at the start of the reaction can be as follows: total concentration of compound B and its salt is 50 mM, D(+)-glucose is 100 mM, total concentration of compound A and its salt is 500 mM, phosphate buffer is 100 mM, β-NADPH is 0.89 mM, GDH solution is 0.002 unit / μL, and the concentration of the polypeptide to be evaluated is 2.5 μM. The reaction can be performed by adding the polypeptide to be evaluated for catalytic activity to a premix described below. The premix can be prepared by dissolving Compound B and / or a salt thereof, D(+)-glucose in a mixed solvent of an aqueous solution of Compound A and / or a salt thereof prepared by adjusting the pH to 7.5 to 8.5 with hydrochloric acid, and a phosphate buffer solution, adjusting the pH to 8.0 with an aqueous sodium hydroxide solution, and then adding ultrapure water to the resulting solution, and adding a β-NADPH aqueous solution (dissolved in ultrapure water to make it 89 mM) and a GDH solution (dissolved in 1x TNG) to the resulting solution.

[0125] In one embodiment, the catalytic activity can be evaluated by determining the yield of amino acid produced by the reductive amination reaction after 19 hours from the start of the reaction under conditions of 50 mM phenylpyruvic acid, 2-oxo-3-(p-tolyl)propanoic acid, or 2-cyclopentyl-2-oxo-acetic acid, 100 mM D(+)-glucose, 500 mM methylamine or ethylamine, 100 mM phosphate buffer, 0.89 mM β-NADPH, 0.002 unit / μL GDH solution, and 2.5 μM polypeptide to be evaluated, at 37°C, pH 8.

[0126] The catalytic activity is calculated based on the amount of amino acid (hereinafter, sometimes referred to as "target product") produced by the reductive amination reaction. The amount of target product can be measured using a liquid chromatograph mass spectrometer (LCMS). Specifically, the measurement can be performed under the conditions described in the Examples below.

[0127] The catalytic activity of the polypeptide according to this embodiment may be 1.2 times or more, 1.5 times or more, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, or 8 times or more than the catalytic activity of the polypeptide having the amino acid sequence represented by SEQ ID NO: 1. The catalytic activity of the polypeptide according to this embodiment relative to the catalytic activity of the polypeptide having the amino acid sequence represented by SEQ ID NO: 1 can be determined by calculating the ratio of the yields of the target product when both peptides are used 19 hours after the start of the reaction. Specifically, the yield when the polypeptide according to this embodiment is used can be determined by dividing the yield when the polypeptide having the amino acid sequence represented by SEQ ID NO: 1 is used. As used herein, the start of the reaction refers to the latest of the times when compound A represented by formula (1) or a salt thereof, compound B represented by formula (2) or a salt thereof, the enzyme disclosed herein, and a reducing agent are added to the system.

[0128] The yield at 19 hours after the start of the reaction is calculated by the following method. First, the reaction solution and a separately prepared calibration curve sample (a compound having the same structure as the target product or a structure showing the same UV absorption wavelength) are subjected to LCMS analysis, and a UV chart or extracted ion chromatogram is obtained for each. The UV peak area or MS peak area derived from the target product is obtained from the UV chart or extracted ion chromatogram obtained from the reaction solution. Similarly, the UV peak area or MS peak area of ​​the standard is obtained from the calibration curve sample. Based on the correspondence between the concentration of the standard contained in the calibration curve sample and the UV peak area or MS peak area, the concentration of the target product contained in the reaction solution is calculated from the UV peak area or MS peak area. When compound B and its salt in the reaction solution are all converted to the target product, the concentration of the target product in the reaction solution is considered to be equal to the sum of the concentrations of compound B and its salt in the reaction solution at the start of the reaction. The yield is calculated by dividing the concentration of the target product actually contained in the reaction solution by the total concentration of compound B and its salt in the reaction solution at the start of the reaction.

[0129] Compound A is a compound represented by the following formula (1).

[0130] In formula (1), R １ and R ２ each independently represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, a heterocyclyl group, or a heteroaryl group, and these groups may be substituted; R １ or R ２ In addition, in formula (1), any one or more of R １ may be a hydrogen atom, and R ２ is C １ ~C ６ Compound A may be an alkyl group. Compound A may be an amine or an amine analogue.

[0131] Compound A is a compound represented by formula (1) in which R １ and R ２wherein one of the alkyl groups is an alkyl group and the other is a hydrogen atom. The alkylamine or a salt thereof may be one or more selected from the group consisting of methylamine, ethylamine, and salts thereof. Compound A may be ammonia. Compound A may be one or more selected from the group consisting of ammonia, methylamine, ethylamine, and salts thereof.

[0132] Compound B is a compound represented by the following formula (2).

[0133] In formula (2), X represents a carbon atom, and Y represents a hydrogen atom, a group represented by formula (1′) above, or a group represented by formula (3) above. ３ ~C ８ or a cycloalkyl group of C ６ ~C ９ The aralkyl group is C １ ~C ３ may be substituted with an alkyl group or a halogen atom.

[0134] n represents an integer of 0 to 2. n may be 0 or 1, or may be 0.

[0135] R ６ represents a hydrogen atom, an aliphatic hydrocarbon group having 1 to 6 carbon atoms which may be substituted, an aryl group having 5 to 12 carbon atoms which may be substituted, a heteroaryl group having 5 to 12 ring atoms which may be substituted, a group containing a nitrogen atom, or a group containing an oxygen atom. ６ Specific examples of the group represented by the formula (1') are as described above. indicates the point of attachment to X. １ａ is R in formula (1) １ It is a group obtained by removing a hydrogen atom from a group represented by the formula: and is not a hydrogen atom.

[0136] In formula (3), indicates the point of attachment to X.

[0137] m represents an integer of 0 or more and 6 or less. The lower limit of m is 0 or more, and may be 1 or more, 2 or more, 3 or more, 4 or more, or 5 or more. The upper limit of m is 6 or less, and may be 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less. p is 0 or 1, q is 0 or 1, and r is 0 or 1.

[0138] Z １ represents an alkylene group which may be substituted, or an ether bond-containing group having 1 to 6 carbon atoms. When m is 2 or more, a plurality of Z １ may be the same or different. Examples of the "ether bond-containing group" include an alkyl group substituted with an alkoxy group, an allyloxy group, etc. Specific examples of the ether bond-containing group having from 1 to 6 carbon atoms include a methoxymethyl group, an ethoxymethyl group, an allyloxymethyl group, an allyloxyethyl group, and an allyloxypropyl group.

[0139] Z ２ indicates a carbon atom.

[0140] R ３ , R ４ and R ５ each independently represents a hydrogen atom, an optionally substituted aliphatic hydrocarbon group having from 1 to 6 carbon atoms, an optionally substituted aryl group having from 5 to 12 carbon atoms, an optionally substituted heteroaryl group having from 5 to 12 ring-constituting atoms, a group containing a nitrogen atom, or a group containing an oxygen atom.

[0141] R ３ , R ４ and R ５ Any two or more of the following may be bonded to each other to form Z ２ together form a ring, and these ring structures may be cycloalkyl, aryl, heterocyclyl, or heteroaryl, and these groups may be substituted.

[0142] R ３ , R ４ , and R ５ Is Z ２ may form a double bond or a triple bond with R ３ , R ４ , and R ５is connected to Z by a double bond or a triple bond. ２ When p is bonded to q, any one or more of p, q, and r is 0.

[0143] In formula (1), R １ and R ２ is a methyl group and the other is a hydrogen atom, in formula (2), Y is a group represented by formula (3), m is 0, and R ３ ~R ５ Two or more of the groups are not hydrogen atoms.

[0144] R ３ ~R ５ In the case where any two of R are hydrogen atoms, the group other than the hydrogen atom may be an aliphatic hydrocarbon group having 1 to 6 carbon atoms, which may be substituted, an aryl group having 5 to 12 carbon atoms, or a heteroaryl group having 5 to 12 ring atoms, which may be substituted, an aryl group having 5 to 12 carbon atoms, which may be substituted, a phenyl group, or a p-tolyl group. １ is a hydrogen atom, and R ２ In the formula (1), R may be an ethyl group. １ is a hydrogen atom, and R ２ In the formula (1), R may be a methyl group. １ and R ２ may be a hydrogen atom.

[0145] Compound B may be a keto acid or a keto acid analogue. Compound B may be a compound represented by the following formula (2').

[0146] In formula (2′), Y′ is C ３ ~C ８ a cycloalkyl group, or C ６ ~C ９ represents an aralkyl group, and the aralkyl group is C １ ~C ３ It may be substituted with an alkyl group or a halogen atom.

[0147] The compound represented by formula (2') or a salt thereof may be one or more selected from the group consisting of phenylpyruvic acid, 2-oxo-3-(p-tolyl)propanoic acid, 2-cyclopentyl-2-oxo-acetic acid, and salts thereof.

[0148] The combination of compound A and compound B is represented by the formula (1), １ is a hydrogen atom, and R ２ is an ethyl group, and a compound A (ethylamine) in which n and m are both 0 and Y is a group represented by formula (3) ３ is a phenyl group, and R ４ , R ５ and R ６ are all hydrogen atoms, and p, q, and r are all 1, and in formula (1), R １ is a hydrogen atom, and R ２ is an ethyl group, and a compound A (ethylamine) in which n and m are both 0 and Y is a group represented by formula (3) ３ is a p-tolyl group, and R ４ , R ５ and R ６ are all hydrogen atoms, and p, q, and r are all 1, and in formula (1), R １ is a hydrogen atom, and R ２ is a methyl group, and a compound A (methylamine) in which n and m are both 0 and Y is a group represented by formula (3) ３ and R ４ are linked together to form a cyclopentane ring, and R ５ and R ６ are all hydrogen atoms, and p, q and r are all 1.

[0149] In measuring catalytic activity, a combination of compound A as ethylamine and compound B as phenylpyruvic acid sodium salt can be used, in which case the catalytic activity of the polypeptide can be evaluated using H-EtPhe-OH synthesis activity as an index. In measuring catalytic activity in another embodiment, a combination of compound A as ethylamine and compound B as 2-oxo-3-(p-tolyl)propanoic acid sodium salt can be used, in which case the catalytic activity of the polypeptide can be evaluated using H-EtPhe(4-Me)-OH synthesis activity as an index. In measuring catalytic activity in another embodiment, a combination of compound A as methylamine and compound B as 2-cyclopentyl-2-oxo-acetic acid can be used, in which case the catalytic activity of the polypeptide can be evaluated using H-MeGly(cPent)-OH synthesis activity as an index.

[0150] A polypeptide according to one embodiment of the present invention may be a polypeptide in which one or more amino acid residues in the amino acid sequence represented by SEQ ID NO: 1 have been modified, and 1 to 20, 1 to 15, 1 to 10, 1 to 7, or 1 to 5 amino acid residues may have been modified. The number of modified amino acid residues may be 3 or less, 2 or less, or 1.

[0151] In the polypeptide according to one embodiment of the present invention, the number of modified amino acid residues in the amino acid sequence represented by SEQ ID NO: 1 is, for example, 1 to 5, preferably 1, more preferably 2 or more, and most preferably 2 or 3.

[0152] The modification may be one or more selected from the group consisting of substitution, deletion, and insertion, and may be a substitution. The modification may be a conservative modification. A conservative modification means a modification of an amino acid residue that does not reduce the desired catalytic activity compared to the polypeptide before modification.

[0153] The modification may be a substitution with a natural amino acid different from the amino acid residue before modification. Furthermore, the natural amino acid used for the substitution may be one or more selected from the group consisting of glycine, alanine, serine, threonine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, histidine, glutamine, asparagine, glutamic acid, aspartic acid, methionine, lysine, arginine, cysteine, and proline.

[0154] A polypeptide according to one embodiment of the present invention may be a polypeptide comprising a sequence in which amino acid residues located at positions corresponding to one or more amino acid residues selected from the group consisting of the histidine residue at position 44, the phenylalanine residue at position 117, the methionine residue at position 141, the threonine residue at position 156, the histidine residue at position 182, the glutamine residue at position 186, the tryptophan residue at position 253, and the lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1 have been modified. Since this further improves the catalytic activity for reductive amination, the polypeptide may be a polypeptide comprising a sequence in which amino acid residues located at positions corresponding to one or more amino acid residues selected from the group consisting of the methionine residue at position 141, the histidine residue at position 182, the tryptophan residue at position 253, and the lysine residue at position 260 have been modified. Furthermore, the polypeptide may be a polypeptide comprising a sequence in which amino acid residues located at positions corresponding to one or more amino acid residues selected from the group consisting of the methionine residue at position 141, the tryptophan residue at position 253, and the lysine residue at position 260 have been modified.

[0155] A polypeptide according to one embodiment of the present invention may be a polypeptide comprising a sequence in which the amino acid residue at the site corresponding to the histidine residue at position 44 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid residue other than histidine. The polypeptide may be a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2. The amino acid residue represented by X in SEQ ID NO: 2 is an alanine residue, aspartic acid residue, glutamic acid residue, phenylalanine residue, glycine residue, isoleucine residue, lysine residue, leucine residue, methionine residue, asparagine residue, proline residue, glutamine residue, arginine residue, serine residue, threonine residue, valine residue, tryptophan residue, or tyrosine residue.

[0156] Since this further improves the catalytic activity in the reductive amination reaction, the polypeptide may contain a sequence in which the amino acid residue at the position corresponding to the histidine residue at position 44 is substituted with a methionine residue. That is, the polypeptide according to this embodiment may contain an amino acid sequence (H44M) in which the amino acid residue at the position corresponding to the histidine residue at position 44 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a methionine residue. In this case, the catalytic activity in the reductive amination reaction of ethylamine with 2-oxo-3-(p-tolyl)propanoic acid is further improved.

[0157] A polypeptide according to one embodiment of the present invention may be a polypeptide comprising a sequence in which the amino acid residue at the position corresponding to the phenylalanine residue at position 117 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid residue other than phenylalanine. The polypeptide may be a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4. The amino acid residue represented by X in SEQ ID NO: 4 is an alanine residue, aspartic acid residue, glutamic acid residue, glycine residue, histidine residue, isoleucine residue, lysine residue, leucine residue, methionine residue, asparagine residue, proline residue, glutamine residue, arginine residue, serine residue, threonine residue, valine residue, tryptophan residue, or tyrosine residue.

[0158] Since the catalytic activity for the reductive amination reaction is further improved, the polypeptide may contain a sequence in which the amino acid residue at the position corresponding to the phenylalanine residue at position 117 is substituted with a leucine residue. That is, the polypeptide according to this embodiment may contain an amino acid sequence (F117L) in which the amino acid residue at the position corresponding to the phenylalanine residue at position 117 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a leucine residue. In this case, the catalytic activity for the reductive amination reaction of methylamine with 2-cyclopentyl-2-oxo-acetic acid is further improved.

[0159] A polypeptide according to one embodiment of the present invention may be a polypeptide comprising a sequence in which the amino acid residue at the position corresponding to the methionine residue at position 141 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid residue other than methionine. The polypeptide may be a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 6. The amino acid residue represented by X in SEQ ID NO: 6 is an alanine residue, aspartic acid residue, glutamic acid residue, phenylalanine residue, glycine residue, histidine residue, isoleucine residue, lysine residue, leucine residue, asparagine residue, proline residue, glutamine residue, arginine residue, serine residue, threonine residue, valine residue, tryptophan residue, or tyrosine residue.

[0160] Since the catalytic activity for the reductive amination reaction is further improved, the amino acid residue located at the site corresponding to the methionine residue at position 141 may comprise a sequence substituted with one or more amino acid residues selected from the group consisting of tyrosine residues, tryptophan residues, valine residues, threonine residues, serine residues, arginine residues, leucine residues, lysine residues, isoleucine residues, histidine residues, phenylalanine residues, and alanine residues. That is, the polypeptide according to this embodiment may comprise an amino acid sequence (M141Y, M141W, M141V, M141T, M141S, M141R, M141L, M141K, M141I, M141H, M141F, M141A) in which the amino acid residue at the site corresponding to the methionine residue at position 141 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a tyrosine residue, tryptophan residue, valine residue, threonine residue, serine residue, arginine residue, leucine residue, lysine residue, isoleucine residue, histidine residue, phenylalanine residue, or alanine residue. Furthermore, the polypeptide may comprise a sequence in which the amino acid residue at the site corresponding to the methionine residue at position 141 is substituted with one or more amino acid residues selected from the group consisting of tyrosine residue, tryptophan residue, valine residue, lysine residue, isoleucine residue, phenylalanine residue, and alanine residue. That is, the polypeptide of this embodiment may include an amino acid sequence (M141Y, M141W, M141V, M141K, M141I, M141H, M141F, M141A) in which the amino acid residue located at the position corresponding to the methionine residue at position 141 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a tyrosine residue, tryptophan residue, valine residue, lysine residue, isoleucine residue, histidine residue, phenylalanine residue, or alanine residue.

[0161] A polypeptide comprising an amino acid sequence in which the amino acid residue at the position corresponding to the methionine residue at position 141 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an alanine residue, a lysine residue, a valine residue, a tryptophan residue, or a tyrosine residue has improved catalytic activity for the reductive amination reaction between methylamine and 2-cyclopentyl-2-oxo-acetic acid, the reductive amination reaction between ethylamine and phenylpyruvic acid, and the reductive amination reaction between ethylamine and 2-oxo-3-(p-tolyl)propanoic acid.

[0162] A polypeptide comprising an amino acid sequence in which the amino acid residue at the site corresponding to the methionine residue at position 141 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an arginine residue, a leucine residue, or a serine residue has improved catalytic activity for the reductive amination reaction between methylamine and 2-cyclopentyl-2-oxo-acetic acid and the reductive amination reaction between ethylamine and phenylpyruvic acid.

[0163] A polypeptide comprising an amino acid sequence in which the amino acid residue at a position corresponding to the methionine residue at position 141 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an isoleucine residue or a threonine residue has improved catalytic activity for the reductive amination reaction between ethylamine and phenylpyruvic acid and the reductive amination reaction between ethylamine and 2-oxo-3-(p-tolyl)propanoic acid.

[0164] A polypeptide containing an amino acid sequence in which the amino acid residue at a position corresponding to the methionine residue at position 141 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a phenylalanine residue or a histidine residue has improved catalytic activity for the reductive amination reaction of ethylamine with 2-oxo-3-(p-tolyl)propanoic acid.

[0165] A polypeptide according to one embodiment of the present invention may be a polypeptide comprising a sequence in which the amino acid residue at a position corresponding to the threonine residue at position 156 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid residue other than threonine. The polypeptide may be a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 7. The amino acid residue represented by X in SEQ ID NO: 7 is an alanine residue, aspartic acid residue, glutamic acid residue, phenylalanine residue, glycine residue, histidine residue, isoleucine residue, lysine residue, leucine residue, asparagine residue, proline residue, glutamine residue, arginine residue, serine residue, threonine residue, valine residue, tryptophan residue, or tyrosine residue.

[0166] Since the catalytic activity for the reductive amination reaction is further improved, the polypeptide may contain a sequence in which the amino acid residue at the position corresponding to the threonine residue at position 156 is substituted with a serine residue. That is, the polypeptide of this embodiment may contain an amino acid sequence (T156S) in which the amino acid residue at the position corresponding to the threonine residue at position 156 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a serine residue. In this case, the catalytic activity for the reductive amination reaction of ethylamine with 2-oxo-3-(p-tolyl)propanoic acid is further improved.

[0167] A polypeptide according to one embodiment of the present invention may be a polypeptide comprising a sequence in which the amino acid residue at the site corresponding to the histidine residue at position 182 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid residue other than histidine. The polypeptide may be a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 8. The amino acid residue represented by X in SEQ ID NO: 8 is an alanine residue, aspartic acid residue, glutamic acid residue, phenylalanine residue, glycine residue, isoleucine residue, lysine residue, leucine residue, methionine residue, asparagine residue, proline residue, glutamine residue, arginine residue, serine residue, threonine residue, valine residue, tryptophan residue, or tyrosine residue.

[0168] Since catalytic activity in reductive amination reactions is further improved, the polypeptide may include a sequence in which the amino acid residue at the position corresponding to the histidine residue at position 182 is substituted with one or more amino acid residues selected from the group consisting of tyrosine, glutamine, methionine, leucine, glycine, phenylalanine, and alanine residues. That is, the polypeptide of this embodiment may include an amino acid sequence in which the amino acid residue at the position corresponding to the histidine residue at position 182 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a tyrosine, glutamine, methionine, leucine, glycine, phenylalanine, or alanine residue (H182Y, H182Q, H182M, H182L, H182G, H182F, H182A). Furthermore, the polypeptide may include a sequence in which the amino acid residue at the position corresponding to the histidine residue at position 182 is substituted with one or more amino acid residues selected from the group consisting of methionine, leucine, and phenylalanine residues. That is, the polypeptide of this embodiment may include an amino acid sequence (H182M, H182L, H182F) in which the amino acid residue located at a position corresponding to the histidine residue at position 182 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a methionine residue, a leucine residue, or a phenylalanine residue.

[0169] A polypeptide comprising an amino acid sequence in which the amino acid residue at a position corresponding to the histidine residue at position 182 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an alanine residue, a phenylalanine residue, a leucine residue, a methionine residue, or a tyrosine residue has improved catalytic activity for the reductive amination reaction between ethylamine and phenylpyruvic acid and the reductive amination reaction between ethylamine and 2-oxo-3-(p-tolyl)propanoic acid.

[0170] A polypeptide containing an amino acid sequence in which the amino acid residue at a position corresponding to the histidine residue at position 182 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a glutamine residue has improved catalytic activity for the reductive amination reaction of methylamine with 2-cyclopentyl-2-oxo-acetic acid.

[0171] A polypeptide containing an amino acid sequence in which the amino acid residue located at a position corresponding to the histidine residue at position 182 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a glycine residue has improved catalytic activity for the reductive amination reaction of ethylamine with phenylpyruvic acid.

[0172] A polypeptide according to one embodiment of the present invention may be a polypeptide comprising a sequence in which the amino acid residue at the site corresponding to the glutamine residue at position 186 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid residue other than glutamine. The polypeptide may be a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 10. The amino acid residue represented by X in SEQ ID NO: 10 is an alanine residue, aspartic acid residue, glutamic acid residue, phenylalanine residue, glycine residue, histidine residue, isoleucine residue, lysine residue, leucine residue, methionine residue, asparagine residue, proline residue, arginine residue, serine residue, threonine residue, valine residue, tryptophan residue, or tyrosine residue.

[0173] Since this further improves the catalytic activity for reductive amination, the polypeptide may comprise a sequence in which the amino acid residue at the site corresponding to the glutamine residue at position 186 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with one or more amino acid residues selected from the group consisting of methionine residues and glutamic acid residues. That is, the polypeptide of this embodiment may comprise an amino acid sequence (Q186M, Q186E) in which the amino acid residue at the site corresponding to the glutamine residue at position 186 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a methionine residue or a glutamic acid residue.

[0174] A polypeptide containing an amino acid sequence in which the amino acid residue at the position corresponding to the glutamine residue at position 186 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a methionine residue has improved catalytic activity for the reductive amination reaction of ethylamine with phenylpyruvic acid.

[0175] A polypeptide comprising an amino acid sequence in which the amino acid residue at a position corresponding to the glutamine residue at position 186 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a glutamic acid residue has improved catalytic activity for the reductive amination reaction between ethylamine and 2-oxo-3-(p-tolyl)propanoic acid and the reductive amination reaction between ethylamine and phenylpyruvic acid.

[0176] A polypeptide according to one embodiment of the present invention may be a polypeptide comprising a sequence in which the amino acid residue at a position corresponding to the tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid residue other than tryptophan. The polypeptide may be a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 11. The amino acid residue represented by X in SEQ ID NO: 11 is an alanine residue, aspartic acid residue, glutamic acid residue, phenylalanine residue, glycine residue, histidine residue, isoleucine residue, lysine residue, leucine residue, methionine residue, asparagine residue, proline residue, glutamine residue, arginine residue, serine residue, threonine residue, valine residue, or tyrosine residue.

[0177] Since this further improves the catalytic activity for reductive amination reactions, the amino acid residue located at the site corresponding to the tryptophan residue at position 253 may comprise a sequence in which the amino acid residue is substituted with one or more amino acid residues selected from the group consisting of tyrosine residues, valine residues, threonine residues, serine residues, arginine residues, glutamine residues, proline residues, asparagine residues, methionine residues, leucine residues, lysine residues, isoleucine residues, histidine residues, phenylalanine residues, and alanine residues. That is, the polypeptide of this embodiment may comprise an amino acid sequence (W253Y, W253V, W253T, W253S, W253R, W253Q, W253P, W253N, W253M, W253L, W253K, W253I, W253H, W253F, W253A) in which the amino acid residue located at a position corresponding to the tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a tyrosine residue, a valine residue, a threonine residue, a serine residue, an arginine residue, a glutamine residue, a proline residue, an asparagine residue, a methionine residue, a leucine residue, a lysine residue, an isoleucine residue, a histidine residue, a phenylalanine residue, or an alanine residue. Furthermore, the polypeptide may comprise a sequence in which the amino acid residue at the position corresponding to the tryptophan residue at position 253 is substituted with one or more amino acid residues selected from the group consisting of leucine, isoleucine, and histidine residues, preferably a sequence in which the amino acid residue is substituted with a histidine residue. That is, the polypeptide according to this embodiment may comprise an amino acid sequence in which the amino acid residue at the position corresponding to the tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a leucine, isoleucine, or histidine residue (W253L, W253I, W253H), preferably an amino acid sequence in which the amino acid residue is substituted with a histidine residue (W253H).

[0178] A polypeptide containing an amino acid sequence in which the amino acid residue at a position corresponding to the tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a histidine residue has improved catalytic activity for the reductive amination reaction between methylamine and 2-cyclopentyl-2-oxo-acetic acid, the reductive amination reaction between ethylamine and phenylpyruvic acid, and the reductive amination reaction between ethylamine and 2-oxo-3-(p-tolyl)propanoic acid.

[0179] A polypeptide comprising an amino acid sequence in which the amino acid residue at a position corresponding to the tryptophan residue at position 253 in the amino acid sequence of SEQ ID NO: 1 is substituted with an alanine residue, a phenylalanine residue, an isoleucine residue, a leucine residue, a methionine residue, a proline residue, a glutamine residue, a threonine residue, a valine residue, or a tyrosine residue has improved catalytic activity for the reductive amination reaction between methylamine and 2-cyclopentyl-2-oxo-acetic acid and the reductive amination reaction between ethylamine and phenylpyruvic acid.

[0180] A polypeptide comprising an amino acid sequence in which the amino acid residue at a position corresponding to the tryptophan residue at position 253 in the amino acid sequence of SEQ ID NO: 1 is substituted with a lysine residue, an arginine residue, or a serine residue has improved catalytic activity for the reductive amination reaction of methylamine with 2-cyclopentyl-2-oxo-acetic acid.

[0181] A polypeptide containing an amino acid sequence in which the amino acid residue located at a position corresponding to the tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an asparagine residue has improved catalytic activity for the reductive amination reaction of ethylamine with phenylpyruvic acid.

[0182] A polypeptide according to one embodiment of the present invention may be a polypeptide comprising a sequence in which the amino acid residue at the site corresponding to the lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid residue other than lysine. The polypeptide may be a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 12. The amino acid residue represented by X in SEQ ID NO: 12 is an alanine residue, aspartic acid residue, glutamic acid residue, phenylalanine residue, glycine residue, histidine residue, isoleucine residue, leucine residue, methionine residue, asparagine residue, proline residue, glutamine residue, arginine residue, serine residue, threonine residue, valine residue, tryptophan residue, or tyrosine residue.

[0183] Since this further improves the catalytic activity for a reductive amination reaction, the amino acid residue located at the site corresponding to the lysine residue at position 260 may comprise a sequence in which the amino acid residue is substituted with one or more amino acid residues selected from the group consisting of tyrosine residues, tryptophan residues, threonine residues, serine residues, arginine residues, glutamine residues, asparagine residues, methionine residues, leucine residues, histidine residues, glycine residues, phenylalanine residues, glutamic acid residues, and alanine residues. That is, the polypeptide according to this embodiment may have an amino acid sequence in which the amino acid residue located at the site corresponding to the lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a tyrosine residue, tryptophan residue, threonine residue, serine residue, arginine residue, glutamine residue, asparagine residue, methionine residue, leucine residue, histidine residue, glycine residue, phenylalanine residue, glutamic acid residue, or alanine residue (K260Y, K260W, K260T, K260S, K260R, K260Q, K260N, K260M, K260L, K260H, K260G, K260F, K260E, K260A). Furthermore, the polypeptide may include a sequence in which the amino acid residue located at the site corresponding to the lysine residue at position 260 is substituted with one or more amino acid residues selected from the group consisting of glutamine residue, methionine residue, glutamic acid residue, and asparagine residue. That is, the polypeptide of this embodiment may have an amino acid sequence (K260Q, K260M, K260E, K260N) in which the amino acid residue located at a position corresponding to the lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a glutamine residue, a methionine residue, a glutamic acid residue, or an asparagine residue.

[0184] A polypeptide containing an amino acid sequence in which the amino acid residue at a position corresponding to the lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a glutamic acid residue or a serine residue has improved catalytic activity for the reductive amination reaction between methylamine and 2-cyclopentyl-2-oxo-acetic acid, the reductive amination reaction between ethylamine and phenylpyruvic acid, and the reductive amination reaction between ethylamine and 2-oxo-3-(p-tolyl)propanoic acid.

[0185] A polypeptide comprising an amino acid sequence in which the amino acid residue at a position corresponding to the lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a phenylalanine residue, a leucine residue, a methionine residue, a glutamine residue, a threonine residue, or a tyrosine residue has improved catalytic activity for the reductive amination reaction between ethylamine and phenylpyruvic acid and the reductive amination reaction between ethylamine and 2-oxo-3-(p-tolyl)propanoic acid.

[0186] A polypeptide containing an amino acid sequence in which the amino acid residue located at a position corresponding to the lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an alanine residue, a glycine residue, a histidine residue, an asparagine residue, an arginine residue, or a tryptophan residue has improved catalytic activity for the reductive amination reaction of ethylamine with phenylpyruvic acid.

[0187] The polypeptide according to this embodiment may include an amino acid sequence a1 (mutation: M141V) represented by SEQ ID NO: 6, in which the amino acid residue represented by X is a valine residue; an amino acid sequence a2 (mutation: M141Y) represented by SEQ ID NO: 6, in which the amino acid residue represented by X is a tyrosine residue; an amino acid sequence a3 (mutation: H182L) represented by SEQ ID NO: 8, in which the amino acid residue represented by X is a leucine residue; an amino acid sequence a4 (mutation: W253H) represented by SEQ ID NO: 11, in which the amino acid residue represented by X is a histidine residue; or an amino acid sequence a5 (mutation: K260E) represented by SEQ ID NO: 12, in which the amino acid residue represented by X is a glutamic acid residue.

[0188] The polypeptide according to this embodiment may comprise a sequence having 90% or more sequence identity with amino acid sequence a1, a2, a3, a4, or a5. The sequence identity may be 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, or may be 100%.

[0189] The polypeptide according to this embodiment may be a polypeptide comprising a sequence in which one or more amino acid residues in the amino acid sequence a1, a2, a3, a4, or a5 have been modified, and 1 to 20, 1 to 15, 1 to 10, 1 to 7, or 1 to 5 amino acid residues may be modified. The number of modified amino acid residues may be 3 or less, 2 or less, or 1.

[0190] The polypeptide according to this embodiment may have two or more alterations in the amino acid sequence represented by SEQ ID NO: 1. Preferred embodiments of the polypeptide having two or more alterations will be described below.

[0191] The polypeptide preferably comprises a sequence in which an amino acid residue located at a position corresponding to one amino acid residue selected from the group consisting of the methionine residue at position 141, the histidine residue at position 182, and the tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 has been modified as the first modification site, and an amino acid residue located at a position corresponding to one amino acid residue selected from the group consisting of the histidine residue at position 182, the tryptophan residue at position 253, and the lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1, but different from the amino acid residue modified as the first modification site, has been modified as the second modification site.

[0192] The polypeptide preferably comprises: a sequence in which the amino acid residues located at positions corresponding to the methionine residue at position 141 and the histidine residue at position 182 in the amino acid sequence represented by SEQ ID NO: 1 have been modified; a sequence in which the amino acid residues located at positions corresponding to the methionine residue at position 141 and the tryptophan residue at position 253 have been modified; a sequence in which the amino acid residues located at positions corresponding to the methionine residue at position 141 and the lysine residue at position 260 have been modified; a sequence in which the amino acid residues located at positions corresponding to the histidine residue at position 182 and the tryptophan residue at position 253 have been modified; a sequence in which the amino acid residues located at positions corresponding to the histidine residue at position 182 and the lysine residue at position 260 have been modified; or a sequence in which the amino acid residues located at positions corresponding to the tryptophan residue at position 253 and the lysine residue at position 260 have been modified; More preferably, the amino acid sequence comprises: a sequence in which the amino acid residues located at positions corresponding to the histidine residue at position 182 and the lysine residue at position 260 have been altered; or a sequence in which the amino acid residues located at positions corresponding to the tryptophan residue at position 253 and the lysine residue at position 260 have been altered.

[0193] The polypeptide preferably comprises a sequence in which the amino acid residue located at a position corresponding to the methionine residue at position 141 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with one amino acid residue selected from the group consisting of tyrosine residues, tryptophan residues, valine residues, threonine residues, arginine residues, lysine residues, isoleucine residues, and alanine residues, and more preferably comprises a sequence in which the amino acid residue is substituted with one amino acid residue selected from the group consisting of tyrosine residues, valine residues, and alanine residues.

[0194] The polypeptide preferably comprises a sequence in which the amino acid residue located at a position corresponding to the histidine residue at position 182 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with one amino acid residue selected from the group consisting of tyrosine residues, methionine residues, leucine residues, and phenylalanine residues, and more preferably comprises a sequence in which the amino acid residue is substituted with one amino acid residue selected from the group consisting of methionine residues and leucine residues.

[0195] The polypeptide preferably comprises a sequence in which the amino acid residue located at a position corresponding to the tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with one amino acid residue selected from the group consisting of tyrosine residues, threonine residues, serine residues, asparagine residues, methionine residues, histidine residues, and alanine residues, and more preferably comprises a sequence in which the amino acid residue is substituted with a histidine residue.

[0196] The polypeptide preferably comprises a sequence in which the amino acid residue located at a position corresponding to the lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with one amino acid residue selected from the group consisting of serine residues, glutamine residues, asparagine residues, leucine residues, isoleucine residues, histidine residues, glycine residues, phenylalanine residues, glutamic acid residues, and alanine residues, and more preferably comprises a sequence in which the amino acid residue is substituted with one amino acid residue selected from the group consisting of glutamine residues and glutamic acid residues.

[0197] In the amino acid sequence represented by SEQ ID NO: 1, m １ Amino acid residue X at position １ The amino acid residue located at the position corresponding to １ Other amino acid residues other than x １ and m ２ Amino acid residue X at position ２ The amino acid residue located at the position corresponding to ２ Other amino acid residues other than x ２ The sequence replaced by X １ m １ x １ _X ２m ２ x ２ For example, the amino acid sequence represented by SEQ ID NO: 23 is represented as M141X_H182X, since the amino acid residues at positions 141 and 182 are each substituted with another amino acid residue X. Specifically, the amino acid sequence represented by SEQ ID NO: 23 in which X at the position corresponding to the amino acid residue at position 141 is a tyrosine residue and X at the position corresponding to the amino acid residue at position 182 is a methionine residue is represented as M141Y_H182M. The amino acid sequences represented by SEQ ID NOs: 24 to 30 are represented in the same manner.

[0198] The sequence in which two amino acid residues in the amino acid sequence represented by SEQ ID NO: 1 have been modified may be, for example, an amino acid sequence selected from the group consisting of Groups a1 to a4 consisting of the amino acid sequences shown below, preferably an amino acid sequence selected from the group consisting of Groups a1 to a3, more preferably an amino acid sequence selected from the group consisting of Groups a1 and a2, and most preferably an amino acid sequence selected from Group a1. Group a1: M141Y_K260E, H182L_K260Q, M141A_K260E, H182M_K260Q, M141A_K260Q, H182M_K260E, M141V_K260E, W253H_K260E, and H182L_K260E. Group a2: M141V_K260Q, W253H_K260Q, W253M_K260E, M141Y_H182M, M141Y_K260Q, H182F_K260Q, M141V_ K260F, M141V_H182L, M141V_K260L, M141Y_K260G, H182L_W253H, H182F_K260E, and M141T_K260Q Group a3: M141Y_H182L, W253M_K260Q, M141V_W253M, M141V_K260S, M141V_K260A, M141A_K260F, M141A_K260H, W253A_K260E, M141V_W253A, M141V_W253H, M141K_K260L, M141V_K260G, and M141A_K260L. Group a4: M141W_K260A, M141V_W253Y, M141K_K260G, M141A_K260G, M141A_K260S, M141A_K260N, M141Y_W253H, M141A_K260A, M141K_K260S, W253A_K260Q, M141V_H182M, M141A_K260I, M141T_K260G, W253M_K260L, M141I_W253A, W253T_K260L, M141R_K260G, W253A_K260L, M141K_W253S, M141A_H182L, W253N_K260L, M141K_K260A, M141K_W253M, H182Y_W253A, H182M_W253H, M141R_K260A, H182M_W253A and M141K_W253A.

[0199] The polypeptide may, for example, comprise a sequence having 90% or more sequence identity with an amino acid sequence selected from the group consisting of groups a1 to a4, preferably comprises a sequence having 90% or more sequence identity with an amino acid sequence selected from the group consisting of groups a1 to a3, more preferably comprises a sequence having 90% or more sequence identity with an amino acid sequence selected from the group consisting of groups a1 and a2, and most preferably comprises a sequence having 90% or more sequence identity with an amino acid sequence selected from group a1.

[0200] The polypeptide may comprise an amino acid sequence selected from the group consisting of groups a1 to a4, preferably comprises an amino acid sequence selected from the group consisting of groups a1 to a3, more preferably comprises an amino acid sequence selected from the group consisting of groups a1 and a2, and most preferably comprises an amino acid sequence selected from group a1.

[0201] The polypeptide according to this embodiment may have, for example, three or more alterations in the amino acid sequence represented by SEQ ID NO: 1. Preferred embodiments of the polypeptide having three or more alterations will be described below.

[0202] The polypeptide preferably comprises a sequence in which an amino acid residue located at a position corresponding to one amino acid residue selected from the group consisting of the methionine residue at position 141 and the histidine residue at position 182 in the amino acid sequence represented by SEQ ID NO: 1 has been modified as the first modification site; an amino acid residue located at a position corresponding to one amino acid residue selected from the group consisting of the histidine residue at position 182 and the tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 and different from the amino acid residue modified as the first modification site has been modified as the second modification site; and an amino acid residue located at a position corresponding to one amino acid residue selected from the group consisting of the tryptophan residue at position 253 and the lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1 and different from the amino acid residues modified as the first and second modification sites has been modified as the third modification site.

[0203] The polypeptide preferably comprises: a sequence in which the amino acid residues located at positions corresponding to the methionine residue at position 141, the histidine residue at position 182 and the tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 have been modified; a sequence in which the amino acid residues located at positions corresponding to the methionine residue at position 141, the histidine residue at position 182 and the lysine residue at position 260 have been modified; a sequence in which the amino acid residues located at positions corresponding to the methionine residue at position 141, the tryptophan residue at position 253 and the lysine residue at position 260 have been modified; a sequence in which the amino acid residues located at positions corresponding to the histidine residue at position 182, the tryptophan residue at position 253 and the lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1 have been modified; More preferably, the sequence comprises: a sequence in which amino acid residues located at positions corresponding to the methionine residue at position 141, the tryptophan residue at position 253, and the lysine residue at position 260 have been modified; or a sequence in which amino acid residues located at positions corresponding to the histidine residue at position 182, the tryptophan residue at position 253, and the lysine residue at position 260 have been modified.

[0204] The polypeptide preferably has three alterations in the amino acid sequence represented by SEQ ID NO: 1, and comprises a sequence in which the amino acid residue at the position corresponding to the methionine residue at position 141 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with one amino acid residue selected from the group consisting of tyrosine residue, valine residue, threonine residue, serine residue, leucine residue, lysine residue, isoleucine residue, and alanine residue; and more preferably has three alterations in the amino acid sequence represented by SEQ ID NO: 1, and comprises a sequence in which the amino acid residue at the position corresponding to the methionine residue at position 141 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with one amino acid residue selected from the group consisting of tyrosine residue, valine residue, and isoleucine residue.

[0205] The polypeptide preferably has three alterations in the amino acid sequence represented by SEQ ID NO: 1, and comprises a sequence in which the amino acid residue located at the position corresponding to the histidine residue at position 182 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with one amino acid residue selected from the group consisting of methionine residues, leucine residues, and phenylalanine residues; and more preferably has three alterations in the amino acid sequence represented by SEQ ID NO: 1, and comprises a sequence in which the amino acid residue located at the position corresponding to the histidine residue at position 182 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with one amino acid residue selected from the group consisting of methionine residues and leucine residues.

[0206] The polypeptide preferably has three alterations in the amino acid sequence represented by SEQ ID NO: 1, and comprises a sequence in which the amino acid residue at the position corresponding to the tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with one amino acid residue selected from the group consisting of valine residue, threonine residue, serine residue, glutamine residue, methionine residue, leucine residue, histidine residue, phenylalanine residue, and glutamic acid; and more preferably has three alterations in the amino acid sequence represented by SEQ ID NO: 1, and comprises a sequence in which the amino acid residue at the position corresponding to the tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with one amino acid residue selected from the group consisting of glutamine residue, methionine residue, leucine residue, and histidine residue.

[0207] It is preferable that the polypeptide has three alterations in the amino acid sequence represented by SEQ ID NO: 1, and contains a sequence in which the amino acid residue located at the site corresponding to the lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with one amino acid residue selected from the group consisting of glutamine residues and glutamic acid residues.

[0208] In the amino acid sequence represented by SEQ ID NO: 1, m １ Amino acid residue X at position １ The amino acid residue located at the position corresponding to１ Other amino acid residues other than x １ is replaced by m ２ Amino acid residue X at position ２ The amino acid residue located at the position corresponding to ２ Other amino acid residues other than x ２ and m ３ Amino acid residue X at position ３ The amino acid residue located at the position corresponding to ３ Other amino acid residues other than x ３ The sequence replaced by X １ m １ x １ _X ２ m ２ x ２ _X ３ m ３ x ３ For example, the amino acid sequence represented by SEQ ID NO: 31 is represented as M141X_W253X_K260X, since each of the amino acid residues located at positions 141, 253, and 260 is substituted with another amino acid residue X. Specifically, the amino acid sequence represented by SEQ ID NO: 31, in which X located at the position corresponding to the amino acid residue at position 141 is a valine residue, X located at the position corresponding to the amino acid residue at position 253 is a methionine residue, and X located at the position corresponding to the amino acid residue at position 260 is a glutamic acid residue, is represented as M141V_W253M_K260E. The amino acid sequences represented by SEQ ID NOs: 32 to 35 are represented in the same manner.

[0209] The sequence in which three amino acid residues in the amino acid sequence represented by SEQ ID NO: 1 have been modified may be, for example, an amino acid sequence selected from the group consisting of Groups b1 to b4 consisting of the amino acid sequences shown below, preferably an amino acid sequence selected from the group consisting of Groups b1 to b3, more preferably an amino acid sequence selected from the group consisting of Groups b1 and b2, and most preferably an amino acid sequence selected from Group b1. Group b1: M141Y_H182M_K260E, M141V_W253M_K260E, H182L_W253H_K260E, M141Y_H182M_K260Q, M141V_H18 2M_K260E, M141V_H182L_K260Q, M141Y_H182L_K260Q, M141V_W253Q_K260E, H182L_W253Q_K260E, M 141I_W253H_K260E, M141V_H182L_K260E, M141Y_H182L_K260E, H182L_W253M_K260E, M141V_W253 Q_K260Q, M141V_W253H_K260Q, M141V_W253L_K260Q, M141I_W253H_K260Q, and M141V_W253M_K260Q. Group b2: H182M_W253H_K260E, M141V_W253V_K260Q, H182L_W253H_K260Q, M141V_W253H_K260E, H182L_W253Q_K260Q, M141L_W25 3H_K260E, H182M_W253Q_K260E, M141V_W253F_K260Q, M141V_W253L_K260E, M141V_W253T_K260Q, M141Y_H182F_K260E, H 182M_W253M_K260E, H182L_W253M_K260Q, M141Y_W253H_K260E, M141Y_W253Q_K260E, M141V_H182M_K260Q, H182M_W253L_K260E, H182F_W253H_K260E, M141A_H182F_K260Q, M141Y_W253M_K260E, H182M_W253Q_K260Q, and H182M_W253M_K260Q.Group b3: H182L_W253T_K260Q, M141Y_W253S_K260E, M141V_H182F_K260E, M141A_H182M_K260E, M141T_H182L_K260Q, M141Y_W25 3T_K260E, H182M_W253L_K260Q, M141V_H182F_K260Q, M141S_W253H_K260E, M141T_W253H_K260E, H182M_W253H_K260Q, M 141A_H182L_K260Q, H182M_W253F_K260Q, M141A_H182M_K260Q, M141A_W253Q_K260E, M141A_H182L_K260E, M141A_W253H_K260E, M141V_W253E_K260Q, M141A_W253M_K260E, M141A_W253H_K260Q, M141K_H182L_W253H, and M141T_H182M_K260Q. Group b4: M141A_W253M_K260Q, and M141A_H182F_K260E.

[0210] The polypeptide may, for example, comprise a sequence having 90% or more sequence identity with an amino acid sequence selected from the group consisting of Groups b1 to b4, preferably comprises a sequence having 90% or more sequence identity with an amino acid sequence selected from the group consisting of Groups b1 to b3, more preferably comprises a sequence having 90% or more sequence identity with an amino acid sequence selected from the group consisting of Groups b1 and b2, and most preferably comprises a sequence having 90% or more sequence identity with an amino acid sequence selected from Group b1.

[0211] The polypeptide may comprise an amino acid sequence selected from the group consisting of Groups b1 to b4, preferably comprises an amino acid sequence selected from the group consisting of Groups b1 to b3, more preferably comprises an amino acid sequence selected from the group consisting of Groups b1 and b2, and most preferably comprises an amino acid sequence selected from Group b1.

[0212] The polypeptide according to this embodiment has a catalytic activity, under at least one reaction condition, for a reductive amination reaction between one or more compounds A represented by the above formula (1) or salts thereof and one or more compounds B represented by the above formula (2) or salts thereof, or for an intramolecular reductive amination reaction between one or more compounds B represented by the above formula (2) or salts thereof, which is higher than the catalytic activity of a polypeptide having the amino acid sequence represented by SEQ ID NO: 1. In one aspect, the catalytic activity of the polypeptide according to this embodiment is higher than the catalytic activity of a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 17, under at least one reaction condition.

[0213] In one aspect, the polypeptide of this embodiment has a higher catalytic activity for the reductive amination reaction between ethylamine or a salt thereof and phenylpyruvic acid or a salt thereof than a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 17 under at least one reaction condition.

[0214] In one aspect, the polypeptide according to this embodiment has a higher catalytic activity for the reductive amination reaction between ethylamine or a salt thereof and 2-oxo-3-(p-tolyl)propanoic acid or a salt thereof than the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 17 under at least one reaction condition.

[0215] In one aspect, the polypeptide according to this embodiment has a higher catalytic activity for the reductive amination reaction between methylamine or a salt thereof and 2-cyclopentyl-2-oxo-acetic acid or a salt thereof than the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 17 under at least one reaction condition.

[0216] In one aspect, the polypeptide according to this embodiment has catalytic activity for the reductive amination reaction of ammonia or a salt thereof with phenylpyruvic acid or a salt thereof under at least one reaction condition.

[0217] The polypeptide according to this embodiment may be fused with another polypeptide or protein. That is, the polypeptide according to this embodiment may have, at either or both of the N-terminus and C-terminus, an amino acid sequence (another amino acid sequence) other than the sequence having 90% or more sequence identity with the sequence in which one amino acid residue in the amino acid sequence represented by SEQ ID NO: 1 has been modified. The other amino acid sequence may be, for example, a tag sequence.

[0218] Examples of polypeptides or proteins having such other amino acid sequences include His tags (6XHis, 10XHis, etc.) that are tags consisting of several (e.g., 6, 10, etc.) His (histidine) residues, streptavidin-binding peptide tags (SBP tags) that contain an amino acid sequence capable of binding to the biotin-binding site of streptavidin, GST (Glutathione S-transferase), HA (influenza agglutinin), immunoglobulin constant regions, B-galactosidase, MBP (maltose-binding protein), FLAG (Hopp, T.P. et al., J. Immunol. Chem. Soc. 1999, 123:111-112, 2001), and the like. al., BioTechnology (1988) 6, 1204-1210), a fragment of human c-myc, a fragment of VSV-GP, a fragment of p18HIV, T7-tag, HSV-tag, E-tag, a fragment of SV40 T antigen, Ick tag, a fragment of α-tubulin, B-tag, a fragment of Protein C, Tag, StrepTag, HaloTag, etc. The His tag may be, for example, the amino acid sequence represented by SEQ ID NO: 14. The SBP tag may be, for example, the amino acid sequence represented by SEQ ID NO: 15.

[0219] The polypeptide according to this embodiment may comprise one or more sequences selected from the group consisting of a streptavidin-binding peptide tag sequence and a His tag sequence at either or both of the N-terminus and the C-terminus, and may comprise a streptavidin-binding peptide tag sequence and a His tag sequence at the C-terminus. The linker sequence may have, for example, an amino acid sequence represented by GGSS or GGS. An example of an amino acid sequence comprising a streptavidin-binding peptide tag sequence, a linker sequence, and a His tag sequence is the amino acid sequence represented by SEQ ID NO: 16.

[0220] The polypeptide according to this embodiment may have the amino acid sequence a1, a2, a3, a4, or a5 and a tag sequence. The polypeptide according to this embodiment may have, in addition to the amino acid sequence a1, a2, a3, a4, or a5 and the tag sequence, a linker sequence connecting the amino acid sequence a1, a2, a3, a4, or a5 and the tag sequence.

[0221] The polypeptide according to one embodiment is an amino acid sequence represented by SEQ ID NO: 18, which is represented by SEQ ID NO: 6 and has an amino acid sequence a1 (mutation: M141V) in which the amino acid residue represented by X is a valine residue, and the amino acid sequence represented by SEQ ID NO: 16 linked to the C-terminus of the amino acid sequence a1; an amino acid sequence represented by SEQ ID NO: 19, which is represented by SEQ ID NO: 6 and has an amino acid sequence a2 (mutation: M141Y) in which the amino acid residue represented by X is a tyrosine residue, and the amino acid sequence represented by SEQ ID NO: 16 linked to the C-terminus of the amino acid sequence a2; an amino acid sequence represented by SEQ ID NO: 8 and has an amino acid sequence a3 (mutation: H182L) in which the amino acid residue represented by X is a leucine residue, and the amino acid sequence represented by SEQ ID NO: 16 linked to the C-terminus of the amino acid sequence a3; an amino acid sequence represented by SEQ ID NO: 21, which is represented by SEQ ID NO: 11 and has an amino acid sequence a4 (mutation: W253H) in which the amino acid residue represented by X is a histidine residue, and the amino acid sequence represented by SEQ ID NO: 16 linked to the C-terminus of the amino acid sequence a4; The amino acid sequence may have an amino acid sequence represented by SEQ ID NO: 22, which has an amino acid sequence a5 (mutation: K260E) represented by SEQ ID NO: 12 in which the amino acid residue represented by X is a glutamic acid residue, and an amino acid sequence represented by SEQ ID NO: 16 linked to the C-terminus of the amino acid sequence a5.

[0222] One or more amino acid residues in the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22 may be modified, and 1 to 20, 1 to 15, 1 to 10, 1 to 7, or 1 to 5 amino acid residues may be modified. In the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22, the number of modified amino acid residues may be 3 or less, 2 or less, or 1.

[0223] The polypeptide according to one embodiment preferably has an amino acid sequence represented by any one of SEQ ID NOs: 36 to 44.

[0224] The polypeptide according to one embodiment preferably has an amino acid sequence represented by any one of SEQ ID NOs: 45 to 62.

[0225] The polypeptide according to the present embodiment may be used as a mixture with other polypeptides, or may be used in an isolated and purified state. When used as a mixture with other polypeptides, the target amino acid can be obtained through a process of isolating and purifying the target product.

[0226] When the polypeptide according to this embodiment exists as a monomer, the number of amino acid residues may be 300 or more, 310 or more, 320 or more, or 325 or more, or may be 330. When the polypeptide according to this embodiment exists as a monomer, the number of amino acid residues may be 400 or less, 390 or less, 380 or less, or 375 or less.

[0227] Furthermore, when the other amino acid sequence is not added to either the N-terminus or the C-terminus, the number of amino acid residues of the polypeptide according to this embodiment may be 300 or more, 310 or more, 320 or more, or 325 or more, or may be 330. Furthermore, when the other amino acid sequence is not added to either the N-terminus or the C-terminus, the number of amino acid residues of the polypeptide according to this embodiment may be 360 ​​or less, 350 or less, 340 or less, or 335 or less.

[0228] Furthermore, when the other amino acid sequence is added to either or both of the N-terminus and the C-terminus, the number of amino acid residues of the polypeptide according to this embodiment may be 340 or more, 350 or more, 360 or more, or 370 or more, or may be 374. Furthermore, when the other amino acid sequence is added to either or both of the N-terminus and the C-terminus, the number of amino acid residues of the polypeptide according to this embodiment may be 400 or less, 390 or less, 380 or less, or 375 or less.

[0229] The polypeptide according to this embodiment may be used as a monomer, or may be used in a form in which two or more monomers are associated with each other.Furthermore, the polypeptide according to this embodiment may be a homodimer.

[0230] When the polypeptide according to this embodiment is a homodimer, the number of amino acid residues is twice that of when the polypeptide is present as a monomer. In one aspect, the polypeptide according to this embodiment has a catalytic activity under at least one reaction condition that is higher than the catalytic activity of a polypeptide having the amino acid sequence represented by SEQ ID NO: 1.

[0231] In one aspect, the polypeptide according to this embodiment has a temperature range of 0°C or higher and 50°C or lower at which the catalytic activity is higher than that of the polypeptide having the amino acid sequence represented by SEQ ID NO: 1.

[0232] Furthermore, the polypeptide according to this embodiment may have catalytic activity for the reductive amination reaction between one or more compounds A or salts thereof and one or more compounds B or salts thereof, or for the intramolecular reductive amination reaction of one or more compounds B or salts thereof, at least at one point under reaction conditions of 0°C to 50°C. The lower limit of the temperature may be 0°C or higher, 10°C or higher, 20°C or higher, or 30°C or higher. The upper limit of the reaction temperature may be 50°C or lower, 40°C or lower, or 30°C or lower. The reaction temperature may be, for example, 0°C to 50°C, 10°C to 40°C, or 20°C to 30°C. In one aspect, the temperature may be 25°C or 37°C. The reaction temperature may be changed stepwise or continuously.

[0233] In one aspect, the polypeptide according to this embodiment has a pH in the range of 7 to 11, at which the catalytic activity is higher than that of the polypeptide having the amino acid sequence represented by SEQ ID NO: 1. Furthermore, the polypeptide according to this embodiment may have catalytic activity at at least one point under reaction conditions of pH 7 to 11. The lower limit of the pH may be 7 or more, 7.5 or more, 8 or more, or 8.5 or more. The upper limit of the pH may be 11 or less, 10 or less, 9.5 or less, or 9 or less. The pH may be, for example, pH 7 to 11 or less, 8 to 10 or less, or 8.5 to 9.5 or less. In one aspect, the pH may be 8 or 9. Note that the pH here refers to the pH at the start of the reaction, and pH fluctuations during the reaction are permitted. The pH fluctuation during the reaction may be within 2, 1.5, or 1.

[0234] In one aspect, the polypeptide according to this embodiment has higher catalytic activity than the polypeptide having the amino acid sequence represented by SEQ ID NO: 1 at least at one point in the range of 100 mM or more and 3000 mM or less of the total concentration of compound A and a salt thereof at the start of the reductive amination reaction in the total volume of the reaction solution. In one aspect, the polypeptide according to this embodiment has higher catalytic activity than the polypeptide having the amino acid sequence represented by SEQ ID NO: 1 under reaction conditions of 37°C and pH 8.

[0235] In one aspect, the polypeptide according to this embodiment has a catalytic activity higher than that of the polypeptide having the amino acid sequence represented by SEQ ID NO:1 under reaction conditions of 25°C and pH 9.

[0236] Furthermore, the polypeptide according to this embodiment may have catalytic activity under reaction conditions in which the total concentration of compound A and its salt is 100 mM or more and 3000 mM or less. The lower limit of the total concentration of compound A and its salt may be 100 mM or more, 300 mM or more, 500 mM or more, 700 mM or more, 900 mM or more, 1100 mM or more, 1300 mM or more, 1500 mM or more, or 1700 mM or more. The upper limit of the total concentration of compound A and its salt may be 3000 mM or less, 2500 mM or less, or 2000 mM or less. The total concentration of compound A and its salt may be 100 mM or more and 3000 mM or less, or 500 mM or more and 2000 mM or less. In one aspect, the total concentration of compound A and its salt may be 500 mM or 1750 mM. The total concentration of compound A and its salt is the total concentration of compound A and its salt at the start of the reaction in the total amount of the reaction solution when the reductive amination reaction is carried out.

[0237] In one aspect, the polypeptide according to this embodiment has a higher catalytic activity than the polypeptide having the amino acid sequence represented by SEQ ID NO: 1 at least at one point in the range where the total concentration of compound B and a salt thereof at the start of the reductive amination reaction is 0.001 mM or more and 1000 mM or less in the total volume of the reaction solution.

[0238] Furthermore, the polypeptide according to this embodiment may have catalytic activity under reaction conditions in which the total concentration of compound B and its salt is 10 mM or more and 500 mM or less. The lower limit of the total concentration of compound B and its salt may be 10 mM or more, 30 mM or more, 50 mM or more, 100 mM or more, 150 mM or more, 200 mM or more, 250 mM or more, 300 mM or more, or 330 mM or more. The upper limit of the total concentration of compound B and its salt may be 500 mM or less, 450 mM or less, 400 mM or less, or 380 mM or less. The total concentration of compound B and its salt may be 10 mM or more and 500 mM or less, or 300 mM or more and 400 mM or less. In one aspect, the total concentration of compound B and its salt may be 50 mM or 350 mM. The total concentration of compound B and its salt is the total concentration of compound B and its salt at the start of the reaction in the total amount of the reaction solution when the reductive amination reaction is carried out.

[0239] In one aspect, the polypeptide according to this embodiment has a catalytic activity higher than that of a polypeptide having the amino acid sequence represented by SEQ ID NO: 1 at least at one point when the ratio of the total number of moles of compound A and a salt thereof to the total number of moles of compound B and a salt thereof at the start of the reductive amination reaction in the total amount of reaction solution is 1 or more.

[0240] Furthermore, the polypeptide according to this embodiment may have catalytic activity under reaction conditions in which the ratio of the total number of moles of compound A and a salt thereof to the total number of moles of compound B and a salt thereof (total number of moles of compound A and a salt thereof / total number of moles of compound B and a salt thereof) is 1 or more.

[0241] The ratio of the total number of moles of compound A and its salt to the total number of moles of compound B and its salt (total number of moles of compound A and its salt / total number of moles of compound B and its salt) may be 1 or more, 3 or more, 5 or more, 8 or more, or 9 or more, and may be 50 or less, 30 or less, 20 or less, or 15 or less, or may be 5 or 10. The ratio of the total number of moles of compound A and its salt to the total number of moles of compound B and its salt is the ratio of the total number of moles of compound A and its salt to the total number of moles of compound B and its salt at the start of the reaction in the total amount of reaction solution when carrying out the reductive amination reaction.

[0242] The polypeptide according to this embodiment can be used in a method for producing an amino acid. The method for producing an amino acid includes the step of reacting one or more compounds selected from the group consisting of amines, amine analogs, and salts thereof with one or more compounds selected from the group consisting of keto acids, keto acid analogs, and salts thereof, or reacting a compound selected from the group consisting of keto acids, keto acid analogs, and salts thereof intramolecularly in the presence of the polypeptide and a reducing agent. When the reaction is carried out intramolecularly, the keto acids and keto acid analogs may have a primary amino group or a secondary amino group.

[0243] In the method for producing an amino acid, the reaction can be carried out under appropriate conditions, where appropriate conditions refer to conditions described in paragraphs

[0231] to

[0241] , in which the temperature, pH, the total concentration of compound A and its salt, the total concentration of compound B and its salt in the reaction solution at the start of the reaction, and the ratio of the total number of moles of compound A and its salt to the total number of moles of compound B and its salt are appropriately set.

[0244] The reaction may be carried out in a reaction solution containing an amine or amine analog, a keto acid or keto acid analog, the polypeptide, and a reducing agent. In one embodiment, compound A can be used as the amine or amine analog. The amine or amine analog may be an alkylamine or ammonia, and the alkylamine may be C １ ~C ６ Alkylamine or C １ ~C ４The keto acid or keto acid analogue may be a keto acid represented by the formula (2) where Y is C. ３ ~C ８ Cycloalkyl or C ６ ~C ９ It may be an aralkyl. More specific examples include 2-cyclopentyl-2-oxo-acetic acid, phenylpyruvic acid, and 2-oxo-3-(p-tolyl)propanoic acid. The amine or amine analog and the keto acid or keto acid analog may be added to the reaction solution in the form of a salt. The substances used in the reaction may be mixed simultaneously or separately in any order. The salt is preferably a chemically or pharmaceutically acceptable salt. Examples of the salt include hydrochloride; hydrobromide; hydroiodide; phosphate; phosphonate; sulfate; sulfonate such as methanesulfonate and p-toluenesulfonate; carboxylate such as acetate, citrate, malate, tartrate, succinate, and salicylate; alkali metal salts such as lithium salt, sodium salt, and potassium salt; alkaline earth metal salts such as magnesium salt and calcium salt; and ammonium salts such as ammonium salt, alkylammonium salt, dialkylammonium salt, trialkylammonium salt, and tetraalkylammonium salt.

[0245] Reducing agents include, for example, reduced nicotinamide adenine dinucleotide phosphate (NADPH), oxidized nicotinamide adenine dinucleotide phosphate (NADP+), reduced nicotinamide adenine dinucleotide (NADH), and oxidized nicotinamide adenine dinucleotide (NAD+). In one embodiment, the reducing agent can be β-NADPH.

[0246] Furthermore, additives that reduce NADP+ and NAD+ to NADPH and NADH, respectively, such as glucose dehydrogenase (GDH) and glucose, can be added together. For example, when NADPH is used as a reducing agent, the amount of NADPH used can be reduced, and therefore, in the reaction step, NADP that may be generated in the reaction solution can be reduced. ＋ A reactant that reduces oxidized nicotinamide adenine dinucleotide phosphate (NADPH) to NADPH may be used. For example, a combination of glucose and GDH (glucose dehydrogenase) may be used. When glucose and GDH are used together with NADPH, the amount of NADPH used can be reduced to a catalytic amount.

[0247] The concentration of the reducing agent in the reaction solution at the start of the reaction may be, for example, 0.001 mM to 100 mM, or 0.01 to 10 mM.

[0248] In the reaction, the reaction conditions such as temperature, pH, and concentration of each compound may be the conditions described above.

[0249] In the method for producing an amino acid, a reductive amination reaction may be carried out in the presence of a compound C represented by the following formula (4):

[0250] In formula (4), v and w each independently represent 0 or 1, provided that at least one of v and w represents 1, and T represents a carbon atom, a phosphorus atom, or a sulfur atom.

[0251] In formula (4), the following formula (4a) The functional group represented by formula (4a) represents ═O, —ORd, or a hydroxy group. Here, when the functional group represented by formula (4a) is ═O, it means that T is bonded to an oxygen atom via a double bond. That is, compound C in which the functional group represented by formula (4a) is ═O has a structure represented by T═O. When the functional group represented by formula (4a) is —ORd, it means that T is bonded to ORd via a single bond. That is, compound C in which the functional group represented by formula (4a) is —ORd has a structure represented by T-ORd. When the functional group represented by formula (4a) is a hydroxy group, it means that T and the hydroxy group are directly bonded. That is, compound C in which the functional group represented by formula (4a) is —OH has a structure represented by T-OH.

[0252] In formula (4), when v and w are both 1, two functional groups represented by formula (4a) present in plural may be the same or different. Ra, Rb, and Rc each independently represent a hydrogen atom, C １ ~C ３ Alkyl group, alkylamino group or -CH ２ -ORd, and any two or more of Ra, Rb, and Rc may be bonded to each other together with T to form a ring structure. １ ~C ３ represents an alkyl group, and d, e, and f each independently represent 0 or 1, provided that at least one of d, e, and f represents 1.

[0253] In formula (4), when v and w are both 1, one or more of Ra, Rb, and Rc are methyl groups, and Ra, Rb, and Rc are not bonded to each other to form a ring structure together with T. For example, when v and w are both 1, T is a sulfur atom, all functional groups represented by formula (4a) are ═O, f is 0, and at least one of Ra and Rb may be a methyl group.

[0254] When any one or more of Ra, Rb, and Rc is a methylamino group, Ra, Rb, and Rc do not bond to each other to form a ring structure with T. When the functional group represented by formula (4a) is a hydroxy group and T is a carbon atom, v is 1, w is 0, d, e, and f are all 1, and Ra, Rb, and Rc are all hydrogen atoms.

[0255] Compound C may be a compound represented by the following formula (4-1), in which T in formula (1) is a carbon atom.

[0256] The compound represented by formula (4-1) may be a compound represented by the following formula (4-1a) in which v is 1 and w is 0.

[0257] In formula (4-1a), when the functional group represented by formula (4a) is ═O, two of d, e, and f are 1, and the others are 0. When the functional group represented by formula (4a) is ═O, the compound represented by formula (4-1a) may be a compound represented by the following formula (4-1b) in which d and e are 1 and f is 0.

[0258] The compound represented by formula (4-1b) is a compound in which one of Ra and Rb is a hydrogen atom or C １ ~C ３ The compound represented by formula (4-1b) may be a compound in which one group is an alkyl group and the other is an alkylamino group. The alkylamino group may be a monomethylamino group or a dimethylamino group. For example, the compound represented by formula (4-1b) may be N,N-dimethylformamide, N,N-dimethylacetamide, or N-methylformamide.

[0259] In formula (4-1a), when the functional group represented by formula (4a) is a hydroxy group, d, e, and f are all 1. When the functional group represented by formula (4a) is a hydroxy group, the compound represented by formula (4-1a) may be a compound represented by the following formula (4-1c) in which d, e, and f are all 1.

[0260] Ra, Rb and Rc each independently represent a hydrogen atom or C１ ~C ３ The compound represented by formula (4-1c) may be methanol in which Ra, Rb and Rc are all hydrogen atoms.

[0261] In formula (4-1a), when the functional group represented by formula (4a) is -ORd, d, e, and f may all be 1, and Rb and Rd may all be hydrogen atoms. When the functional group represented by formula (4a) is -ORd, the compound represented by formula (4-1a) may be a compound represented by the following formula (4-1d) in which d, e, and f are all 1, and Rb and Rd are all hydrogen atoms. In the compound represented by formula (4-1d), Ra is -CH ２ In the compound represented by the following formula (4-1d), Ra may be —CH ２ -ORd, and each Rd is -CH ３ It may be dimethoxyethane,

[0262] Compound C may be a compound represented by the following formula (4-2), in which T in formula (4) is a phosphorus atom.

[0263] The compound represented by formula (4-2) may be a compound represented by the following formula (4-2a) in which v is 1, w is 0, and the functional group represented by formula (4a) is ═O.

[0264] In formula (4-2a), Ra, Rb and Rc are all C １ ~C ３ The compound represented by formula (4-2a) may be trimethylphosphine oxide in which Ra, Rb and Rc are all methyl groups.

[0265] Compound C may be a compound represented by the following formula (4-3), in which T in formula (4) is a sulfur atom.

[0266] The compound represented by formula (4-3) may be a compound represented by the following formula (4-3a), in which v is 1, w is 0, the functional group represented by formula (4a) is ═O, d and e are both 1, and f is 0.

[0267] In formula (4-3a), Ra and Rb are both C １ ~C ３ They may be alkyl groups, and Ra and Rb may be linked to each other to form a ring structure together with the sulfur atom (S).

[0268] In the compound represented by formula (4-3a), Ra and Rb may each independently be a methyl group or an ethyl group. In the compound represented by formula (4-3a), Ra and Rb may each independently be a methyl group, such as dimethyl sulfoxide, or Ra and Rb may each independently be an ethyl group, such as diethyl sulfoxide. In the compound represented by formula (4-3a), Ra and Rb may each independently be C １ ~C ３ It may be tetramethylene sulfoxide, which is an alkyl group in which the alkyl groups are linked to each other to form a ring structure together with S.

[0269] The compound represented by formula (4-3) may be a compound represented by the following formula (4-3b), in which v and w are both 1, the functional group represented by formula (4a) is ═O, d and e are both 1, and f is 0.

[0270] In the compound represented by formula (4-3b), at least one of Ra and Rb is a methyl group. The compound represented by formula (4-3b) may be dimethyl sulfone in which both Ra and Rb are methyl groups.

[0271] Compound C may be one or more compounds selected from the group consisting of dimethyl sulfoxide, dimethyl sulfone, trimethylphosphine oxide, dimethoxyethane, N,N-dimethylformamide, N,N-dimethylacetamide, tetramethylene sulfoxide, diethyl sulfoxide, methanol, and methylformamide, because compound C allows for the production of amino acids in a higher yield. Compound C may be one or more compounds selected from the group consisting of dimethyl sulfoxide, dimethyl sulfone, and trimethylphosphine oxide, or may be dimethyl sulfoxide, because compound C allows for the production of amino acids in a higher yield.

[0272] Compound C may be liquid or solid under conditions of 25°C and 1 atmosphere, and may be liquid under conditions of 25°C and 1 atmosphere, since this further improves the production efficiency of the amino acid. Examples of compound C that is liquid under conditions of 25°C and 1 atmosphere include dimethyl sulfoxide, dimethoxyethane, N,N-dimethylformamide, N,N-dimethylacetamide, methanol, and N-methylformamide.

[0273] The method for producing an amino acid may include the following steps (A) and (B): step (A): contacting a target product for purification, which is a mixture of the following (i) as a target product for purification and the following (ii) as an impurity, obtained by the above-described method, with a lithium-containing substance, (i) the amino acid having a protecting group at the N-terminus, and (ii) a compound other than the target product for purification, and step (B): precipitating a lithium salt of the target product for purification.

[0274] Another embodiment of the present invention is a method for removing impurities from a material to be purified, comprising the above steps (A) and (B).

[0275] Yet another embodiment of the present invention is a lithium salt of an amino acid or a solvate thereof, wherein the lithium salt of an amino acid is represented by the following general formula (5) at a purity of 99 mol % or more or an apparent purity of 99% or more:

[0276] In the general formula (5), n5 represents a number of 1 or more and 3 or less, and R５ａ When there are multiple ５ａ may be the same or different, and R ５ａ and R ５ｂ each independently represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, a heterocyclyl group, or a heteroaryl group, or a substituent to which two or more groups selected from the group consisting of these substituents are bonded, and these groups may have a substituent, and these groups may be saturated or unsaturated hydrocarbon groups; A represents a carbon atom; B represents a hydrogen atom or a bonding point to an amino acid or peptide compound; Z ５ represents a fluorenylmethoxycarbonyl group, a tert-butoxycarbonyl group, a benzyloxycarbonyl group, an allyloxycarbonyl group, a 2,2,2-trichloroethoxycarbonyl group, or a 2-(trimethylsilyl)ethoxycarbonyl group; R ５ａ and R ５ｂ may be bonded together with N and A to form a ring, in which case R ５ａ and R ５ｂ is a substituent having a structure in which one hydrogen atom is removed from the structure of a substituent not involved in ring formation, and when n5 is 2, R ５ｂ and two R ５ａ At least one of the groups is not a hydrogen atom.

[0277] In the past, when purifying the above-mentioned purification target substances, it was difficult to purify them to a high purity. Generally, the purification target substances are used as raw materials for the synthesis of peptide compounds and proteins, etc. Obtaining highly pure peptide compounds and proteins is important for suppressing side effects caused by impurities when they are pharmaceuticals. In other words, it is important to purify the raw materials that constitute them to a high purity. As a result of extensive research, the present inventors surprisingly discovered that the purification target substances can be purified to a high purity by converting them into lithium salts, further improving the usefulness of the purification target substances and their usefulness in the synthesis of peptide compounds and proteins.

[0278] The amino acid salt or solvate thereof, more preferably the amino acid salt, and even more preferably the lithium salt of the amino acid, of the present invention can be obtained by treating a purification target through steps (A) and (B). The purification target herein refers to a mixture of the purification target and the impurities. The purification target herein refers to the amino acid having a protecting group at the N-terminus. An example of the purification target is an amino acid represented by the general formula (5) above. Furthermore, the impurities herein refer to compounds contained in the purification target other than the purification target. Examples of the impurities include quasi-specific impurities (amino acids having a protecting group at the N-terminus other than the purification target). More specifically, β-alanine or a derivative thereof is exemplified. The term "derivative" as used herein refers to a compound that has been modified, when a certain organic compound is considered as the parent, by introducing a functional group, oxidizing, reducing, or substituting atoms, without significantly altering the structure or properties of the parent. An example of a β-alanine derivative is a specific impurity (β-alanine having a protecting group at the N-terminus).

[0279] Step (A) is a step of contacting a lithium-containing material with the target material for purification. Step (A) may form a lithium salt of the target material, or may form a lithium salt of the impurity. Step (B) is a step of precipitating the lithium salt of the target material for purification. Part of step (A) and part of step (B) may be performed overlapping in time. Prior to step (A), the target material for purification may be purified by a method other than that of the present invention. For example, the target material for purification may be purified by precipitating the target material in its free form without forming a lithium salt of the target material. At least one of steps (A) and (B) may be performed in the presence of a solvent, and in this specification, the generation of a solid from a solvent is referred to as "precipitation."

[0280] The protecting group of the purification target may be a carbamate group. The carbamate group refers to a fluorenylmethoxycarbonyl group, a tert-butoxycarbonyl group, a benzyloxycarbonyl group, an allyloxycarbonyl group, a 2,2,2-trichloroethoxycarbonyl group, or a 2-(trimethylsilyl)ethoxycarbonyl group, and may be a fluorenylmethoxycarbonyl group, a tert-butoxycarbonyl group, or a benzyloxycarbonyl group, or may be a fluorenylmethoxycarbonyl group or a benzyloxycarbonyl group.

[0281] The protecting group of the impurity may be a carbamate group. The carbamate group refers to a fluorenylmethoxycarbonyl group, a tert-butoxycarbonyl group, a benzyloxycarbonyl group, an allyloxycarbonyl group, a 2,2,2-trichloroethoxycarbonyl group, or a 2-(trimethylsilyl)ethoxycarbonyl group, and may be a fluorenylmethoxycarbonyl group, a tert-butoxycarbonyl group, or a benzyloxycarbonyl group, or may be a fluorenylmethoxycarbonyl group or a benzyloxycarbonyl group. These protecting groups may be protecting groups for the N-terminus of the impurity.

[0282] The protecting group of the product to be purified and the protecting group of the impurity may be different or the same, but it is preferred that the protecting group of the product to be purified and the protecting group of the impurity are the same.

[0283] Step (A) may be carried out in the presence of a first organic solvent. From the viewpoint of efficiently bringing the target product into contact with the lithium-containing material, the first organic solvent is a solvent capable of dissolving the target product. The solubility of the target product in the first organic solvent is preferably 20 g / L or more. The solubility of the lithium salt of the target product in the first organic solvent is preferably 20 g / L or less. The solubility of the impurities in the first organic solvent is preferably 20 g / L or more.

[0284] Examples of the first organic solvent include nitriles such as acetonitrile, alcohols such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 2-methyl-2-propanol, and ethylene glycol, ethers such as tetrahydrofuran, 1,4-dioxane, 2-methyltetrahydrofuran, methyl tert-butyl ether, diisopropyl ether, diethyl ether, and 1,2-dimethoxyethane, ketones such as acetone, methyl ethyl ketone, and methyl isobutyl ketone, dimethylformamide, dimethylacetamide, N-methyl- Examples of the first solvent include at least one selected from the group consisting of amides such as 2-pyrrolidone, ureas such as 1,3-dimethyl-2-imidazolidinone, sulfoxides such as dimethyl sulfoxide, sulfones such as sulfolane, alkanes such as heptane, methylcyclohexane, hexane, cyclohexane, and pentane, aromatic compounds such as benzene, toluene, 1,2-dimethylbenzene, 1,3-dimethylbenzene, and 1,4-dimethylbenzene, esters such as ethyl acetate and isopropyl acetate, and alkyl halides such as dichloroethane, dichloromethane, chloroform, and carbon tetrachloride. The first solvent may be of multiple types, but is preferably of one type from the viewpoint of simplifying step (A).

[0285] Step (A) may be carried out in the presence of water.

[0286] Step (B) may be carried out in the presence of a second organic solvent. The second organic solvent is a solvent in which the lithium salt of the purification target has a low solubility, i.e., a solvent that functions as a poor solvent for the lithium salt of the purification target. The solubility of the lithium salt of the purification target in the second organic solvent is preferably 10 g / L or less.

[0287] Examples of the second organic solvent include nitriles such as acetonitrile, alcohols such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 2-methyl-2-propanol, and ethylene glycol, ethers such as tetrahydrofuran, 1,4-dioxane, 2-methyltetrahydrofuran, methyl tert-butyl ether, diisopropyl ether, diethyl ether, and 1,2-dimethoxyethane, ketones such as acetone, methyl ethyl ketone, and methyl isobutyl ketone, dimethylformamide, dimethylacetamide, N-methyl- Examples of the second solvent include at least one selected from the group consisting of amides such as 2-pyrrolidone, ureas such as 1,3-dimethyl-2-imidazolidinone, sulfoxides such as dimethyl sulfoxide, sulfones such as sulfolane, alkanes such as heptane, methylcyclohexane, hexane, cyclohexane, and pentane, aromatic compounds such as benzene, toluene, 1,2-dimethylbenzene, 1,3-dimethylbenzene, and 1,4-dimethylbenzene, esters such as ethyl acetate and isopropyl acetate, and alkyl halides such as dichloroethane, dichloromethane, chloroform, and carbon tetrachloride. The second solvent may be of multiple types, but is preferably of one type from the viewpoint of simplifying step (B).

[0288] Step (B) may be carried out in the presence of water, and is preferably carried out in the presence of water from the viewpoint of dissolving the lithium salt of the impurity, the solubility of which in water is preferably 10 g / L or more.

[0289] The second organic solvent may be used together with the first organic solvent in step (A). In step (B), the lithium salt of the purification target product is precipitated. During the precipitation, the precipitation of the lithium salt of the purification target product can be promoted by adding the second organic solvent and increasing its proportion. When the proportion of the second organic solvent is increased, the second organic solvent may be added to the first organic solvent used in step (A).

[0290] Furthermore, the ratio of the first organic solvent or the ratio of the second organic solvent means a volume / mass ratio (ml / g, hereinafter also referred to as v / w) calculated by dividing the volume of the first organic solvent or the second organic solvent by the mass of the target substance to be purified.

[0291] Furthermore, if the solubility of the lithium salt of the purification target in the first organic solvent used in step (A) is sufficiently low, the first organic solvent and the second organic solvent may be the same solvent. In this case, it is not necessary to add the second organic solvent in step (B). In this case, step (B) is started immediately after the purification target is contacted with the lithium-containing material in step (A), and the lithium salt of the purification target is precipitated.

[0292] The lithium salt to be purified is preferably precipitated as a solid. Here, the term "solid" includes crystalline and amorphous. It is more preferable to precipitate (crystallize) the lithium salt as a crystal. In this case, step (B) can also be said to be a step of crystallizing the lithium salt to be purified.

[0293] The volume of the first organic solvent per 1 g of the purification target may be 1 ml or more, 2 ml or more, or 3 ml or more, and may be 100 ml or less, 80 ml or less, 60 ml or less, or 40 ml or less throughout steps (A) and (B). The volume of the first organic solvent per 1 g of the purification target may be 1 ml or more and 100 ml or less, 1 ml or more and 80 ml or less, 2 ml or more and 60 ml or less, or 3 ml or more and 40 ml or less throughout steps (A) and (B).

[0294] The volume of the first organic solvent per gram of the purification target material at the end of step (A) (initial concentration of the first organic solvent) may be 1 ml or more, 2 ml or more, or 3 ml or more, and may be 50 ml or less, 40 ml or less, 30 ml or less, or 20 ml or less, when the proportion of the second organic solvent is increased in step (B). The initial concentration of the first organic solvent may be 1 ml or more and 50 ml or less, 1 ml or more and 40 ml or less, 2 ml or more and 3 ml or more and 20 ml or less.

[0295] The volume of the first organic solvent per gram of the purification target material at the end of step (A) (initial concentration of the first organic solvent) may be 1 ml or more, 10 ml or more, 20 ml or more, 30 ml or more, or 35 ml or more, and may be 100 ml or less, 75 ml or less, 50 ml or less, or 37 ml or less, when the proportion of the second organic solvent is not increased in step (B). The initial concentration of the first organic solvent may be 1 ml or more and 100 ml or less, 10 ml or more and 75 ml or less, 20 ml or more and 50 ml or less, 30 ml or more and 37 ml or less, or 35 ml or more and 37 ml or less.

[0296] The volume of the second organic solvent per 1 g of the target substance may be 0 ml or more, 40 ml or less, 60 ml or less, 80 ml or less, or 100 ml or less throughout steps (A) and (B). The volume of the second organic solvent per 1 g of the target substance may be 0 ml to 100 ml, 0 ml to 80 ml, 1 ml to 60 ml, or 1 ml to 40 ml throughout steps (A) and (B).

[0297] The volume of the second organic solvent per gram of the target substance at the end of step (B) may be 0 ml or more, 40 ml or less, 60 ml or less, 80 ml or less, or 100 ml or less. The volume of the second organic solvent per gram of the target substance at the end of step (B) may be 0 ml or more and 100 ml or less, 0 ml or more and 80 ml or less, 0 ml or more and 60 ml or less, or 0 ml or more and 40 ml or less.

[0298] The volume of water per 1 g of the purification target may be 0.1 ml or more, 0.3 ml or more, or 0.5 ml or more, and may be 50 ml or less, 30 ml or less, 10 ml or less, or 5 ml or less throughout steps (A) and (B). The volume of water in the solvent may be 0.1 ml or more and 50 ml or less, 0.1 ml or more and 30 ml or less, 0.3 ml or more and 10 ml or less, or 0.5 ml or more and 5 ml or less throughout steps (A) and (B).

[0299] The volume of water (initial concentration of water) per gram of the purification target at the end of step (A) may be 0.1 ml or more, 0.3 ml or more, or 0.5 ml or more, and may be 50 ml or less, 30 ml or less, 10 ml or less, or 4 ml or less. The initial concentration of water may be 0.1 ml or more and 50 ml or less, 0.1 ml or more and 30 ml or less, 0.3 ml or more and 10 ml or less, or 0.5 ml or more and 4 ml or less.

[0300] At the end of step (B), the volume of water (initial concentration of water) per gram of the purification target may be 0.1 ml or more, 0.3 ml or more, or 0.5 ml or more, and may be 50 ml or less, 30 ml or less, 10 ml or less, or 4 ml or less. The initial concentration of water may be 0.1 ml or more and 50 ml or less, 0.1 ml or more and 30 ml or less, 0.3 ml or more and 10 ml or less, or 0.5 ml or more and 4 ml or less.

[0301] Step (A) and step (B) may be carried out in the presence of a solvent other than the first organic solvent and the second organic solvent, such as a phosphate buffer solution, CHES (N-cyclohexyl-2-aminoethanesulfonic acid), Tris (trishydroxymethylaminomethane), or bicine (N,N-di(2-hydroxyethyl)glycine).

[0302] The temperature at which step (A) is carried out may be -20°C or higher, 0°C or higher, 10°C or higher, or 20°C or higher. The temperature at which step (A) is carried out may be 100°C or lower, 80°C or lower, 60°C or lower, 40°C or lower, or 30°C or lower. The temperature at which step (A) is carried out may be -20 to 100°C, 0 to 80°C, 10 to 60°C, 10 to 40°C, or 20 to 30°C.

[0303] The temperature at which step (B) is carried out may be -20°C or higher, 0°C or higher, 10°C or higher, 20°C or higher, 30°C or higher, or 35°C or higher. The temperature at which step (B) is carried out may be 100°C or lower, 80°C or lower, 60°C or lower, 40°C or lower, or 30°C or lower. The temperature at which step (A) is carried out may be -20 to 100°C, 0 to 80°C, 10 to 60°C, 10 to 40°C, or 20 to 30°C.

[0304] The conditions in step (A) and step (B), such as temperature and the concentrations of substances contained in the various solutions, may be the same or different. When these conditions are different between the two, conditions selected from the various conditions for step (A) described above may be combined. Furthermore, the amount of the lithium-containing substance at the end of step (A) may be 0.5 equivalents or more, 0.8 equivalents or more, or 1.0 equivalents or more, based on the amount of the substance to be purified, and may be 2.0 equivalents or less, 1.5 equivalents or less, or 1.1 equivalents or less. The amount of the lithium-containing substance based on the amount of the substance to be purified may be 0.5 equivalents or more, 0.8 equivalents or more, or 1.5 equivalents or more, or 1.0 equivalents or more, or 1.1 equivalents or less.

[0305] The lithium-containing substance in this specification refers to a substance containing lithium that is to be brought into contact with the purification target product in step (A). Examples of the lithium-containing substance used in step (A) include at least one selected from the group consisting of lithium hydroxide, lithium tert-butoxide, lithium hydroxide, lithium carbonate, lithium hydride, trilithium phosphate, lithium methoxide, lithium ethoxide, lithium isopropoxide, lithium tert-butoxide, methyllithium, n-butyllithium, sec-butyllithium, tert-butyllithium, lithium amide, lithium diisopropylamide, lithium hexamethyldisilazide, and lithium tetramethylpiperidide.

[0306] Although there is no limitation on the method for contacting the target material with the lithium-containing substance, the target material and the lithium-containing substance can be contacted in the presence of the first organic solvent. In this case, the target material, the lithium-containing substance, and the first organic solvent may be mixed in the same container from the beginning, or the target material and the first organic solvent may be mixed and the lithium-containing substance may be added to the resulting mixture, or the lithium-containing substance and the first organic solvent may be mixed and the target material may be added to the resulting mixture.

[0307] The content of the target substance in the target substance, which serves as a basis for determining the amounts of the first organic solvent, the second organic solvent, and the lithium-containing substance, may be an actually measured value, an estimated value, or a calculated value, preferably an actually measured value or a calculated value, and most preferably an actually measured value.

[0308] Examples of the amino acid having a protecting group at the N-terminus or the amino acid constituting a peptide compound having a protecting group at the N-terminus contained in the purification target include the amino acid represented by the above general formula (5) (also referred to as amino acid (1)), hydrophobic amino acids, aliphatic amino acids, and aromatic amino acids.

[0309] n5 represents a number of 1 or more and 3 or less, and may be an integer of 1 or more and 3 or less. n5 may be 1, 2, or 3. When n5 is 2, R ５ｂ and two R ５ａ At least one of R is not a hydrogen atom, ５ｂ and two R ５ａ Two or more of R may not be hydrogen atoms, ５ｂ and two R ５ａ All three of these do not have to be hydrogen atoms.

[0310] R ５ａ and R ５ｂ represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, a heterocyclyl group, or a heteroaryl group, or a substituent to which two or more selected from the group consisting of these substituents are bonded (for example, a benzyl group, etc.), and these groups may have a substituent and may be saturated or unsaturated.

[0311] A represents a carbon atom, and B represents a hydrogen atom or a point of attachment to an amino acid or peptide compound.

[0312] Z ５ represents a fluorenylmethoxycarbonyl group, a tert-butoxycarbonyl group, a benzyloxycarbonyl group, an allyloxycarbonyl group, a 2,2,2-trichloroethoxycarbonyl group, or a 2-(trimethylsilyl)ethoxycarbonyl group.

[0313] R ５ａ and R ５ｂ may be bonded together with N and A to form a ring. In that case, R ５ａ and R ５ｂ is a substituent having a structure in which one hydrogen atom is removed from the structure of the substituent when it is not involved in ring formation.

[0314] In step (B), the purified product may be precipitated so that the content of quasi-specific impurities contained in the precipitate (purified product) obtained in step (B) is reduced. In step (B), the purified product may be precipitated so that the purity of the purified product is 95 mol% or more. The purity is more preferably 96 mol% or more, even more preferably 97 mol% or more, even more preferably 98 mol% or more, particularly preferably 99 mol% or more, and most preferably 100 mol%. In this specification, "purity" broadly means the proportion of the target substance contained in the substance whose purity is to be measured, and narrowly means the proportion of the amount of substance of the lithium salt of the target substance based on the total amount of substance of the lithium salt of the target substance and the lithium salt of the specific impurity contained in the purified product.

[0315] In step (B), the purified product may be precipitated so that the impurity content in the purified product is 5 mol% or less. The impurity content is preferably 4 mol% or less, more preferably 3 mol% or less, even more preferably 2 mol% or less, particularly preferably 1 mol% or less, and most preferably 0 mol%. In this specification, the "impurity content" refers to the amount of the lithium salt of the specific impurity contained in the purified product based on the total amount of the lithium salt of the target product and the lithium salt of the specific impurity.

[0316] The purity or impurity content can be measured, for example, by a nuclear magnetic resonance spectrometer, gas chromatography, or high performance liquid chromatography. When high performance liquid chromatography is used, the purity or impurity content can be calculated from the area ratio of UV absorption peaks. The following formula 1a shows a formula for calculating the purity, and the following formula 2a shows a formula for calculating the impurity content.

[0317] Here, the amount of substance per unit UV absorption peak area is a value obtained by dividing the UV absorption peak area of ​​the target product or specific impurity by the amount of substance.

[0318] In step (B), the purified product may be precipitated so that the apparent purity of the purified product is 95% or more. The apparent purity is more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, particularly preferably 99% or more, and most preferably 100%. As used herein, "apparent purity" refers to the ratio of the UV absorption peak area of ​​the target product at a specific wavelength to the sum of the UV absorption peak areas of the target product and specific impurities at a specific wavelength, as measured after separating the purified product into the target product and specific impurities by high performance liquid chromatography.

[0319] In step (B), the purified product may be precipitated so that the apparent impurity content of the purified product is 5% or less. The apparent impurity content is preferably 4% or less, more preferably 3% or less, even more preferably 2% or less, particularly preferably 1% or less, and most preferably 0%. As used herein, the term "apparent impurity content" refers to the ratio of the UV absorption peak area of ​​a specific impurity at a specific wavelength to the sum of the UV absorption peak areas of the target product and the specific impurity at a specific wavelength, as measured after separating the purified product into the target product and the specific impurity by high performance liquid chromatography.

[0320] The following formula 1b shows a formula for calculating the apparent purity, and the following formula 2b shows a formula for calculating the apparent impurity content.

[0321] The specific wavelength in this specification refers to a wavelength within 10% of the absorption maximum wavelength exhibited by the protecting group of the product to be purified. Furthermore, when there are multiple absorption maximum wavelengths, any absorption maximum wavelength may be selected as the absorption maximum wavelength as long as it exhibits an absorption intensity of 30% or more based on the absorption intensity at the maximum absorption wavelength among them. Furthermore, the influence of noise can be reduced by selecting a longer wavelength among the multiple absorption maximum wavelengths. Furthermore, the sample concentration can be adjusted according to the absorption intensity of the selected absorption maximum wavelength. During measurement, the absorption maximum wavelength that is most preferable from the standpoint of ease of sample concentration adjustment and noise reduction can be selected.

[0322] For example, the specific wavelength of UV light can be 254 nm for an Fmoc group, 197 nm for a Boc group, or 210 nm for a Cbz group.

[0323] In the production method according to one embodiment, the content of β-alanine or a derivative thereof in the object to be purified can be reduced by obtaining a lithium salt. In other words, the production method according to this embodiment may include a step of reducing the content of β-alanine or a derivative thereof.

[0324] The production method according to this embodiment may further include a step of converting the amino acid or peptide compound into a salt other than the lithium salt. Specifically, for example, the lithium salt obtained by the production method according to one embodiment may be desalted to obtain a free amino acid or a free peptide compound. Desalting may be performed by a known method. Furthermore, a salt other than the lithium salt may be produced from the obtained free amino acid or peptide compound. Examples of salts other than the lithium salt include salts with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid; salts with organic acids such as acetic acid, succinic acid, fumaric acid, maleic acid, tartaric acid, citric acid, lactic acid, stearic acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, and p-toluenesulfonic acid; salts with alkali metals (sodium or potassium); salts with alkaline earth metals such as calcium and magnesium; and ammonium salts.

[0325] The polypeptide according to this embodiment can be used in a method for producing a peptide compound. In one aspect, the method for producing the peptide compound can include the following steps: (1) producing an amino acid or an amino acid derivative by the method described above; and (2) linking the amino acid to one or more selected from the group consisting of other amino acids and peptides. Step (2) may be performed under the conditions described in "SOLID PHASE PEPTIDE SYNTHESIS" (https: / / www.bachem.com / wpfd_file / solid-phase-peptide-synthesis / ,2002363 published by Global Marketing, Bachem AG, December 2019).

[0326] The reaction may be carried out in an aqueous medium or a mixture of an aqueous medium and an organic solvent. The aqueous medium may be water or a buffer solution. Examples of the buffer solution include phosphate buffer, CHES (N-cyclohexyl-2-aminoethanesulfonic acid), Tris (trishydroxymethylaminomethane), and bicine (N,N-di(2-hydroxyethyl)glycine). Examples of the organic solvent include dimethyl sulfoxide.

[0327] The peptide compound obtained by the method for producing a peptide compound according to this embodiment may be, for example, a peptide compound having a cyclic portion (cyclic peptide compound).

[0328] The number of amino acid residues in the cyclic peptide compound of this embodiment may be 30 or less, 25 or less, 20 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. Furthermore, the cyclic peptide compound of this embodiment may be a peptide compound consisting of 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or 15 or more amino acids. Furthermore, the number of amino acid residues in the cyclic peptide compound of this embodiment may be 2 or more and 10 or less, 2 or more and 8 or less, 2 or more and 6 or less, or 2 or more and 4 or less. Furthermore, the number of amino acid residues in the cyclic peptide compound of this embodiment may be 5 or more and 30 or less, 7 or more and 25 or less, 8 or more and 15 or less, 9 or more and 14 or less, 10 or more and 13 or less, or 11.

[0329] The "cyclic portion" of a cyclic peptide compound refers to a cyclic portion formed by linking two or more amino acid residues. The number of amino acid residues constituting the cyclic portion of the cyclic peptide compound may be 5 to 14, 6 to 14, 7 to 14, 8 to 14, 9 to 14, 10 to 14, 5 to 13, 6 to 13, 7 to 13, 8 to 13, 9 to 13, 10 to 13, 5 to 12, 6 to 12, 7 to 12, 8 to 12, 9 to 12, 10 to 12, 5 to 11, 6 to 11, 7 to 11, 8 to 11, 9 to 11, 10 to 11, or 11. The cyclic moiety is preferably formed via a covalent bond such as an amide bond, a carbon-carbon bond, an S-S bond, a thioether bond, or a triazole bond, and can be formed by bonding a group at the N-terminus and a group at the C-terminus of a linear peptide compound to which amino acid residues are linked. Specifically, for example, it can be formed by bonding an amino group at the N-terminus and a carboxyl group at the C-terminus of a linear peptide compound to which amino acid residues are linked. The cyclization may be performed in any manner, such as via a carbon-nitrogen bond such as an amide bond, via a carbon-oxygen bond such as an ester bond or an ether bond, via a carbon-sulfur bond such as a thioether bond, via a carbon-carbon bond, via a sulfur-sulfur bond, or via a heterocyclic ring structure. Among these, cyclization via a covalent bond such as an amide bond or a carbon-carbon bond is preferred, and cyclization via an amide bond between a carboxy group in a side chain and an amino group in the main chain is more preferred. The position of the carboxy group and amino group used for cyclization may be on the main chain or on the side chain, and is not particularly limited as long as it is in a position that allows cyclization.

[0330] The cyclic peptide compound according to this embodiment may have 15 to 46 ring atoms. As used herein, the term "number of ring atoms" refers to the number of atoms (ring atoms) in a cyclic compound, including the innermost ring. When a compound has multiple rings, the term is defined as the number of atoms in the ring with the largest number of atoms. When two rings share some atoms, the ring with the fewer shared atoms is used to calculate the number of ring atoms in each ring. To further illustrate the "number of ring atoms" with a specific example, using this method, tetrahydrofuran (THF) has 5 ring atoms, and tacrolimus (FK506) has 21 ring atoms.

[0331] The number of ring atoms in the cyclic peptide compound according to this embodiment may be, for example, 34 to 46, 34 to 43, 34 to 40, 34 to 37, 34 to 36, or 34. The ring atoms used to calculate the number of ring atoms may be selected from the group consisting of carbon atoms, hydrogen atoms, nitrogen atoms, oxygen atoms, sulfur atoms, phosphorus atoms, and silicon atoms, or may be selected from the group consisting of carbon atoms, hydrogen atoms, nitrogen atoms, and oxygen atoms.

[0332] The cyclic peptide compound according to this embodiment may have a linear portion in addition to a cyclic portion. Specific embodiments of the number of amino acid residues in the cyclic peptide compound are the same as those of the peptide compound described above. When the cyclic peptide compound has a linear portion, it is preferable that the total number of amino acid residues in the cyclic portion and the linear portion be within the same range. Furthermore, when the cyclic peptide compound has a linear portion, the number of amino acid residues constituting the cyclic portion is preferably 5 to 15, 6 to 15, 6 to 14, 7 to 14, 8 to 14, or 7 to 13, more preferably 7 to 12, or 8 to 11, even more preferably 9 to 11, and particularly preferably 10 or 11. The number of amino acid residues constituting the linear portion is preferably 1 to 8, 1 to 7, 1 to 6, 1 to 5, or 1 to 4, and more preferably 1 to 3.

[0333] The molecular weight of the cyclic peptide compound according to this embodiment is not particularly limited, but may be, for example, 500 or more, 550 or more, 600 or more, 650 or more, 700 or more, 750 or more, 800 or more, 850 or more, 900 or more, or 950 or more, with 1000 or more, 1100 or more, 1200 or more, 1300 or more, or 1400 or more being preferred. The upper limit of the molecular weight of the peptide compound according to this embodiment is not particularly limited, but is preferably 5000 or less, 4000 or less, 3000 or less, 2500 or less, or 2000 or less. As used herein, the term "molecular weight" refers to the sum of the atomic weights of the atoms constituting the compound molecule (unit: "g / mol") and is obtained by calculating the sum of the atomic weights of the atoms contained in the molecular structure (unit: "g / mol"). In this specification, the molecular weight unit may be omitted. The molecular weight of the peptide compound can be measured by liquid chromatography mass spectrometry (LC / MS) as described in the Examples.

[0334] The cyclic peptide compound according to this embodiment may contain one or more N-substituted amino acid residues, preferably at least three N-substituted amino acid residues, more preferably at least four N-substituted amino acid residues, and even more preferably at least five N-substituted amino acid residues. The N-substituted amino acid residues may be present consecutively or discontinuously in the N-substituted cyclic peptide compound.

[0335] One embodiment of the present invention is a DNA encoding the above polypeptide. This DNA includes, for example, genomic DNA, cDNA, and DNA artificially created based on them. Genomic DNA includes exons and introns. That is, genomic DNA may or may not contain introns, and may or may not contain untranslated regions (5'UTR and / or 3'UTR), transcriptional regulatory regions, etc. Furthermore, cDNA may contain a nucleic acid sequence derived from a portion of an intron sequence that encodes an amino acid sequence. Furthermore, this DNA also includes degenerate polynucleotides composed of any codons that encode the same amino acid. The DNA according to this embodiment may be DNA derived from a desired organism.

[0336] The DNA of this embodiment may be obtained by any method. For example, it includes complementary DNA (cDNA) prepared from mRNA, DNA prepared from genomic DNA, DNA obtained by chemical synthesis, DNA obtained by PCR amplification using RNA or DNA as a template, and DNA constructed by an appropriate combination of these methods. The DNA of this embodiment can be prepared by cloning the DNA from genomic DNA or RNA encoding the above polypeptide and introducing a mutation according to standard methods.

[0337] For example, a method for cloning a cDNA from mRNA encoding the polypeptide encoded by the DNA of this embodiment involves first preparing mRNA encoding the polypeptide from any tissue or cell that expresses and produces the polypeptide according to a standard method, such as by subjecting total RNA prepared using the guanidine thiocyanate method, hot phenol method, or AGPC (Acid Guanidine-Phenol-Chloroform) method to affinity chromatography using oligo(dT) cellulose, poly U-Sepharose, or the like.

[0338] Next, using the obtained mRNA as a template, a cDNA strand is synthesized by a known method such as using reverse transcriptase (Mil. Cell. Biol., Vol. 2, p. 161, 1982; Mol. Cell. Biol., Vol. 3, p. 280, 1983; Gene, Vol. 25, p. 263, 1983), and the cDNA is converted into double-stranded cDNA. This cDNA is then inserted into a plasmid vector, a phage vector, a cosmid vector, or the like, and transformed into Escherichia coli, or after in vitro packaging, transfected into Escherichia coli, thereby preparing a cDNA library.

[0339] The target gene can be obtained by screening the prepared cDNA library using the DNA according to this embodiment or a portion thereof as a probe. Alternatively, the DNA according to this embodiment or a portion thereof can be used as a primer to directly amplify the gene by PCR. The sites and lengths of the probe and primer may be determined appropriately.

[0340] One embodiment of the present invention is a recombinant vector comprising the above DNA. The recombinant vector is not particularly limited as long as it can maintain replication or self-replicate in a prokaryotic and / or eukaryotic host, and includes plasmid vectors, phage vectors, viral vectors, and the like.

[0341] Examples of cloning vectors include pUC19, λgt10, λgt11, etc. When isolating cells capable of expressing the polypeptide according to this embodiment in host cells, the vector preferably has a promoter capable of expressing the DNA according to this embodiment.

[0342] The recombinant vector can be prepared by simply ligating the DNA encoding the polypeptide according to this embodiment to a recombinant vector (plasmid DNA or bacteriophage DNA) available in the art using a conventional method.

[0343] Examples of the recombinant vector to be used include plasmids derived from Escherichia coli (pBR322, pBR325, pUC12, pUC13, pUC19, etc.), yeast-derived plasmids (pSH19, pSH15, etc.), and Bacillus subtilis-derived plasmids (pUB110, pTP5, pC194, etc.).

[0344] Examples of phages include bacteriophages such as λ phage, and animal and insect viruses such as retroviruses, vaccinia viruses, nuclear polyhedrosis viruses, and lentiviruses (pVL1393, manufactured by Impitrogen).

[0345] For the purpose of expressing DNA encoding the polypeptide of this embodiment and producing the polypeptide, an expression vector is useful. The expression vector is not particularly limited as long as it has the function of expressing DNA encoding the polypeptide in either prokaryotic and / or eukaryotic host cells and producing the polypeptide.

[0346] Examples include pMAL C2, pEF-BOS (Nucleic Acid Research, Vol. 18, 1990, p. 5322, etc.) and pME18S (Experimental Medicine Special Issue "Genetic Engineering Handbook," 1992, etc.).

[0347] When bacteria, particularly E. coli, are used as host cells, the recombinant vector preferably contains at least a promoter-operator region, an initiation codon, DNA encoding the polypeptide, a termination codon, a terminator region, and a replicable unit.

[0348] When yeast, animal cells, or insect cells are used as hosts, the recombinant vector preferably contains at least a promoter, an initiation codon, a DNA encoding the polypeptide, and a termination codon.

[0349] The recombinant vector may contain DNA encoding a signal peptide, an enhancer sequence, untranslated regions on the 5' and 3' sides of the gene encoding the polypeptide, a splicing junction, a polyadenylation site, a selection marker region, or a replicable unit, etc.

[0350] The recombinant vector may, if necessary, contain a marker gene (such as a gene amplification gene or a drug resistance gene) that enables gene amplification and selection of transformed hosts.

[0351] Examples of marker genes include the dihydrofolate reductase (DHFR) gene, thymidine kinase gene, neomycin resistance gene, glutamate synthetase gene, adenosine deaminase gene, ornithine decarboxylase gene, hygromycin-B-phosphotransferase gene, and aspartate transcarbamylase gene.

[0352] The promoter-operator region for expressing the above-mentioned polypeptide in bacteria can comprise a promoter, an operator, and a Shine-Dal garno (SD) sequence (for example, AAGG, etc.).

[0353] When the host is a bacterium of the genus Escherichia, examples of the promoter include the Trp promoter, the lac promoter, the recA promoter, the λPL promoter, the lPP promoter, and the tac promoter.

[0354] Examples of promoters for expressing the above polypeptides in yeast include the PH05 promoter, PGK promoter, GAP promoter, and ADH promoter. When the host is a Bacillus bacterium, examples of promoters include the SLO1 promoter, SP02 promoter, and penP promoter.

[0355] When the host is a eukaryotic cell such as a mammalian cell, examples of the promoter include an SV40-derived promoter, a retrovirus promoter, and a heat shock promoter. SV40 and retrovirus promoters are preferred. However, the promoter is not particularly limited to these. Furthermore, the use of an enhancer is also an effective method for expression.

[0356] A suitable initiation codon is, for example, a methionine codon (ATG). A suitable termination codon is, for example, a commonly used termination codon (e.g., TAG-TGA, TM). A commonly used natural or synthetic terminator can be used as the terminator region.

[0357] A replicable unit refers to DNA capable of replicating its entire DNA sequence in a host cell, and includes natural plasmids, artificially modified plasmids (DNA fragments prepared from natural plasmids), synthetic plasmids, etc. Suitable plasmids include the plasmid pBR322 or its artificially modified products (DNA fragments obtained by treating pBR322 with an appropriate restriction enzyme) for E. coli, the yeast 2μ plasmid or yeast chromosomal DNA for yeast, and the plasmid pRSVneo (ATCC 37198), the plasmid pSV2dhfr (ATCC 37145), the plasmid pdBPV-MMTneo (ATCC 37224), the plasmid pSV2neo (ATCC 37149), etc. for mammalian cells.

[0358] The enhancer sequence, polyadenylation site and splicing junction site may be any of those commonly used by those skilled in the art, such as those derived from SV40.

[0359] The expression vector can be prepared by ligating at least the above-mentioned promoter, initiation codon, DNA encoding the above-mentioned polypeptide, termination codon, and terminator region consecutively and circularly into an appropriate replicable unit, and if desired, an appropriate DNA fragment (e.g., a linker, other restriction enzyme cleavage site, etc.) can be used by a conventional method such as digestion with a restriction enzyme or ligation using T4 DNA ligase.

[0360] One embodiment of the present invention is a transformant having DNA encoding the above-mentioned polypeptide. The transformant can be obtained as a transformed recombinant cell, for example, by introducing the above-mentioned expression vector into a host cell.

[0361] The host cell is not particularly limited as long as it is compatible with the expression vector and can be transformed with it. Examples include various cells such as natural cells or artificially established recombinant cells commonly used in the technical field of the present invention, for example, bacteria (such as bacteria of the genus Escherichia or Bacillus), yeast (such as those of the genus Saccharomyces or Pichia), animal cells, or insect cells.

[0362] The host cell is preferably Escherichia coli or an animal cell. Examples of the Escherichia coli or animal cell include Escherichia coli (DH5a, TB1, HB101, etc.), mouse-derived cells (Cop, L, C127, Sp2 / 0, NS-1, NIH3T3, etc.), rat-derived cells (PC12, PC12h), hamster-derived cells (BHK, CHO, etc.), monkey-derived cells (COS1, COS3, COS7, CV1, Velo, etc.), and human-derived cells (Hela, diploid fibroblast-derived cells, myeloma cells, HepG2, etc.).

[0363] Introduction of an expression vector into a host cell (transformation (transfection)) can be carried out by a conventional method ([in the case of E. coli, Bacillus subtilis, etc.]: Proc. Natl. Acad. Sci. USA, Vol. 69, p. 2110, 1972; Mil. Gen. Genet., Vol. 168, p. 111, 1979; J. Mol. Biol., Vol. 56, p. 209, 1971; [in the case of Saccharomyces cerevisiae case]: Proc. Natl. Acad. Sci. USA. , Vol. 75, p. 1927, 1978; J. Bacteriol. , Vol. 153, p. 163, 1983); [In the case of animal cells]: Virology, Vol. 52, p. 456, 1973; [In the case of insect cells]: Mol. Cell. Biol. , Vol. 3, p. 2156-2165, 1983).

[0364] The polypeptide according to this embodiment can be produced, for example, by a method comprising culturing a recombinant cell that is a transformant according to a conventional method and obtaining the desired polypeptide from the culture obtained by the culturing.

[0365] The target polypeptide can be obtained from the culture by any commonly used isolation and purification method, including, for example, methods that utilize solubility such as salting out and solvent precipitation, methods that utilize differences in molecular weight such as dialysis, ultrafiltration, gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, methods that utilize charge such as ion exchange chromatography and hydroxylapatite chromatography, methods that utilize specific affinity such as affinity chromatography, methods that utilize differences in hydrophobicity such as reversed-phase high performance liquid chromatography, and methods that utilize differences in isoelectric point such as isoelectric focusing.

[0366] When the target polypeptide is present in the periplasm or cytoplasm of cultured recombinant cells (such as E. coli), the culture is subjected to a conventional method such as filtration or centrifugation to collect the bacterial bodies or cells, which are then suspended in an appropriate buffer solution. The cell walls and / or cell membranes of the cells are disrupted by, for example, ultrasonic waves, lysozyme, or freeze-thawing, and a fraction containing the target polypeptide is obtained by centrifugation, filtration, or the like. The fraction is then purified by Triton X-ray diffraction. ＴＭ The target polypeptide can be isolated and purified from the crude solution by conventional methods such as those described above.

[0367] The polypeptide according to this embodiment can be produced using known gene recombination techniques as described above, but can generally be prepared as follows: First, a plasmid containing a wild-type polypeptide gene is used as a template, and site-specific mutations are introduced into specific positions and specific amino acids by PCR using primers from the template. The template plasmid is then digested with a restriction enzyme and transformed into Escherichia coli or the like, and the desired mutation-introduced plasmid is cloned.

[0368] To introduce a second amino acid mutation, the plasmid containing the mutation at a specific position is used as a template, and site-directed mutagenesis using a primer is repeated in the same manner as above to construct a plasmid DNA encoding a two-amino acid substitution.

[0369] The prepared DNA is transformed into Escherichia coli BL21 strain or the like together with a plasmid such as pREP4 encoding the lac repressor (Laci), and the resulting transformant is isolated and cultured, followed by induction of expression with IPTG. The resulting strain is then disrupted, and the target polypeptide is purified by passing the supernatant through an affinity column utilizing a His tag, for example.

[0370] Alternatively, the polypeptide can be prepared by the following method: a nucleotide sequence encoding the desired polypeptide is gene-synthesized, transferred into an expression vector, and then the protein is expressed and purified using an affinity column utilizing various purification tags.

[0371] The method for producing the polypeptide according to this embodiment is not limited to the above-described method, and various gene manipulation techniques can be used, such as well-known point mutation techniques, gene synthesis techniques, and methods for introducing modified fragments using restriction enzymes. Expression is not limited to E. coli, and animal cells and cell-free translation systems can also be used. Furthermore, the purification method is not limited to affinity columns using polyhistidine, and various peptide tags and purification columns can be used.

[0372] The present invention is further illustrated by, but not limited to, the following examples.

[0373] The following abbreviations are used in the examples:

[0374] The LCMS analysis conditions are as follows.

[0375] Reagents not specifically mentioned as to their source were purchased from Sigma-Aldrich Japan LLC, Fujifilm Wako Pure Chemical Industries, Ltd., Tokyo Chemical Industry Co., Ltd., Nacalai Tesque Inc., and Watanabe Chemical Industry Co., Ltd.

[0376] Comparative Example 1: Expression and purification of wild-type enzyme A gene was synthesized in which a streptavidin-binding peptide tag sequence (GTDEKTTGWRGGHVVEGLAGEELEQLRARLEHHPQ) and a His tag sequence (HHHHHH) were added to the C-terminus of the wild-type enzyme sequence (SEQ ID NO: 1), and the gene was cloned into an E. coli expression vector. This expression vector was introduced into the BL21(DE3) E. coli strain (Novagen), and the target protein was expressed by culturing it at 18°C ​​for 2 days using Overnight Express Instant TB Medium (Novagen).

[0377] The resulting cells were collected by centrifugation and disrupted by sonication. The lysate was fractionated by centrifugation, and the supernatant was purified by affinity chromatography using Ni Sepharose 6 Fast Flow (Cytiva). The fraction containing the target protein was collected and used as the final preparation (#02-001 (SEQ ID NO: 17)).

[0378] Example 1: Expression and purification of modified enzymes Genes were synthesized by adding a streptavidin-binding peptide tag sequence (GTDEKTTGWRGGHVVEGLAGEELEQLRARLEHHPQ), a linker sequence (GGS), and a His tag sequence (HHHHHH) to the C-terminus of each modified enzyme sequence (SEQ ID NOS: 2 to 13 and 23 to 35), and the genes were cloned into an E. coli expression vector. This expression vector was introduced into the BL21(DE3) E. coli strain (Novagen), and the target protein was expressed by culturing it at 18°C ​​for 2 days using Overnight Express Instant TB Medium (Novagen).

[0379] The resulting cells were collected by centrifugation and disrupted by sonication. The lysate was fractionated by centrifugation, and the supernatant was purified by affinity chromatography using Ni Sepharose 6 Fast Flow (Cytiva). Fractions containing the target protein were collected and used as the final preparations (#02-002 to #217, #03-001 to #130, and #04-001 to #0072).

[0380] Example 2: Expression and purification of modified enzyme The supernatant fraction was purified using cComplete instead of Ni Sepharose 6 Fast Flow (Cytiva). ＴＭ The same procedure as in Example 1 was repeated except that the target protein was purified by affinity chromatography using His-Tag Purification Resin (Roche) and that after collecting fractions containing the target protein, the sample was dialyzed against 50 mM Tris-HCl (pH 8.0) / 150 mM sodium chloride / 10% glycerol to obtain the final preparation (#02-169).

[0381] Synthesis Example 1: Synthesis of H-MeGly(cPent)-OH To a suspension of Fmoc-MeGly(cPent)-OH (CAS No. 187475-29-2) (250 mg, 0.659 mmol) in dichloromethane (KANTO, cat. No. 11338-25) (1.0 mL), 4-(3-phenylpropyl)piperidine (TCI, cat. No. P0760) (0.419 mL, 1.977 mmol) was added and stirred at room temperature for 1 minute. Water (1.0 mL) was then added and the mixture was stirred overnight at room temperature. The aqueous layer from the reaction solution was collected and purified by reverse-phase silica gel column chromatography (0.1% formic acid aqueous solution / 0.1% formic acid acetonitrile solution = 100 / 0 → 95 / 5) to obtain H-MeGly(cPent)-OH (88.2 mg, 85%). LCMS (ESI) m / z=158.1 (M+H) ＋ Retention time: 0.25 minutes (Analysis conditions FA05-1) １ H-NMR (JNM-ECZ400S, 400MHz, D ２ O) δppm3.28 (1H, d, 7.6Hz), 2.53 (3H, s), 2.07 (1H, m), 1.69 (1H, m), 1.60-1.38 (5H, m), 1.22 (2H, m)

[0382] Evaluation Example 1: Screening of modified enzymes using H-MeGly(cPent)-OH synthesis activity as an index To screen the modified enzymes (#02-002 to 217) prepared in Example 1 using H-MeGly(cPent)-OH synthesis activity as an index, a solution containing a substrate (hereinafter referred to as Premix 1) was prepared. Specifically, two compounds, 2-cyclopentyl-2-oxo-acetic acid (purchased from Enamine Ltd.) and D(+)-glucose (purchased from Fujifilm Wako Pure Chemical Corporation), were dissolved in a mixed solvent of aqueous methylamine solution (an aqueous solution purchased from Fujifilm Wako Pure Chemical Corporation adjusted to pH 8.5 with hydrochloric acid) and phosphate buffer (400 mM, pH 7.4). The pH was adjusted to 8.0 using aqueous sodium hydroxide solution, and the solution was diluted with ultrapure water. To this solution were added an aqueous β-NADPH solution (powder purchased from Oriental Yeast Co., Ltd. dissolved in ultrapure water at 89 mM) and a GDH solution (powder purchased from Fujifilm Wako Pure Chemical Corporation dissolved in 1x TNG) to obtain Premix 1. The concentrations of each compound at the time of Premix 1 were 55.6 mM for 2-cyclopentyl-2-oxo-acetic acid, 111 mM for D(+)-glucose, 556 mM for methylamine, 111 mM for phosphate buffer, 0.99 mM for β-NADPH, and 0.0022 units / μL for the GDH solution.

[0383] To the prepared Premix 1, any one of #02-001 to #217 was added, and the H-MeGly(cPent)-OH synthesis reaction was carried out. Specifically, any one of #02-001 to #217 was added to Premix 1. For the negative control, Enzyme (-), an imidazole-containing buffer solution (250 mM imidazole, 50 mM HEPES (pH 7.5), 150 mM sodium chloride) was added. After thorough mixing, the mixture was incubated at 37°C for 19 hours. The concentrations of each compound at the start of the reaction were 50 mM for 2-cyclopentyl-2-oxo-acetic acid, 100 mM for D(+)-glucose, 500 mM for methylamine, 100 mM for phosphate buffer, 0.89 mM for β-NADPH, 0.002 unit / μL for GDH solution, and 2.5 μM for any one of #02-001 to #02-217.

[0384] The reaction solution after incubation was post-treated to prepare a sample for LCMS analysis. Specifically, hydrochloric acid was added in an amount 9 times the volume of the reaction solution so that the final concentration after addition of hydrogen chloride was 30 mM (hereinafter, this solution is referred to as post-treated reaction solution 1).

[0385] For the purpose of quantifying the concentration of H-MeGly(cPent)-OH dissolved in the post-treated reaction solution 1 by LCMS, a dilution series of H-MeGly(cPent)-OH synthesized in Synthesis Example 1 was prepared. Specifically, H-MeGly(cPent)-OH was dissolved in a 1 M aqueous sodium hydroxide solution to a concentration of 1000 mM, and then the solution was repeatedly diluted two-fold with ultrapure water.

[0386] Because ion suppression by the various compounds dissolved in post-treated reaction solution 1 made it difficult to quantify the concentrations using this dilution series, the dilution series was added to a solution simulating the composition of post-treated reaction solution 1. First, two compounds, 2-cyclopentyl-2-oxo-acetic acid and D(+)-glucose, were dissolved in a mixed solvent of aqueous methylamine solution and phosphate buffer, and the pH was adjusted to 8.0 using aqueous sodium hydroxide solution and hydrochloric acid, followed by dilution with ultrapure water (hereinafter, this solution will be referred to as Premix 2). Next, the β-NADPH aqueous solution, 1x TNG, imidazole-containing buffer solution, and dilution series were added to Premix 2 and mixed thoroughly. The concentrations of each compound at this stage were 25 mM for 2-cyclopentyl-2-oxo-acetic acid, 75 mM for D(+)-glucose, 475 mM for methylamine, 100 mM for phosphate buffer, and 0.89 mM for β-NADPH, and the 1x TNG and imidazole-containing buffer were each 10% by volume. Finally, hydrochloric acid in an amount 9 times the reaction solution was added so that the final concentration after addition of hydrogen chloride was 30 mM, to obtain a calibration curve sample for LCMS analysis (hereinafter, this solution is referred to as post-treated calibration curve sample 1).

[0387] Considering the possibility that post-treated reaction solution 1 contained dissolved H-MeGly(cPent)-OH at levels exceeding the calibration curve range of post-treated calibration curve sample 1, a dilution operation was performed. First, to address the aforementioned ion suppression, a dilution solution was prepared that mimicked the composition of the various compounds dissolved in post-treated reaction solution 1. Specifically, premix 2, 1x TNG, imidazole-containing buffer, β-NADPH aqueous solution, and hydrochloric acid were mixed and diluted with ultrapure water. After dilution, the concentrations of β-NADPH and hydrogen chloride were 0.089 mM and 30 mM, respectively, and the final volume ratios of premix 2, 1x TNG, and imidazole-containing buffer were 6%, 1%, and 1%, respectively. Post-treated reaction solution 1 was diluted 32-fold using this dilution solution to obtain post-treated reaction solution 2. The post-treated reaction solution 1, post-treated reaction solution 2, and post-treated calibration curve sample 1 were all passed through a 0.2 μm PVDF membrane filter (purchased from Corning, Inc.) and then subjected to LCMS measurement. The volume of solution used for the measurement was 1 μL. The extracted ion chromatogram peak area detection function (TargetLynx) installed in MassLynx was used to evaluate the H-MeGly(cPent)-OH synthesis activity of each of #02-001 to #217. The results are shown in the table below, and several modified enzymes were found to have higher H-MeGly(cPent)-OH synthesis activity than the wild-type enzyme (#02-001). LCMS (ESI) m / z = 158.10 (M+H) ＋ Retention time: 0.23 minutes (analysis condition FA05-1). The synthetic activity was determined by dividing the yield of the target product when #02-001 to #02-002-217 were used by the yield of the target product when the wild-type enzyme (#02-001) was used. The calculated synthetic activity ratios are shown in Tables 4 and 5. The yield when the wild-type enzyme was used was 21%.

[0388] Evaluation Example 2: Screening of Modified Enzymes Using H-EtPhe-OH Synthase Activity as an Indicator (Single Mutant Screening) To screen the modified enzymes (#02-002 to 217) prepared in Example 1 using H-EtPhe-OH synthase activity as an indicator, a solution containing a substrate (hereinafter referred to as Premix 3) was prepared. Specifically, two compounds, phenylpyruvate sodium salt (purchased from Sigma-Aldrich Japan LLC) and D(+)-glucose, were dissolved in a mixed solvent of an ethylamine aqueous solution (an aqueous solution purchased from Tokyo Chemical Industry Co., Ltd., adjusted to pH 7.7 with hydrochloric acid) and a phosphate buffer. The pH was adjusted to 8.0 using aqueous sodium hydroxide and hydrochloric acid, and then the solution was diluted with ultrapure water. To this solution, a β-NADPH aqueous solution and a GDH solution were added to obtain Premix 3. The concentrations of each compound at the time of premix 3 were as follows: phenylpyruvate sodium salt 55.6 mM, D(+)-glucose 111 mM, ethylamine 556 mM, phosphate buffer 111 mM, β-NADPH 0.99 mM, and GDH solution 0.0022 units / μL.

[0389] To the prepared Premix 3, any one of #02-001 to #217 was added, and the H-EtPhe-OH synthesis reac...

Claims

1. A sequence having 90% or more sequence identity with a sequence in which one amino acid residue in the amino acid sequence represented by SEQ ID NO: 1 has been modified, and a catalytic activity for a reductive amination reaction between any one or more of compounds A represented by the following formula (1) or salts thereof and any one or more of compounds B represented by the following formula (2) or salts thereof, or for an intramolecular reductive amination reaction of any one or more of said compounds B or salts thereof, which is higher than the catalytic activity of a polypeptide having the amino acid sequence represented by SEQ ID NO: 1 under at least one reaction condition; A polypeptide comprising a sequence in which amino acid residues located at positions corresponding to one or more amino acid residues selected from the group consisting of the histidine residue at position 44, the threonine residue at position 156, the histidine residue at position 182, the glutamine residue at position 186, the tryptophan residue at position 253, and the lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1 have been modified. 【Chemistry 1】 [In formula (1), R １ and R ２ each independently represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, a heterocyclyl group, or a heteroaryl group, and these groups may be substituted; R １ or R ２ and at least one of the groups is a hydrogen atom. 【Chemistry 2】 【Transformation 3】 【Chemistry 4】 [In formula (2), X represents a carbon atom; Y represents a hydrogen atom, a group represented by the above formula (1') or a group represented by the above formula (3), n represents an integer of 0 to 2, R ６ represents a hydrogen atom, an aliphatic hydrocarbon group having from 1 to 6 carbon atoms which may be substituted, an aryl group having from 5 to 12 carbon atoms which may be substituted, a heteroaryl group having from 5 to 12 ring atoms which may be substituted, a group containing a nitrogen atom, or a group containing an oxygen atom, In formula (1′), 【Transformation 5】 indicates the point of attachment to X, R １ａ is R in formula (1) １ It is a group obtained by removing a hydrogen atom from a group represented by the formula: In formula (3), 【Transformation 6】 indicates the point of attachment to X, m represents an integer of 0 to 6, p is 0 or 1; q is 0 or 1; r is 0 or 1; Z １ represents an alkylene group which may be substituted, or an ether bond-containing group having 1 to 6 carbon atoms; when m is 2 or more, a plurality of Z １ may be the same or different, Z ２ indicates a carbon atom, R ３ , R ４ and R ５ each independently represents a hydrogen atom, an optionally substituted aliphatic hydrocarbon group having from 1 to 6 carbon atoms, an optionally substituted aryl group having from 5 to 12 carbon atoms, an optionally substituted heteroaryl group having from 5 to 12 ring-constituting atoms, a group containing a nitrogen atom, or a group containing an oxygen atom; R ３ , R ４ and R ５ Any two or more of the following may be bonded to each other to form Z ２ together form a ring structure, which may be a cycloalkyl group, an aryl group, a heterocyclyl group, or a heteroaryl group, which may be substituted; R ３ , R ４ , and R ５ Is Z ２ may form a double bond or a triple bond with R ３ , R ４ , and R ５ is connected to Z by a double bond or a triple bond. ２ When the group is bonded to p, q, or r, at least one of p, q, or r is 0; In formula (1), R １ and R ２ is a methyl group and the other is a hydrogen atom, in formula (2), Y is a group represented by formula (3), m is 0, and R ３ ~R ５ Two or more of these are not hydrogen atoms.

2. An amino acid residue located at a site corresponding to one amino acid residue selected from the group consisting of a methionine residue at position 141, a histidine residue at position 182, and a tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 is modified as a first modification site; A polypeptide as described in claim 1, comprising an amino acid sequence in which an amino acid residue located at a position corresponding to one amino acid residue selected from the group consisting of a histidine residue at position 182, a tryptophan residue at position 253, and a lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1, and which is modified as a second modification site, is different from the amino acid residue modified as the first modification site.

3. An amino acid residue located at a site corresponding to one amino acid residue selected from the group consisting of a methionine residue at position 141 and a histidine residue at position 182 in the amino acid sequence represented by SEQ ID NO: 1 is modified as a first modification site, an amino acid residue located at a position corresponding to one amino acid residue selected from the group consisting of a histidine residue at position 182 and a tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 and different from the amino acid residue modified as the first modification site is modified as the second modification site; A polypeptide as described in claim 1, comprising an amino acid sequence in which an amino acid residue located at a position corresponding to one amino acid residue selected from the group consisting of the tryptophan residue at position 253 and the lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1 and different from the amino acid residues modified as the first and second modification positions is modified as the third modification position.

4. In the formula (1), R １ is a hydrogen atom, and R ２ is C １ ~C ６ The polypeptide according to any one of claims 1 to 3, which is an alkyl group.

5. In formula (2), Y is C ３ ~C ８ Cycloalkyl group or C ６ ~C ９ an aralkyl group, the aralkyl group being C １ ~C ３ The polypeptide according to any one of claims 1 to 3, which may be substituted with an alkyl group or a halogen atom.

6. A polypeptide comprising a sequence having 90% or more sequence identity with a sequence in which one amino acid residue in the amino acid sequence represented by SEQ ID NO: 1 has been modified, and whose catalytic activity for a reductive amination reaction between an alkylamine or a salt thereof and one or more compounds represented by the following formula (2') or salts thereof is higher than the catalytic activity of a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 17 under at least one reaction condition: 【Transformation 7】 wherein Y′ is C ３ ~C ８ a cycloalkyl group, or C ６ ~C ９ represents an aralkyl group, and the aralkyl group is C １ ~C ３ It may be substituted with an alkyl group or a halogen atom.]

7. The polypeptide according to claim 6, wherein the alkylamine or its salt is one or more selected from the group consisting of methylamine, ethylamine, and salts thereof.

8. The polypeptide according to claim 6 or 7, wherein the compound represented by formula (2') or a salt thereof is one or more selected from the group consisting of phenylpyruvic acid, 2-oxo-3-(p-tolyl)propanoic acid, 2-cyclopentyl-2-oxo-acetic acid, and salts thereof.

9. 2. The polypeptide according to claim 1, wherein the sequence in which one amino acid residue in the amino acid sequence represented by SEQ ID NO:1 has been modified is the amino acid sequence represented by SEQ ID NO:6, in which X is a valine residue; the amino acid sequence represented by SEQ ID NO:6, in which X is a tyrosine residue; the amino acid sequence represented by SEQ ID NO:8, in which X is a leucine residue; the amino acid sequence represented by SEQ ID NO:11, in which X is a histidine residue; or the amino acid sequence represented by SEQ ID NO:12, in which X is a glutamic acid residue.

10. 2. The polypeptide of claim 1, comprising an amino acid sequence in which the amino acid residue located at the position corresponding to the tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with one or more amino acid residues selected from the group consisting of tyrosine residues, valine residues, threonine residues, serine residues, arginine residues, glutamine residues, proline residues, asparagine residues, methionine residues, leucine residues, lysine residues, isoleucine residues, histidine residues, phenylalanine residues and alanine residues.

11. 2. The polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:6, wherein X is a valine residue, the amino acid sequence of SEQ ID NO:6, wherein X is a tyrosine residue, the amino acid sequence of SEQ ID NO:8, wherein X is a leucine residue, the amino acid sequence of SEQ ID NO:11, wherein X is a histidine residue, or the amino acid sequence of SEQ ID NO:12, wherein X is a glutamic acid residue.

12. In the presence of the polypeptide of claim 1 and a reducing agent, reacting one or more selected from the group consisting of amines, amine analogs, and salts thereof with one or more selected from the group consisting of keto acids, keto acid analogs, and salts thereof; or A method for producing an amino acid, comprising a step of intramolecularly reacting a compound selected from the group consisting of keto acids, keto acid analogs, and salts thereof.

13. The amine or amine analogue is represented by formula (1), and R １ and R ２ is R according to formula (1) of claim 1 １ and R ２ The method according to claim 12, wherein

14. The keto acid or keto acid analogue is represented by formula (2), and in formula (2), the formula (1′), the formula (3), X, Y, Z １ , Z ２ , R １ａ , R ３ , R ４ , R ５ , R ６ , m, n, p, q and r are the same as those of formula (1'), formula (3), X, Y, Z １ , Z ２ , R １ａ , R ３ , R ４ , R ５ , R ６ , m, n, p, q and r have the same meanings as each other.

15. The method according to claim 12, wherein the reaction is carried out in the presence of a compound C represented by the following formula (4): 【Transformation 8】 [In formula (4), v and w each independently represent 0 or 1; At least one of v and w represents 1; T represents a carbon atom, a phosphorus atom, or a sulfur atom; The following formula (4a) 【Chemistry 9】 The functional group represented by the formula: represents ═O, —ORd, or a hydroxy group, When v and w are both 1, two functional groups represented by formula (4a) present in plural may be the same or different, Ra, Rb and Rc each independently represent a hydrogen atom, C １ ~C ３ Alkyl group, alkylamino group or -CH ２ represents —ORd, any two or more of Ra, Rb, and Rc may be bonded to each other and T to form a ring structure; Rd is C １ ~C ３ represents an alkyl group, d, e, and f each independently represent 0 or 1; At least one of d, e, and f represents 1; when v and w are both 1, any one or more of Ra, Rb, and Rc are a methyl group, and Ra, Rb, and Rc do not bond to each other to form a ring structure together with T; when any one or more of Ra, Rb, and Rc is a methylamino group, Ra, Rb, and Rc do not combine with each other to form a ring structure together with T, When the functional group represented by formula (4a) is a hydroxy group and T is a carbon atom, v is 1, w is 0, d, e, and f are all 1, and Ra, Rb, and Rc are all hydrogen atoms.

16. The method according to claim 12, comprising the following steps (A) and (B): Step (A): A step of contacting a lithium-containing substance with a target product to be purified, which is a mixture of the following (i) as a target product to be purified and the following (ii) as an impurity, obtained by the method according to claim 12; (i) the amino acid having a protecting group at the N-terminus (ii) a compound other than the target product; Step (B): A step of precipitating the lithium salt that is the target of purification.

17. A method for producing a peptide compound, comprising the steps of: (1) Producing an amino acid by the method according to claim 12; and (2) A step of linking the amino acid to one or more selected from the group consisting of other amino acids and peptides to produce a peptide compound.

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