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WO2025142009A1 - L-lysine position-5 hydroxylase, and method for producing 5-hydroxy-l-lysine using same - Google Patents

L-lysine position-5 hydroxylase, and method for producing 5-hydroxy-l-lysine using same Download PDF

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Publication number
WO2025142009A1
WO2025142009A1 PCT/JP2024/035265 JP2024035265W WO2025142009A1 WO 2025142009 A1 WO2025142009 A1 WO 2025142009A1 JP 2024035265 W JP2024035265 W JP 2024035265W WO 2025142009 A1 WO2025142009 A1 WO 2025142009A1
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Prior art keywords
lysine
amino acid
polypeptide
acid sequence
seq
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French (fr)
Japanese (ja)
Inventor
康夫 大西
陽平 勝山
誠司 川合
剛 阪本
美紀 若松
杏壮 高松
崇敦 井浦
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API Corp
University of Tokyo NUC
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API Corp
University of Tokyo NUC
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine

Definitions

  • the present invention relates to an L-lysine 5-hydroxylase, a method for producing 5-hydroxy-L-lysine, and a catalyst for producing 5-hydroxy-L-lysine.
  • 5-Hydroxy-L-lysine is useful as an intermediate for pharmaceuticals.
  • 5-hydroxy-L-lysine can be used as an intermediate for Bengamide B, which has antitumor activity. It has also been reported that 5-hydroxy-L-lysine can be used as a raw material for 5-hydroxy-L-pipecolic acid (Non-Patent Document 1, Non-Patent Document 2).
  • 5-Hydroxy-L-pipecolic acid is known to be usable as a precursor for antibacterial agents (Patent Document 1).
  • Patent Document 2 reports that 2-ketoglutarate-dependent dioxygenase has activity for hydroxylating lysine at the 3- or 4-position, and describes an efficient method for producing 3-hydroxy-L-lysine and/or 4-hydroxy-L-lysine using this enzyme.
  • Patent Document 2 it can be predicted that if an L-lysine hydroxylase having hydroxylation activity at the 5-position of L-lysine can be found, a method for producing 5-hydroxy-L-lysine from L-lysine can be established.
  • Non-Patent Document 7 suggests that by knocking out a part of the arazopeptin biosynthetic gene cluster (hereinafter referred to as the "azp gene cluster") in the Streptacidiphilus griseoplanus JCM4300 strain, the AzpK protein of SEQ ID NO: 136 or SEQ ID NO: 138 (the base sequences of the DNA encoding the amino acid sequence of the protein are SEQ ID NO: 135 and SEQ ID NO: 137, respectively) encoded by the azpK gene in the azp gene cluster may have the activity of hydroxylating the 5-position of L-lysine.
  • the AzpK protein of SEQ ID NO: 136 or SEQ ID NO: 138 the base sequences of the DNA encoding the amino acid sequence of the protein are SEQ ID NO: 135 and SEQ ID NO: 137, respectively
  • Non-Patent Document 2 a polypeptide having L-lysine 5-hydroxylation activity exists among the polypeptides encoded by the azpK gene or an azpK gene homologue with high sequence identity thereto, and it can be easily imagined by a person skilled in the art that a method for producing 5-hydroxy-L-lysine from L-lysine could be established by using such a polypeptide.
  • Non-Patent Document 7 reports that the AzpK protein of SEQ ID NO: 136 or 138 has no similarity in amino acid sequence to known hydroxylases, the reaction mechanism is unknown, and that the AzpK protein was obtained from the azpK gene and its L-lysine 5-hydroxylation activity was evaluated, but the activity could not be detected.
  • Non-Patent Document 8 also reports on an L-lysine halogenating (chlorinating) enzyme called BesD, which has sequence similarity to the amino acid sequence of the polypeptide of the present invention described below. However, the function of the polypeptide of the present invention is not described.
  • BesD L-lysine halogenating (chlorinating) enzyme
  • a DNA encoding the L-lysine 5-hydroxylase [3] The DNA is the following (G), (H), (I), (J), (K) or (L): (G) DNA containing the base sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 57, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133; (H) a DNA encoding a polypeptide having an L-lysine 5-hydroxylating activity, comprising a nucleotide sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33
  • fast or slow alignment is used with default settings, with slow alignment being preferred.
  • default values refers to a set of values or parameters established by the software manufacturer. Default values are originally loaded into the software when it is first initialized.
  • a homology search was performed for the azpK2 gene and the Pp_azpK2 gene using databases such as the DNA Databank of JAPAN (DDBJ), and gene sequence information for 1,000 homologous genes of each of the azpK2 gene and the Pp_azpK2 gene was obtained.
  • DDBJ DNA Databank of JAPAN
  • the amino acid sequences encoded by these homolog genes were classified based on a molecular phylogenetic tree created using the Neighbor-joining method, and were classified into 10 Clades. Each classified Clade was named Clades 1 to 10. Representative proteins encoded by the 1,115 homolog genes (including the azpK2 and Pp_azpK2 genes) classified into the 10 Clades are shown in Figure 2.
  • the signature motif associated with enzymes that mainly catalyze hydroxylation reactions is known to contain the following two conserved motifs: The positions of amino acid residues are numbered based on the amino acid sequence of the AzpK2 protein (SEQ ID NO: 2). 1) His139-X140-Asp141: "HXD”motif; 2) Tyr197-Arg216: “Y-R” motif (unless otherwise specified herein, X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V).
  • the L-lysine 5-hydroxylase of the present invention is, for example, a polypeptide belonging to Clade 1, 2, 3, 4, and 10, and has L-lysine 5-hydroxylation activity.
  • polypeptides having the amino acid sequence of group (a) are capable of selectively S-hydroxylating the 5-position of L-lysine and can be suitably used for producing (2S,5S)-5-hydroxy-L-lysine.
  • polypeptides belong to Class 1.
  • polypeptides having the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16 or 22 are preferred, and the polypeptides having the amino acid sequence shown in SEQ ID NO: 8, 10 or 16 are more preferred, because they have high L-lysine 5-position hydroxylation activity and high yield of (2S,5S)-5-hydroxy-L-lysine.
  • polypeptides having the amino acid sequence (a) group the polypeptides having the amino acid sequence shown in SEQ ID NO: 4, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134 can R-selectively hydroxylate the 5-position of L-lysine, and can be suitably used for producing (2S, 5R)-5-hydroxy-L-lysine.
  • Polypeptide (B), (C), (D), (E) or (F) is a polypeptide that has a homologue of the amino acid sequence (a) group and has L-lysine 5-hydroxylation activity.
  • Polypeptide (C) is a polypeptide that has a homologue of the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20, or 22 among the homologues of the amino acid sequence (a) group, and has L-lysine 5-hydroxylation activity.
  • polypeptide (C) belongs to Clade 1.
  • Polypeptide (D) is capable of R-selectively hydroxylating the 5-position of L-lysine and can be suitably used to produce (2S,5R)-5-hydroxy-L-lysine.
  • Polypeptide (E) is capable of R-selectively hydroxylating the 5-position of L-lysine and can be suitably used to produce (2S,5R)-5-hydroxy-L-lysine.
  • Polypeptide (F) is a polypeptide that has a consecutive amino acid sequence motif "HGWHWDD” among homologs of the amino acid sequence (a) group; and any consecutive amino acid sequence motif selected from the group consisting of "HETMEXLF”, “HETMDXLW”, “HETMEXLW”, “HETNDXLF” and “HETNNXLF”; and retains L-lysine 5-hydroxylation activity. More specifically, polypeptide (F) has a consecutive amino acid sequence motif "HGWHWDD”; and any one consecutive amino acid sequence motif selected from the group consisting of "HETMEXLF”, “HETMDXLW”, “HETMEXLW”, “HETNDXLF” and "HETNNXLF".
  • HETMEXLF plays an important role in expressing S-selective L-lysine 5-hydroxylation activity
  • HTMDXLW plays an important role in expressing S-selective L-lysine 5-hydroxylation activity
  • HETMEXLW plays an important role in expressing S-selective L-lysine 5-hydroxylation activity
  • HETMDXLW plays an important role in expressing S-selective L-lysine 5-hydroxylation activity
  • HETMEXLW “HETMEXLW”
  • HETNDXLF” and HETNNXLF” play an important role in expressing R-selective L-lysine 5-hydroxylation activity.
  • polypeptides (F) the following polypeptides (F-1) to (F-3) are preferred.
  • Polypeptide (F-1) is a polypeptide that is a homolog of the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20, or 22 among the homologs of the amino acid sequence (a) group, has an amino acid sequence having the consecutive amino acid sequence motifs "HGWHWDD” and "HETMEXLF", and has L-lysine 5-position hydroxylation activity.
  • Polypeptide (F-1) is capable of selectively S-hydroxylating the 5-position of L-lysine and can be suitably used for producing (2S,5S)-5-hydroxy-L-lysine.
  • polypeptide (F-1) belongs to Clade 1.
  • Polypeptide (F-2-1) is a polypeptide that is a homolog of the amino acid sequence shown in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110 among the homologs of amino acid sequence (a), has an amino acid sequence having consecutive amino acid sequence motifs "HGWHWDD" and either "HETMDXLW" or "HETMEXLW", and has L-lysine 5-position hydroxylation activity.
  • Polypeptide (F-2-1) is capable of R-selectively hydroxylating the 5-position of L-lysine, and can be suitably used for producing (2S,5R)-5-hydroxy-L-lysine.
  • polypeptide (F-2-1) belongs to Class 2 or 3.
  • Polypeptide (F-2-2) is a polypeptide that is a homolog of the amino acid sequence shown in SEQ ID NO: 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134 among the homologs of the amino acid sequence (a) group, has an amino acid sequence having the consecutive amino acid sequence motifs "HGWHWDD” and either "HETNDXLF” or "HETNNXLF", and has L-lysine 5-position hydroxylation activity.
  • Polypeptide (F-2-2) is capable of R-selectively hydroxylating the 5-position of L-lysine and can be suitably used for producing (2S,5R)-5-hydroxy-L-lysine.
  • polypeptide (F-2-2) belongs to Class 4.
  • the L-lysine 5-hydroxylase of the present invention has the following two conserved motifs 1) and 2), which are signature motifs associated with enzymes that mainly catalyze hydroxylation reactions among 2-oxoglutarate-dependent dioxygenases, and is considered to be classified as a 2-oxoglutarate-dependent dioxygenase belonging to the E.C. number 1.14.11.
  • the positions of amino acid residues are numbered based on the amino acid sequence of the AzpK2 protein (SEQ ID NO: 2). 1) His139-X140-Asp141: "HXD” motif 2) Tyr197-Arg216: "Y-R" motif
  • the L-lysine 5-hydroxylase of the present invention can be obtained, for example, by purification from the origin organisms shown in Table 1 above. It can also be obtained by cloning the DNA encoding the L-lysine 5-hydroxylase by known methods such as PCR or hybridization, or by using chemically synthesized nucleic acid, and expressing it in an appropriate host.
  • a DNA encoding an L-lysine 5-hydroxylase having an amino acid sequence in the amino acid sequence (a) group there can be mentioned a DNA having the base sequence shown in Table 2 above.
  • it encodes the L-lysine 5-hydroxylase of the present invention it may be a homologue of the DNA having the base sequence shown in Table 2.
  • (G) is a DNA encoding an L-lysine 5-hydroxylase having the amino acid sequence (a) group, and has the base sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 57, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133 (hereinafter, sometimes referred to as "base sequence (g) group").
  • (H) to (L) are DNAs that are homologs of DNAs having the base sequence group (g) and encode polypeptides having L-lysine 5-hydroxylation activity.
  • (H) is a DNA having a base sequence in the base sequence group (g) in which one or more bases have been substituted, deleted, and/or added, and encoding a polypeptide having L-lysine 5-hydroxylation activity.
  • the one or more bases referred to here are usually 1 to 150, preferably 1 to 60, more preferably 1 to 30, even more preferably 1 to 15, particularly preferably 1 to 5 bases, even more particularly preferably 1 to 3, even more particularly preferably 1 to 2, and most preferably 1.
  • (I) is a DNA that has 55% or more identity with a polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19 or 21 among homologues of DNA having the nucleotide sequence (g) group, and encodes a polypeptide having L-lysine 5-hydroxylation activity.
  • 55% or more identity means usually 55% or more, preferably 70% or more, more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, particularly preferably 95% or more, even more particularly preferably 97% or more, even more particularly preferably 98% or more, and most preferably 99% or more.
  • (J) is a DNA that has 50% or more identity to a polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, or 109 among homologues of DNA having the nucleotide sequence (g) group, and encodes a polypeptide having L-lysine 5-position hydroxylation activity.
  • (K) is a DNA that has 58% or more identity with an amino acid sequence encoded by any of DNAs having the base sequence shown in SEQ ID NO: 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133 among homologs of DNAs having the base sequence (g) group, and encodes a polypeptide having L-lysine 5-hydroxylation activity.
  • 58% or more identity means usually 58% or more, preferably 70% or more, more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, particularly preferably 95% or more, even more particularly preferably 97% or more, even more particularly preferably 98% or more, and most preferably 99% or more.
  • the culture medium obtained by culturing cells containing L-lysine 5-hydroxylase is a culture medium that contains L-lysine 5-hydroxylase and has the enzyme activity, and may be, for example, a suspension of the cells and a liquid medium, or, if the cells are secretory expression cells, the supernatant obtained by removing the cells by centrifugation or the like, or a concentrate thereof.
  • Methods for producing cells (transformants) of microorganisms or the like transformed with DNA encoding L-lysine 5-hydroxylase include, for example, a method of introducing DNA encoding L-lysine 5-hydroxylase into a plasmid vector, phage vector, or virus vector that is stable in a host cell of a microorganism, and introducing the constructed expression vector into the host cell, and a method of directly introducing the DNA into the host genome and transcribing and translating the genetic information.
  • a transformant having DNA encoding L-lysine 5-hydroxylase introduced therein can be obtained by transforming a host cell with an L-lysine hydroxylase expression vector obtained by expressibly inserting DNA encoding L-lysine 5-hydroxylase into a known expression vector.
  • a transformant can be obtained by expressibly incorporating DNA encoding L-lysine 5-hydroxylase into the chromosomal DNA of a host cell by a method such as homologous recombination.
  • the host cell (microorganism) to be transformed to express L-lysine 5-hydroxylase is not particularly limited as long as it does not adversely affect the hydroxylation reaction of L-lysine.
  • Specific examples of the host cell include the following microorganisms: Bacteria belonging to the genera Escherichia, Bacillus, Pseudomonas, Serratia, Brevibacterium, Corynebacterium, Streptococcus, Lactobacillus, etc., for which a host-vector system has been established. Actinomycetes belonging to the genera Rhodococcus and Streptomyces, etc., for which host-vector systems have been established.
  • examples of plasmid vectors include pBR and pUC-based plasmids
  • examples of promoters include those derived from lac ( ⁇ -galactosidase), trp (tryptophan operon), tac, trc (lac and trp fusion), and ⁇ phages PL and PR.
  • examples of terminators include those derived from trpA, phages, and rrnB ribosomal RNA.
  • examples of vectors include pUB110-based plasmids and pC194-based plasmids, and they can also be integrated into chromosomes.
  • promoters and terminators that can be used include promoters and terminators of enzyme genes such as alkaline protease, neutral protease, and ⁇ -amylase.
  • examples of vectors include general host vector systems established for Pseudomonas putida, Pseudomonas cepacia, etc., plasmids involved in the decomposition of toluene compounds, and the broad host range vector pKT240 (Gene 26, 273-282 (1983)) based on the TOL plasmid (containing genes necessary for autonomous replication derived from RSF1010, etc.).
  • host-vector systems have been established for various microorganisms, and these can be used as appropriate.
  • L-lysine 5-hydroxylases of the present invention those containing the polypeptide shown in (A-1), (B-1), (C-1) or (F-1) below are S-selective L-lysine 5-hydroxylases.
  • A-1) A polypeptide having an amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22.
  • B-1) A polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted and/or added in the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22, and having S-selective L-lysine 5-hydroxylation activity.
  • C-1) A polypeptide having an amino acid sequence having 55% or more sequence identity with the full length amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22, and having S-selective L-lysine 5-hydroxylation activity.
  • F-1) A polypeptide having consecutive amino acid sequence motifs "HGWHWDD” and "HETMEXLF", and having S-selective L-lysine 5-hydroxylation activity.
  • S-selective L-lysine 5-hydroxylases can be obtained, for example, by cloning the DNA shown in (G-1), (H-1), (I-1) or (L-1) below by a known method such as PCR or hybridization or by using a chemically synthesized nucleic acid, and expressing the clone in an appropriate host.
  • H-1 A DNA encoding a polypeptide having a nucleotide sequence in which one or more nucleotides are substituted, deleted, and/or added in the nucleotide sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, or 21, and having an activity of hydroxylating the 5-position of L-lysine.
  • (I-1) A DNA encoding a polypeptide having 55% or more sequence identity with a polypeptide encoded by the base sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19 or 21 and having S-selective L-lysine 5-hydroxylation activity.
  • L-1) DNA encoding a polypeptide having the consecutive amino acid sequence motifs "HGWHWDD" and "HETMEXLF" and having S-selective L-lysine 5-hydroxylation activity.
  • L-lysine 5-hydroxylases of the present invention those containing the polypeptide shown in (A-2), (B-2), (D-2), (E-2) or (F-2) below are R-selective L-lysine 5-hydroxylases.
  • SEQ ID NO: 112, 114, 116, 120, 122, 124, 126, 128 130, 132 or 134, and having R-selective L-lysine 5-hydroxylation activity F-2
  • R-selective L-lysine 5-hydroxylases can be obtained, for example, by cloning the DNA shown in (G-2), (H-2), (J-2), (K-2) or (L-2) below by a known method such as PCR or hybridization or by using a chemically synthesized nucleic acid, and expressing the clone in an appropriate host.
  • a DNA encoding a polypeptide having an R-selective L-lysine 5-hydroxylation activity comprising a base sequence in which one or more bases are substituted, deleted, and/or added in the base sequence shown in SEQ ID NO: 3, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85,
  • (K-2) DNA encoding a polypeptide having 58% or more sequence identity with a polypeptide encoded by the base sequence shown in SEQ ID NO: 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133 and having R-selective L-lysine 5-hydroxylation activity.
  • (L-2) A DNA encoding a polypeptide having a consecutive amino acid sequence motif of "HGWHWDD” and any consecutive amino acid sequence motif selected from the group consisting of "HETMDXLW”, “HETMEXLW”, “HETNDXLF” and “HETNNXLF”, and having R-selective L-lysine 5-hydroxylation activity.
  • the enzyme composition of the present invention contains the L-lysine 5-hydroxylase of the present invention, or a cell containing it, a preparation of the cell, or a culture solution obtained by culturing the cell (hereinafter, these may be collectively referred to as "the L-lysine 5-hydroxylase of the present invention, etc.”), and has the L-lysine 5-hydroxylase activity.
  • the enzyme composition of the present invention can inexpensively produce 5-hydroxy-L-lysine from L-lysine with high yield and low cost.
  • the enzyme composition of the present invention has high regioselectivity and stereoselectivity when hydroxylating L-lysine, and therefore can efficiently obtain the desired optical isomer of 5-hydroxy-L-lysine.
  • the enzyme composition of the present invention may contain, in addition to the active ingredient L-lysine 5-hydroxylase of the present invention, excipients, buffers, suspending agents, stabilizers, preservatives, antiseptics, physiological saline, etc.
  • excipients include lactose, sorbitol, D-mannitol, sucrose, etc.
  • buffers that can be used include phosphates, citrates, acetates, etc.
  • stabilizers include propylene glycol, ascorbic acid, etc.
  • preservatives include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, etc.
  • the method is characterized by producing 5-hydroxy-L-lysine represented by the formula:
  • L-lysine comes into contact with and reacts with the L-lysine 5-hydroxylase of the present invention, L-lysine is hydroxylated to produce 5-hydroxy-L-lysine.
  • R-selective L-lysine 5-hydroxylation activity F-2
  • the pCold I vector (Takara Bio) treated with the restriction enzymes NdeI and XhoI (Takara Bio) and the above PCR product were ligated using the In-Fusion (registered trademark) HD Cloning Kit (Takara Bio) in a standard manner.
  • the E. coli JM109 strain (Takara Bio) was then transformed in a standard manner. Plasmid DNA was extracted from the resulting colonies using the miniprep method in a standard manner to obtain the pCold I-AzpK2 vector.
  • coli and synthetic genes in which TCGAAGGTAGGCATATG (SEQ ID NO: 154) was added upstream of the gene and TAACTCGAGGGATCCGAAT (SEQ ID NO: 155) was added downstream of the gene as sequences homologous to the pCold I vector in the nucleic acid sequence of each SEQ ID NO were purchased from Thermo Fisher Scientific.
  • the obtained culture medium was inoculated at 1% volume into Terrific Broth (hereinafter referred to as "TB (Amp50) medium”) containing a final concentration of 50 ⁇ g/mL sodium ampicillin, and cultured at 37°C with shaking (150 rpm).
  • T (Amp50) medium containing a final concentration of 50 ⁇ g/mL sodium ampicillin
  • the medium was cooled to 15°C, and induction was performed by adding IPTG to a final concentration of 20 ⁇ M or 50 ⁇ M.
  • the bacterial cells were collected from the culture medium by centrifugation (20,000 x g, 10 minutes, 4°C).
  • the cells were resuspended in lysis buffer (20 mM HEPES, 10% W/V glycerol, 200 mM sodium chloride (hereinafter referred to as "NaCl”), pH 8.0) and ultrasonically disrupted using a Q500 Sonicator (QSonica), followed by centrifugation (20,000 x g, 60 minutes, 4°C) to obtain an aqueous solution containing the recombinant protein. His60 Ni Superflow Resin (Takara Bio) was added to this aqueous solution and stirred to bind the recombinant protein, which was then transferred to a column.
  • lysis buffer (20 mM HEPES, 10% W/V glycerol, 200 mM sodium chloride (hereinafter referred to as "NaCl”), pH 8.0
  • QSonica Q500 Sonicator
  • His60 Ni Superflow Resin was added to this aqueous solution and stirred to bind the recombinant protein, which was then
  • Elution buffer (20 mM HEPES, 10% glycerol, 200 mM NaCl, 500 mM imidazole, pH 8.0) was mixed with lysis buffer to prepare aqueous solutions with imidazole concentrations of 20 mM, 50 mM, 100 mM, and 250 mM, respectively.
  • the recombinant protein was eluted from the resin in the column by gradually adding aqueous solutions with low to high imidazole concentrations to the column.
  • the eluted fractions at each imidazole concentration were examined for the presence or absence of recombinant protein by polyacrylamide gel electrophoresis (SDS-PAGE), and the eluted fractions in which the presence was confirmed were concentrated and desalted by ultrafiltration (5000 x g, 4°C) using Amicon Ultra-15 Centrifugal Filters (10 kDa) (Merck).
  • the protein concentration of the resulting protein solution was measured using a NanoDrop (registered trademark) microspectrophotometer (Thermo Fisher Scientific) and then frozen and stored.
  • the concentration of the protein solution prepared by this method was calculated using the approximate absorption coefficient at A280 nm calculated from the amino acid sequence and the predicted molecular weight of the amino acid.
  • Example 5 (Confirmation of Lysine 5-hydroxylation Activity of AzpK2, Pp_AzpK2, and AzpK2 Homologs)
  • the reaction was carried out at 30° C. for 1 hour using 100 ⁇ L of a reaction solution prepared with the following composition: 0.1 mM L-lysine (hereinafter referred to as "L-Lys”), 0.1 mM ⁇ -ketoglutaric acid (hereinafter sometimes referred to as " ⁇ KG”), 1.0 mM ascorbic acid, 25 ⁇ M FeSO 4 , 20 mM HEPES, 10 W/V% glycerol, 200 mM NaCl (pH 8.0), and 5.0 ⁇ M of the recombinant protein of AzpK2, Pp_AzpK2, or AzpK2 homolog (AzpK2 Homolog 1-1 to 1-9, 2-1 to 2-3, 3-1 to 3-2) obtained in Example 4.
  • the reaction was stopped by adding 100 ⁇ L of
  • Fmoc-Cl 1 mM fluorenylmethyloxycarbonyl chloride
  • acetonitrile 50 ⁇ L of 1 mM fluorenylmethyloxycarbonyl chloride (hereinafter sometimes referred to as "Fmoc-Cl") dissolved in acetonitrile, and reacted at 30°C for 30 minutes.
  • 50 ⁇ L of 1 M HCl was added to this to stop the reaction, and the reaction product was extracted from this with 150 ⁇ L of ethyl acetate.
  • the solvent was removed using a centrifugal evaporator, and the remaining solid was dissolved in 20 ⁇ L of methanol to obtain an Fmoc-treated sample in which the amino acid was Fmoc-treated.
  • This Fmoc-treated sample was analyzed by LC-MS (analysis condition 2). Details of analysis condition 2 are as follows.
  • Figure 3 shows the LC-MS detection results of the reaction product when AzpK2 was used (detection results of di-Fmoc-5-hydroxy-L-lysine with MS +607.3).
  • PCR was carried out according to standard methods using PrimeStar max DNA polymerase (Takara Bio) with each primer sequence designed to provide restriction enzyme EcoRI and XbaI sites to each gene shown in Table 8 below, to amplify gene fragments having restriction enzyme EcoRI and XbaI sites.
  • LB LB agar medium
  • kanamycin sulfate Fujifilm Wako Pure Chemical Industries, Ltd.
  • PCR-amplified fragments were ligated using the In-Fusion (registered trademark) HD Cloning Kit (Takara Bio Inc.) in the usual manner.
  • the E. coli JM109 strain was then transformed in the usual manner and cultured overnight at 30°C on LB (Km50) agar medium. Plasmid DNA was extracted from the resulting colonies by the miniprep method, and the pCold-PS2K vector was obtained, in which the drug resistance gene of the pCold-ProS2 vector was changed to kanamycin.
  • the resulting pCold-PS2K vector was cleaved with the restriction enzymes NdeI and XhoI (both manufactured by Takara Bio Inc.), and then dephosphorylated in the same manner as in Example 12-1.
  • the inserted gene fragments of AzpK2 or AzpK2 Homolog1-2 were then used for ligation in the same manner as in Example 12-1 to obtain the respective expression vectors pCold-PS2K-AzpK2 and pCold-PS2K-1-2.
  • Example 12-3 Flask culture> Using the expression vectors obtained in Examples 12-1 and 12-2, the pKW32 plasmid was introduced into E. coli JM109 strain, and the pCold-PS2K plasmid was introduced into E. coli BL21 star (DE3) (Thermo Fisher Scientific) according to a conventional method.
  • Example 12-4 Reactivity evaluation using recombinant Escherichia coli>
  • the culture solution obtained in Example 12-3 was centrifuged (6500 rpm, 10 minutes, 4°C) to collect the bacteria, and the wet weight of the collected bacteria was measured and suspended in 50 mM MES-NaOH buffer (pH 7.0) (hereinafter sometimes referred to as "MES buffer (pH 7.0)”) so as to be 100 g/L, followed by ultrasonic disruption using an ultrasonic homogenizer SONIFIER (registered trademark) 250D (manufactured by BRANSON).
  • MES buffer (pH 7.0) 50 mM MES-NaOH buffer
  • SONIFIER registered trademark
  • reaction solutions were prepared in 14 mL Falcon (registered trademark) round tubes for each of reaction solution compositions 1 to 3 in Reaction Solution Composition Table-1.
  • reaction solution was taken from each round tube, diluted 5-fold with purified water, and ultrafiltered at room temperature for 10 minutes at 10,000 x g using a filter unit (Amicon (registered trademark) Ultra-0.5 10 kDa (Merck)).
  • filter unit Amicon (registered trademark) Ultra-0.5 10 kDa (Merck)
  • HPLC HPLC
  • the enzyme activity that accumulates 1 ⁇ mol of 5-hydroxy-L-lysine in a hydroxylation reaction for 1 minute is defined as 1 unit (hereinafter referred to as "U"), and is expressed in U/g-cell.
  • U 1 unit
  • AzpK2 Homolog1-1, AzpK2 Homolog1-2, and AzpK2 Homolog1-3 showed higher specific activity than AzpK2 and Pp_AzpK2.
  • AzpK2 Homolog1-1 and AzpK2 Homolog1-2 showed particularly high specific activity.
  • the degree of influence of substrate inhibition on the decrease in reaction rate is such that the higher the relative value when the substrate concentration is increased, the less the influence of substrate inhibition is, and conversely, the lower the relative value when the substrate concentration is increased, the greater the influence of substrate inhibition is.
  • each preculture solution was inoculated into 40 mL of another TB (Amp100) medium and cultured with shaking at 30°C. Culture was continued until the OD630nm reached 0.5, and after standing at 15°C for 30 minutes, IPTG was added to a final concentration of 0.02 mM, and cultured with shaking at 15°C and 200 rpm for 24 hours to obtain each culture solution.
  • Example 14 Reactivity evaluation of S-selective lysine hydroxylase using recombinant Escherichia coli-3) The effect of substrate inhibition on the enzyme and the hydroxylation activity of 5-hydroxy-L-lysine were evaluated using each cell lysate of recombinant E. coli transformed with the pCold I vector, pCold I-1-2, pCold I-1-3, or pCold I-1-6 obtained in Example 13.
  • Example 14-2 Evaluation of 5-hydroxy-L-lysine hydroxylation activity of S-selective lysine hydroxylase L-lysine 5-hydroxylase has the activity of hydroxylating L-lysine, but also has the activity of further hydroxylating the 5-position of the product, 5-hydroxylysine, as shown in Example 10. Furthermore, if 5-oxolidine is produced here, the yield of the target product, 5-hydroxylysine, decreases. Therefore, when attempting to establish a method for synthesizing 5-hydroxylysine, it is preferable that the hydroxylase has a low hydroxylating activity of 5-hydroxylysine.
  • a 0.4 mL reaction solution was prepared with a composition of 20 mM racemic 5-hydroxylysine, 30 mM ⁇ -ketoglutaric acid, 2 mM ascorbic acid, 0.5 mM FeSO 4 , 2.5 mM citric acid, 50 mM HEPES (pH 7.0), and 50 g/L of cell lysate, and the reaction was carried out for 3 hours under the same conditions as in Example 13, and the 5-hydroxy-L-lysine concentration was measured. The measured 5-hydroxy-L-lysine concentration was divided by the reaction time to calculate the 5-hydroxy-L-lysine accumulation rate per unit time.
  • the remaining amount of 5-hydroxy-L-lysine after 3 hours of reaction with each enzyme was subtracted by the difference in the remaining amount of 5-hydroxy-L-lysine in the negative control pCold I vector-introduced strain to calculate the 5-hydroxy-L-lysine consumption rate per unit time.
  • the 5-hydroxy-L-lysine consumption rate was divided by the 5-hydroxy-L-lysine accumulation rate measured in Example 14-1 to calculate the 5-hydroxy-L-lysine/L-Lys hydroxylation activity ratio, which is shown in Table 12.
  • Example 15 (Study of reaction conditions) ⁇ Example 15-1>
  • the pKW32-1-3 vector prepared in Example 12-1 was introduced into the E. coli JM109 strain according to a conventional method to prepare the E. coli JM109-pKW32-1-3 strain.
  • the obtained E. coli JM109-pKW32-1-3 strain was cultured by inducing enzyme expression with IPTG to a final concentration of 0.2 mM according to a conventional method, and cultured cells of the E. coli JM109-pKW32-1-3 strain were obtained.
  • Example 15-2 Examination of cell disruption conditions The cultured cells of E. coli JM109-pKW32-1-3 strain obtained in Example 15-1 were suspended in each of the buffer solutions under the following conditions I to III to a concentration of 100 g/L, and were ultrasonically disrupted using a SONIFIER (registered trademark) 250D (manufactured by BRANSON).
  • SONIFIER registered trademark
  • Example 14-1 The resulting cell disruption solution and the undisrupted cell suspension were used to react in the same manner as in Example 13 for 0.5 hours at 15°C, using reaction composition 5 of Example 14-1.
  • Example 15-4 Examination of substrate concentration-1>
  • the cultured cells of E. coli JM109-pKW32-1-3 strain obtained in Example 15-1 were suspended in the buffer solution of Condition II in Example 15-2 to a concentration of 100 g/L or 200 g/L, and then ultrasonically disrupted using a SONIFIER (registered trademark) 250D (manufactured by BRANSON).
  • SONIFIER registered trademark
  • 250D manufactured by BRANSON
  • areas with a light gray background indicate areas with an identity value of 58% or more
  • areas with a dark gray background indicate areas with an identity value of 50% or more.
  • Example 18 (Crystal structure analysis of AzpK2)
  • Example 18-1 Purification of AzpK2 protein
  • the recombinant protein aqueous solution of AzpK2 obtained in the same manner as in Example 4 was purified by anion exchange chromatography using AKTA pure (manufactured by Cytiva). Elution was performed using a NaCl concentration gradient (solution A (20 mM HEPES, 10 W/V% glycerol, pH 8.0) and solution B (20 mM HEPES, 10 W/V% glycerol, 1 M NaCl, pH 8.0)) and a RESOURCE (registered trademark) Q 1 mL (manufactured by Cytiva) column.
  • solution A (20 mM HEPES, 10 W/V% glycerol, pH 8.0
  • solution B (20 mM HEPES, 10 W/V% glycerol, 1 M NaCl, pH 8.0
  • RESOURCE registered trademark
  • Example 18-3 X-ray structure analysis of AzpK2 protein crystal>
  • the AzpK2 single crystal obtained in Example 18-2 was scooped up with a litho loop and frozen with liquid nitrogen. Thereafter, the frozen crystal was irradiated with X-rays under a nitrogen gas flow at 100 K using the beamline PF BL1A at the High Energy Accelerator Research Organization to obtain a diffraction image. As a result, a diffraction image with a maximum resolution of 1.60 angstroms was obtained.
  • the data obtained was processed using XDS software (Max Planck Institute for Medical Research) and further scaled using AIMLESS software. Phase determination was then performed by molecular replacement using Phoenix phaser, and an initial model was constructed using Autobuild.
  • circles represent residues that interact with the carboxylic acid of L-lysine
  • circles represent residues that interact with the iron atom
  • double circles represent residues that interact with the water molecules that coordinate around the ⁇ -amino group of L-lysine
  • ⁇ represent residues that interact with ⁇ KG.
  • the top left figure shows the model construction results from X-ray structural analysis of AzpK2 single crystals (near the active center)
  • the top right figure shows the model construction results from X-ray structural analysis of AzpK2, ⁇ KG, and 5-hydroxylysine co-crystals (near the active center)
  • the bottom left figure shows the predicted structure of BesD in AlphaFold2
  • the bottom right figure shows the predicted structure of AzpK2 in AlphaFold2 (BesD-derived lysine and ⁇ KG are superimposed).
  • the gray sphere near the center represents a region of high electron density, which is generally known to correspond to an iron atom (Fe2 + ) in ⁇ KG-dependent dioxygenases
  • the light gray sphere near the center represents an iron atom ion in BesD.
  • region (2) of AzpK2 corresponds to the carbonyl group and amino group of L-lysine in BesD, and the region close to ⁇ KG.
  • the His136-Asp141 region is suggested to be close to the amino group of lysine, carboxylic acid, or iron atom, and is shown to be an important region that contributes to substrate selectivity and activity.
  • Example 21 Measurement of activity of AzpK2 amino acid substituted derivatives
  • a primer set for introducing amino acid substitution mutations shown in Table 15 was used, and PrimeStar HS DNA polymerase (manufactured by Takara Bio Inc.) was used according to a standard method to obtain pCold I-AzpK2 vectors encoding the corresponding amino acid substitution mutants.
  • a protein aqueous solution of each of the obtained amino acid substituted AzpK2 was prepared in the same manner as in Example 4. Using the obtained protein solution, an enzyme assay was performed in the same manner as in Example 5, and the Fmoc-conjugated sample was analyzed by LC-MS (analysis condition 2). The analysis results are shown in Figure 16. In Figure 16, WT represents wild-type AzpK2.
  • Example 22 (Identification of important motifs for enzyme activity in AzpK2 and homologues) Regarding important amino acid residues related to the enzyme activity of AzpK2 clarified in Example 20, the amino acid sequences of Pp_AzpK2 belonging to Clades 1 to 4 and Clade 10, which are L-lysine 5-hydroxylase, and Clade 5, which is adjacent to Clade 4 but does not have L-lysine 5-hydroxylation activity, were compared.
  • the amino acid sequences belonging to Clades 1 to 5 were compared for identity within each Clade using ClustalW. From the results of this identity comparison, the occurrence frequency of amino acids in each of the amino acid sequences belonging to Clades 1 to 5 for the amino acid sequences of His136 to Asp142 and His235 to Phe242, which are important regions of AzpK2 identified in Example 20, is shown in Figures 17 and 18 as logo plots.
  • the amino acid sequence of Pp_AzpK2 was compared with the amino acid sequences belonging to Clades 1 to 5 without comparing identity within the Clade.
  • amino acids 136 to 142 of Pp_AzpK2 were “HGWHLDD", while the amino acids 136 to 142 of Clade5 were “HGWHWGD”.
  • Non-Patent Document 8 reports that the combination of amino acids at positions 139 and 141 is characterized as "HXD” in hydroxylases and "HXG” in halogenases. Furthermore, Non-Patent Document 8 reports that the "H” located third amino acid on the N-terminal side from “HXD” or "HXG” is important for enzyme activity. Also, Example 21 above reveals that introducing an amino acid mutation into His136 of AzpK2 causes the enzyme activity to be lost.
  • HGWHWDD or "HGWHLDD” motif, which is a group of amino acid residues from His136 to Asp142, is important for the expression of the hydroxylation activity of L-lysine 5-hydroxylase.

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Abstract

The present invention addresses the problem of providing a novel method for inexpensively producing 5-hydroxy-L-lysine with high yield and at low cost. As a solution for the problem, the present invention provides: a novel L-lysine position-5 hydroxylase; an enzyme agent composition for producing 5-hydroxy-L-lysine, the composition containing the hydroxylase; and a method for producing 5-hydroxy-L-lysine using the enzyme agent composition.

Description

Ｌ－リジン５位水酸化酵素及びそれを利用した５－ヒドロキシ－Ｌ－リジンの製造方法L-lysine 5-hydroxylase and method for producing 5-hydroxy-L-lysine using the same

　本発明は、Ｌ－リジン５位水酸化酵素、５－ヒドロキシ－Ｌ－リジンの製造方法、及び５－ヒドロキシ－Ｌ－リジン生成用触媒に関するものである。 The present invention relates to an L-lysine 5-hydroxylase, a method for producing 5-hydroxy-L-lysine, and a catalyst for producing 5-hydroxy-L-lysine.

　５－ヒドロキシ－Ｌ－リジンは医薬品の中間体として有用である。例えば、５－ヒドロキシ－Ｌ－リジンは抗腫瘍活性のあるBengamide Bの中間体として利用可能であることが知られている。また、５－ヒドロキシ－Ｌ－リジンが５－ヒドロキシ－Ｌ－ピペコリン酸の原料として利用可能なことが報告されている（非特許文献１、非特許文献２）。５－ヒドロキシ－Ｌ－ピペコリン酸は抗菌剤の前駆体として利用可能であることが知られている（特許文献１）。 5-Hydroxy-L-lysine is useful as an intermediate for pharmaceuticals. For example, it is known that 5-hydroxy-L-lysine can be used as an intermediate for Bengamide B, which has antitumor activity. It has also been reported that 5-hydroxy-L-lysine can be used as a raw material for 5-hydroxy-L-pipecolic acid (Non-Patent Document 1, Non-Patent Document 2). 5-Hydroxy-L-pipecolic acid is known to be usable as a precursor for antibacterial agents (Patent Document 1).

　５－ヒドロキシ－Ｌ－リジンの合成法は、例えば、非特許文献３～６において報告されている。しかしながら、いずれの合成法も、製造工程が長い若しくは複雑な工程を含む、又は高額な原料若しくは触媒が必要であるため、５－ヒドロキシ－Ｌ－リジンを高収率かつ低コストで安価に製造する方法が望まれていた。 Methods for synthesizing 5-hydroxy-L-lysine have been reported, for example, in Non-Patent Documents 3 to 6. However, all of these synthesis methods involve long or complicated manufacturing steps, or require expensive raw materials or catalysts, and therefore there has been a demand for a method for inexpensively producing 5-hydroxy-L-lysine at high yield and low cost.

　ここで、特許文献２において、２－ケトグルタル酸依存型ジオキシゲナーゼがリジンの３位又は４位水酸化活性を有すること、及び当該酵素を利用した３－ヒドロキシ－Ｌ－リジン及び／又は４－ヒドロキシ－Ｌ－リジンの効率的な製造法が報告されている。 Patent Document 2 reports that 2-ketoglutarate-dependent dioxygenase has activity for hydroxylating lysine at the 3- or 4-position, and describes an efficient method for producing 3-hydroxy-L-lysine and/or 4-hydroxy-L-lysine using this enzyme.

　特許文献２から、Ｌ－リジンの５位に水酸化活性を有するＬ－リジン水酸化酵素を見出すことができれば、Ｌ－リジンから５－ヒドロキシ－Ｌ－リジンを製造する方法が確立できることが予想できる。 From Patent Document 2, it can be predicted that if an L-lysine hydroxylase having hydroxylation activity at the 5-position of L-lysine can be found, a method for producing 5-hydroxy-L-lysine from L-lysine can be established.

　ここで、非特許文献７において、Ｓｔｒｅｐｔａｃｉｄｉｐｈｉｌｕｓ　ｇｒｉｓｅｏｐｌａｎｕｓ　ＪＣＭ４３００株におけるアラゾペプチン生合成遺伝子クラスター（以下、「ａｚｐ遺伝子クラスター」と称する。）の一部をノックアウトする手法により、ａｚｐ遺伝子クラスター中のａｚｐＫ遺伝子がコードする、配列番号１３６又は配列番号１３８のＡｚｐＫタンパク質（同タンパク質のアミノ酸配列をコードするＤＮＡの塩基配列は、それぞれ配列番号１３５及び配列番号１３７）がＬ－リジンの５位水酸化活性を有する可能性があることが示唆されている。 Here, Non-Patent Document 7 suggests that by knocking out a part of the arazopeptin biosynthetic gene cluster (hereinafter referred to as the "azp gene cluster") in the Streptacidiphilus griseoplanus JCM4300 strain, the AzpK protein of SEQ ID NO: 136 or SEQ ID NO: 138 (the base sequences of the DNA encoding the amino acid sequence of the protein are SEQ ID NO: 135 and SEQ ID NO: 137, respectively) encoded by the azpK gene in the azp gene cluster may have the activity of hydroxylating the 5-position of L-lysine.

　したがって、非特許文献２及び非特許文献７の知見を組み合わせることにより、ａｚｐＫ遺伝子又はこれと配列同一性の高いａｚｐＫ遺伝子ホモログがコードするポリペプチドの中にはＬ－リジン５位水酸化活性を有するポリペプチドが存在する可能性があり、それを使用することにより、Ｌ－リジンから５－ヒドロキシ－Ｌ－リジンを製造する方法が確立できるだろうことは当業者ならば容易に想像し得る。 Therefore, by combining the findings of Non-Patent Document 2 and Non-Patent Document 7, it is possible that a polypeptide having L-lysine 5-hydroxylation activity exists among the polypeptides encoded by the azpK gene or an azpK gene homologue with high sequence identity thereto, and it can be easily imagined by a person skilled in the art that a method for producing 5-hydroxy-L-lysine from L-lysine could be established by using such a polypeptide.

　しかしながら、非特許文献７には、配列番号１３６又は１３８のＡｚｐＫタンパク質は既知の水酸化酵素とのアミノ酸配列の配列類似性が無く、反応メカニズムは不明であること、ａｚｐＫ遺伝子からＡｚｐＫタンパク質を取得してＬ－リジン５位水酸化活性を評価したが当該活性を検出できなかったことが報告されている。 However, Non-Patent Document 7 reports that the AzpK protein of SEQ ID NO: 136 or 138 has no similarity in amino acid sequence to known hydroxylases, the reaction mechanism is unknown, and that the AzpK protein was obtained from the azpK gene and its L-lysine 5-hydroxylation activity was evaluated, but the activity could not be detected.

　また非特許文献８では、後述する本発明のポリペプチドのアミノ酸配列と配列類似性のあるＢｅｓＤというＬ－リジンハロゲン化（クロロ化）酵素について報告されている。しかしながら、当該本発明のポリペプチドの機能については記載されていない。 Non-Patent Document 8 also reports on an L-lysine halogenating (chlorinating) enzyme called BesD, which has sequence similarity to the amino acid sequence of the polypeptide of the present invention described below. However, the function of the polypeptide of the present invention is not described.

特許第４５９０９８１号公報Patent No. 4590981 特許第６４７６１１０号公報Patent No. 6476110

Ｙａｓｕｄａら、Ｔｅｔｒａｈｅｄｒｏｎ　Ａｓｙｍｍ．，２００６，１７，１７７５Yasuda et al., Tetrahedron Asymm., 2006, 17, 1775 Ｔｓｏｔｓｏｕら、Ｂｉｏｃｈｅｍｉｅ，２００７，８９，５９１Tsotsou et al., Biochemie, 2007, 89, 591 Ａｌｌｅｖｉら、Ｔｅｔｒａｈｅｄｒｏｎ　Ａｓｙｍｍｅｔｒｙ，Ｖｏｌ　１１，Ｉｓｓｕｅ　１５，２０００，３１５１－３１６０Allevi et al., Tetrahedron Asymmetry, Vol. 11, Issue 15, 2000, 3151-3160 Ｇｕｏら、Ｏｒｇ．　Ｂｉｏｍｏｌ．　Ｃｈｅｍ．，２０１４，１２，７３１０－７３１７Guo et al., Org. Biomol. Chem., 2014, 12, 7310-7317 Ｎｉｅｕｗｅｎｄｉｊｋら、Ｅｕｒ．　Ｊ．　Ｏｒｇ．　Ｃｈｅｍ．　２０００,３６８３．３６９１Nieuwendijk et al., Eur. J. Org. Chem.　2000,3683.3691 Ｊｏｈａｎｎｅｓら、Ｊ．　Ｏｒｇ．　Ｃｈｅｍ．　２０１３，７８，１２８０９－１２８１３Johannes et al., J. Org. Chem. 2013, 78, 12809-12813 Ｋａｗａｉら、Ａｎｇｅｗ．Ｃｈｅｍ．　Ｉｎｔ．　Ｅｄ．　２０２１，６０,１０３１９－１０３２５Kawai et al., Angew. Chem. Int. Ed.　2021,60,10319-10325 Ｎｅｕｇｅｂａｕｅｒら、Ｎａｔ．　Ｃｈｅｍ．　Ｂｉｏ．，Ｖｏｌ１５，Ｏｃｔ２０１９，１００９－１０１６Neugebauer et al., Nat. Chem. Bio., Vol. 15, Oct. 2019, 1009-1016

　以上のように、従来から５－ヒドロキシ－Ｌ－リジンの製造法は知られていたものの、より高収率かつ低コストで安価に５－ヒドロキシ－Ｌ－リジンを製造する方法が望まれていた。また、効率的な製造法として考え得るＬ－リジン５位水酸化酵素を用いたＬ－リジンから５－ヒドロキシ－Ｌ－リジンを製造する方法については、Ｌ－リジン５位水酸化酵素が見出されていないために技術的に実現できなかった。 As described above, although methods for producing 5-hydroxy-L-lysine have been known for some time, there has been a demand for a method for producing 5-hydroxy-L-lysine inexpensively with a higher yield and at a lower cost. Furthermore, a method for producing 5-hydroxy-L-lysine from L-lysine using L-lysine 5-hydroxylase, which could be considered as an efficient production method, has not been technically feasible because L-lysine 5-hydroxylase has not yet been discovered.

　すなわち、本発明は、高収率かつ低コストで安価に５－ヒドロキシ－Ｌ－リジンを製造する方法を提供することを課題とする。特に、Ｌ－リジンから５－ヒドロキシ－Ｌ－リジンを製造することのできるＬ－リジン５位水酸化酵素を見出し、これを用いて５－ヒドロキシ－Ｌ－リジンを高収率かつ低コストで安価に製造する新規な方法を提供することを課題とする。 In other words, the objective of the present invention is to provide a method for inexpensively producing 5-hydroxy-L-lysine at high yield and low cost. In particular, the objective of the present invention is to discover an L-lysine 5-hydroxylase capable of producing 5-hydroxy-L-lysine from L-lysine, and to provide a novel method for inexpensively producing 5-hydroxy-L-lysine at high yield and low cost using the same.

　本発明者らは、上記課題を解決するために、非特許文献７の情報を基にＬ－リジン５位水酸化酵素活性を有する遺伝子について鋭意検討した結果、これまでタンパク質として単離された報告が無く機能が不明であった２－オキソグルタル酸依存型水酸化酵素がＬ－リジン５位水酸化酵素活性を有することを見出し、さらに、Ｌ－リジン５位水酸化酵素の発現に重要なアミノ酸配列モチーフを特定した。そして、これらのポリペプチドをコードするＤＮＡを用いて形質転換体を作製し、当該形質転換体細胞、その調製物及び／又は培養液をＬ－リジンに作用させることにより、高い光学純度かつ高濃度で５－ヒドロキシ－Ｌ－リジンを製造できることを見出した。本発明はこれらの知見に基づいて成し遂げられたものである。
　すなわち、本発明の要旨は、以下のとおりである。 In order to solve the above problems, the present inventors have intensively investigated genes having L-lysine 5-hydroxylase activity based on the information in Non-Patent Document 7, and as a result, have found that 2-oxoglutarate-dependent hydroxylase, the function of which has not been reported as being isolated as a protein, has L-lysine 5-hydroxylase activity, and further identified an amino acid sequence motif important for the expression of L-lysine 5-hydroxylase. Then, the present inventors have found that 5-hydroxy-L-lysine can be produced in high optical purity and high concentration by preparing a transformant using DNA encoding these polypeptides and allowing the transformant cells, preparations thereof and/or culture solutions thereof to act on L-lysine. The present invention has been accomplished based on these findings.
That is, the gist of the present invention is as follows.

［１］
　以下の（Ａ）、（Ｂ）、（Ｃ）、（Ｄ）、（Ｅ）又は（Ｆ）に示すポリペプチドを含むＬ－リジン５位水酸化酵素：
（Ａ）配列番号２、４、６、８、１０、１２、１４、１６、１８、２０、２２、２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８、１１０、１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列を有するポリペプチド；
（Ｂ）配列番号２、４、６、８、１０、１２、１４、１６、１８、２０、２２、２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８、１１０、１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換及び／又は付加されたアミノ酸配列を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチド；
（Ｃ）配列番号２、６、８、１０、１２、１４、１６、１８、２０又は２２に示すアミノ酸配列全長と５５％以上の配列同一性を有するアミノ酸配列を有し、かつ、Ｌ－リジン５位水酸化酵素活性を有するポリペプチド；
（Ｄ）配列番号２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８又は１１０に示すアミノ酸配列全長と５０％以上の配列同一性を有するアミノ酸配列を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチド；
（Ｅ）配列番号１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列全長と５８％以上の配列同一性を有するアミノ酸配列を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチド；
（Ｆ）連続したアミノ酸配列のモチーフである「ＨＧＷＨＷＤＤ」；と、「ＨＥＴＭＥＸＬＦ」、「ＨＥＴＭＤＸＬＷ」、「ＨＥＴＭＥＸＬＷ」、「ＨＥＴＮＤＸＬＦ」及び「ＨＥＴＮＮＸＬＦ」よりなる群から選ばれるいずれかの連続したアミノ酸配列のモチーフ；を有し、かつ、Ｌ－リジン５位水酸化活性を保持するポリペプチド（ここで、Ｘは、Ａ、Ｒ、Ｎ、Ｄ、Ｃ、Ｑ、Ｅ、Ｇ、Ｈ、Ｉ、Ｌ、Ｋ、Ｍ、Ｆ、Ｐ、Ｓ、Ｔ、Ｗ、Ｙ又はＶのいずれかのアミノ酸を示す）。
［２］
　前記のＬ－リジン５位水酸化酵素をコードするＤＮＡ。
［３］
　前記ＤＮＡが、以下の（Ｇ）、（Ｈ）、（Ｉ）、（Ｊ）、（Ｋ）又は（Ｌ）である、前記のＤＮＡ：
（Ｇ）配列番号１、３、５、７、９、１１、１３、１５、１７、１９、２１、２３、２５、２７、２９、３１、３３、３５、３７、３９、４１、４３、４５、４７、４９、５１、５５、５７、６７、６９、７１、７３、７５、７９、８１、８３、８５、８７、８９、９１、９３、９５、９７、９９、１０１、１０３、１０５、１０７、１０９、１１１、１１３、１１５、１１９、１２１、１２３、１２５、１２７、１２９、１３１又は１３３に示す塩基配列を含むＤＮＡ；
（Ｈ）配列番号１、３、５、７、９、１１、１３、１５、１７、１９、２１、２３、２５、２７、２９、３１、３３、３５、３７、３９、４１、４３、４５、４７、４９、５１、５５、５７、６７、６９、７１、７３、７５、７９、８１、８３、８５、８７、８９、９１、９３、９５、９７、９９、１０１、１０３、１０５、１０７、１０９、１１１、１１３、１１５、１１９、１２１、１２３、１２５、１２７、１２９、１３１又は１３３に示す塩基配列において、１又は複数個の塩基が置換、欠失、及び／又は付加された塩基配列を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ；
（Ｉ）配列番号１、５、７、９、１１、１３、１５、１７、１９又は２１に示す塩基配列がコードするポリペプチドと５５％以上の配列同一性を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ；
（Ｊ）配列番号２３、２５、２７、２９、３１、３３、３５、３７、３９、４１、４３、４５、４７、４９、５１、５５、６７、６９、７１、７３、７５、７９、８１、８３、８５、８７、８９、９１、９３、９５、９７、９９、１０１、１０３、１０５、１０７又は１０９に示す塩基配列がコードするポリペプチドと５０％以上の配列同一性を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ；
（Ｋ）配列番号１１１、１１３、１１５、１１９、１２１、１２３、１２５、１２７、１２９、１３１又は１３３に示す塩基配列がコードするポリペプチドと５８％以上の配列同一性を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ；
（Ｌ）連続したアミノ酸配列のモチーフである「ＨＧＷＨＷＤＤ」；と、「ＨＥＴＭＥＸＬＦ」、「ＨＥＴＭＤＸＬＷ」、「ＨＥＴＭＥＸＬＷ」、「ＨＥＴＮＤＸＬＦ」及び「ＨＥＴＮＮＸＬＦ」よりなる群から選ばれるいずれかの連続したアミノ酸配列のモチーフ；を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ（ここで、Ｘは、Ａ、Ｒ、Ｎ、Ｄ、Ｃ、Ｑ、Ｅ、Ｇ、Ｈ、Ｉ、Ｌ、Ｋ、Ｍ、Ｆ、Ｐ、Ｓ、Ｔ、Ｗ、Ｙ又はＶのいずれかのアミノ酸を示す）。
［４］
　Ｌ－リジンに、以下の（Ａ）、（Ｂ）、（Ｃ）、（Ｄ）、（Ｅ）又は（Ｆ）に示すポリペプチド又はそれを含む細胞、前記細胞の調製物若しくは前記細胞を培養して得られた培養液を接触させて、５－ヒドロキシ－Ｌ－リジンを生成させることを特徴とする、５－ヒドロキシ－Ｌ－リジンの製造方法：
（Ａ）配列番号２、４、６、８、１０、１２、１４、１６、１８、２０、２２、２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８、１１０、１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列を有するポリペプチド；
（Ｂ）配列番号２、４、６、８、１０、１２、１４、１６、１８、２０、２２、２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８、１１０、１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換及び／又は付加されたアミノ酸配列を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチド；
（Ｃ）配列番号２、６、８、１０、１２、１４、１６、１８、２０又は２２に示すアミノ酸配列全長と５５％以上の配列同一性を有するアミノ酸配列を有し、かつ、Ｌ－リジン５位水酸化酵素活性を有するポリペプチド；
（Ｄ）配列番号２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８又は１１０に示すアミノ酸配列全長と５０％以上の配列同一性を有するアミノ酸配列を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチド；
（Ｅ）配列番号１１２，１１４，１１６，１２０，１２２，１２４，１２６，１２８，１３０，１３２又は１３４に示すアミノ酸配列と５８％以上の同一性を有するアミノ酸配列を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチド；
（Ｆ）連続したアミノ酸配列のモチーフである「ＨＧＷＨＷＤＤ」；と、「ＨＥＴＭＥＸＬＦ」、「ＨＥＴＭＤＸＬＷ」「ＨＥＴＭＥＸＬＷ」、「ＨＥＴＮＤＸＬＦ」及び「ＨＥＴＮＮＸＬＦ」よりなる群から選ばれるいずれかの連続したアミノ酸配列のモチーフ；を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチド（ここで、Ｘは、Ａ、Ｒ、Ｎ、Ｄ、Ｃ、Ｑ、Ｅ、Ｇ、Ｈ、Ｉ、Ｌ、Ｋ、Ｍ、Ｆ、Ｐ、Ｓ、Ｔ、Ｗ、Ｙ又はＶのいずれかのアミノ酸を示す）。
［５］
　前記細胞が、前記Ｌ－リジン５位水酸化酵素をコードするＤＮＡで形質転換された細胞である、前記の５－ヒドロキシ－Ｌ－リジンの製造方法。
［６］
　前記ＤＮＡが、以下の（Ｇ）、（Ｈ）、（Ｉ）、（Ｊ）、（Ｋ）又は（Ｌ）である、前記の５－ヒドロキシ－Ｌ－リジンの製造方法：
（Ｇ）配列番号１、３、５、７、９、１１、１３、１５、１７、１９、２１、２３、２５、２７、２９、３１、３３、３５、３７、３９、４１、４３、４５、４７、４９、５１、５５、５７、６７、６９、７１、７３、７５、７９、８１、８３、８５、８７、８９、９１、９３、９５、９７、９９、１０１、１０３、１０５、１０７、１０９、１１１、１１３、１１５、１１９、１２１、１２３、１２５、１２７、１２９、１３１又は１３３に示す塩基配列を含むＤＮＡ；
（Ｈ）配列番号１、３、５、７、９、１１、１３、１５、１７、１９、２１、２３、２５、２７、２９、３１、３３、３５、３７、３９、４１、４３、４５、４７、４９、５１、５５、５７、６７、６９、７１、７３、７５、７９、８１、８３、８５、８７、８９、９１、９３、９５、９７、９９、１０１、１０３、１０５、１０７、１０９、１１１、１１３、１１５、１１９、１２１、１２３、１２５、１２７、１２９、１３１又は１３３に示す塩基配列において、１又は複数個の塩基が置換、欠失、及び／又は付加された塩基配列を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ；
（Ｉ）配列番号１、５、７、９、１１、１３、１５、１７、１９又は２１に示す塩基配列がコードするポリペプチドと５５％以上の配列同一性を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ；
（Ｊ）配列番号２３、２５、２７、２９、３１、３３、３５、３７、３９、４１、４３、４５、４７、４９、５１、５５、６７、６９、７１、７３、７５、７９、８１、８３、８５、８７、８９、９１、９３、９５、９７、９９、１０１、１０３、１０５、１０７又は１０９に示す塩基配列がコードするポリペプチドと５０％以上の配列同一性を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ；
（Ｋ）配列番号１１１、１１３、１１５、１１９、１２１、１２３、１２５、１２７、１２９、１３１又は１３３に示す塩基配列がコードするポリペプチドと５８％以上の配列同一性を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ；
（Ｌ）連続したアミノ酸配列のモチーフである「ＨＧＷＨＷＤＤ」；と、「ＨＥＴＭＥＸＬＦ」、「ＨＥＴＭＤＸＬＷ」、「ＨＥＴＭＥＸＬＷ」、「ＨＥＴＮＤＸＬＦ」及び「ＨＥＴＮＮＸＬＦ」よりなる群から選ばれるいずれかの連続したアミノ酸配列のモチーフ；とを有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ（ここで、Ｘは、Ａ、Ｒ、Ｎ、Ｄ、Ｃ、Ｑ、Ｅ、Ｇ、Ｈ、Ｉ、Ｌ、Ｋ、Ｍ、Ｆ、Ｐ、Ｓ、Ｔ、Ｗ、Ｙ又はＶのいずれかのアミノ酸を示す）。
［７］
　以下の（Ａ）、（Ｂ）、（Ｃ）、（Ｄ）、（Ｅ）又は（Ｆ）に示すポリペプチド又はそれを含む細胞、前記細胞の調製物若しくは前記細胞を培養して得られた培養液を含み、Ｌ－リジン５位水酸化活性を有する酵素剤組成物：
（Ａ）配列番号２、４、６、８、１０、１２、１４、１６、１８、２０、２２、２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８、１１０、１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列を有するポリペプチド；
（Ｂ）配列番号２、４、６、８、１０、１２、１４、１６、１８、２０、２２、２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８、１１０、１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換及び／又は付加されたアミノ酸配列を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチド；
（Ｃ）配列番号２、６、８、１０、１２、１４、１６、１８、２０又は２２に示すアミノ酸配列全長と５５％以上の配列同一性を有するアミノ酸配列を有し、かつ、Ｌ－リジン５位水酸化酵素活性を有するポリペプチド；
（Ｄ）配列番号２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８又は１１０に示すアミノ酸配列全長と５０％以上の配列同一性を有するアミノ酸配列を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチド；
（Ｅ）配列番号１１２，１１４，１１６，１２０，１２２，１２４，１２６，１２８，１３０，１３２又は１３４に示すアミノ酸配列と５８％以上の同一性を有するアミノ酸配列を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチド；
（Ｆ）連続したアミノ酸配列のモチーフである「ＨＧＷＨＷＤＤ」；と、「ＨＥＴＭＥＸＬＦ」、「ＨＥＴＭＤＸＬＷ」「ＨＥＴＭＥＸＬＷ」、「ＨＥＴＮＤＸＬＦ」及び「ＨＥＴＮＮＸＬＦ」よりなる群から選ばれるいずれかの連続したアミノ酸配列のモチーフ；を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチド（ここで、Ｘは、Ａ、Ｒ、Ｎ、Ｄ、Ｃ、Ｑ、Ｅ、Ｇ、Ｈ、Ｉ、Ｌ、Ｋ、Ｍ、Ｆ、Ｐ、Ｓ、Ｔ、Ｗ、Ｙ又はＶのいずれかのアミノ酸を示す）。
［８］
　以下の（Ａ－１）、（Ｂ－１）、（Ｃ－１）又は（Ｆ－１）に示すポリペプチドを含むＳ選択的Ｌ－リジン５位水酸化酵素：
（Ａ－１）配列番号２、６、８、１０、１２、１４、１６、１８、２０又は２２に示すアミノ酸配列を有するポリペプチド；
（Ｂ－１）配列番号２、６、８、１０、１２、１４、１６、１８、２０又は２２に示すアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換及び／又は付加されたアミノ酸配列を有し、かつ、Ｓ選択的Ｌ－リジン５位水酸化活性を有するポリペプチド；
（Ｃ－１）配列番号２、６、８、１０、１２、１４、１６、１８、２０又は２２に示すアミノ酸配列全長と５５％以上の配列同一性を有するアミノ酸配列を有し、かつ、Ｓ選択的Ｌ－リジン５位水酸化酵素活性を有するポリペプチド；
（Ｆ－１）連続したアミノ酸配列のモチーフである「ＨＧＷＨＷＤＤ」及び「ＨＥＴＭＥＸＬＦ」を有し、かつ、Ｓ選択的Ｌ－リジン５位水酸化活性を有するポリペプチド（ここで、Ｘは、Ａ、Ｒ、Ｎ、Ｄ、Ｃ、Ｑ、Ｅ、Ｇ、Ｈ、Ｉ、Ｌ、Ｋ、Ｍ、Ｆ、Ｐ、Ｓ、Ｔ、Ｗ、Ｙ又はＶのいずれかのアミノ酸を示す）。
［９］
　前記のＳ選択的Ｌ－リジン５位水酸化酵素をコードするＤＮＡ。
［１０］
　前記ＤＮＡが、以下の（Ｇ－１）、（Ｈ－１）、（Ｉ－１）又は（Ｌ－１）である、前記のＤＮＡ：
（Ｇ－１）配列番号１、５、７、９、１１、１３、１５、１７、１９又は２１に示す塩基配列を含むＤＮＡ；
（Ｈ－１）配列番号１、５、７、９、１１、１３、１５、１７、１９又は２１に示す塩基配列において、１又は複数個の塩基が置換、欠失、及び／又は付加された塩基配列を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ；
（Ｉ－１）配列番号１、５、７、９、１１、１３、１５、１７、１９又は２１に示す塩基配列がコードするポリペプチドと５５％以上の配列同一性を有し、かつ、Ｓ選択的Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ；
（Ｌ－１）連続したアミノ酸配列のモチーフである「ＨＧＷＨＷＤＤ」及び「ＨＥＴＭＥＸＬＦ」を有し、かつ、Ｓ選択的Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ（ここで、Ｘは、Ａ、Ｒ、Ｎ、Ｄ、Ｃ、Ｑ、Ｅ、Ｇ、Ｈ、Ｉ、Ｌ、Ｋ、Ｍ、Ｆ、Ｐ、Ｓ、Ｔ、Ｗ、Ｙ又はＶのいずれかのアミノ酸を示す）。
［１１］
　Ｌ－リジンに、以下の（Ａ－１）、（Ｂ－１）、（Ｃ－１）又は（Ｆ－１）に示すポリペプチド又はそれを含む細胞、前記細胞の調製物若しくは前記細胞を培養して得られた培養液を接触させて、（２Ｓ，５Ｓ）－５－ヒドロキシ－Ｌ－リジンを生成させることを特徴とする、（２Ｓ，５Ｓ）－５－ヒドロキシ－Ｌ－リジンの製造方法：
（Ａ－１）配列番号２、６、８、１０、１２、１４、１６、１８、２０又は２２に示すアミノ酸配列を有するポリペプチド；
（Ｂ－１）配列番号２、６、８、１０、１２、１４、１６、１８、２０又は２２に示すアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換及び／又は付加されたアミノ酸配列を有し、かつ、Ｓ選択的Ｌ－リジン５位水酸化活性を有するポリペプチド；
（Ｃ－１）配列番号２、６、８、１０、１２、１４、１６、１８、２０又は２２に示すアミノ酸配列全長と５５％以上の配列同一性を有するアミノ酸配列を有し、かつ、Ｓ選択的Ｌ－リジン５位水酸化酵素活性を有するポリペプチド；
（Ｆ－１）連続したアミノ酸配列のモチーフである「ＨＧＷＨＷＤＤ」及び「ＨＥＴＭＥＸＬＦ」を有し、かつ、Ｓ選択的Ｌ－リジン５位水酸化活性を保持するポリペプチド（ここで、Ｘは、Ａ、Ｒ、Ｎ、Ｄ、Ｃ、Ｑ、Ｅ、Ｇ、Ｈ、Ｉ、Ｌ、Ｋ、Ｍ、Ｆ、Ｐ、Ｓ、Ｔ、Ｗ、Ｙ又はＶのいずれかのアミノ酸を示す）。
［１２］
　前記細胞が、前記Ｌ－リジン５位水酸化酵素をコードするＤＮＡで形質転換された細胞である、前記の（２Ｓ，５Ｓ）－５－ヒドロキシ－Ｌ－リジンの製造方法。
［１３］
　前記ＤＮＡが、以下の（Ｇ－１）、（Ｈ－１）、（Ｉ－１）又は（Ｌ－１）である、前記の（２Ｓ，５Ｓ）－５－ヒドロキシ－Ｌ－リジンの製造方法：
（Ｇ－１）配列番号１、５、７、９、１１、１３、１５、１７、１９又は２１に示す塩基配列を含むＤＮＡ；
（Ｈ－１）配列番号１、５、７、９、１１、１３、１５、１７、１９又は２１に示す塩基配列において、１又は複数個の塩基が置換、欠失、及び／又は付加された塩基配列を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ；
（Ｉ－１）配列番号１、５、７、９、１１、１３、１５、１７、１９又は２１に示す塩基配列がコードするポリペプチドと５５％以上の配列同一性を有し、かつ、Ｓ選択的Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ；
（Ｌ－１）連続したアミノ酸配列のモチーフである「ＨＧＷＨＷＤＤ」及び「ＨＥＴＭＥＸＬＦ」を有し、かつ、Ｓ選択的Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ（ここで、Ｘは、Ａ、Ｒ、Ｎ、Ｄ、Ｃ、Ｑ、Ｅ、Ｇ、Ｈ、Ｉ、Ｌ、Ｋ、Ｍ、Ｆ、Ｐ、Ｓ、Ｔ、Ｗ、Ｙ又はＶのいずれかのアミノ酸を示す）。
［１４］
　以下の（Ａ－１）、（Ｂ－１）、（Ｃ－１）又は（Ｆ－１）に示すポリペプチド又はそれを含む細胞、前記細胞の調製物若しくは前記細胞を培養して得られた培養液を含み、かつ、Ｓ選択的Ｌ－リジン５位水酸化活性を有する酵素剤組成物：
（Ａ－１）配列番号２、６、８、１０、１２、１４、１６、１８、２０又は２２に示すアミノ酸配列を有するポリペプチド；
（Ｂ－１）配列番号２、６、８、１０、１２、１４、１６、１８、２０又は２２に示すアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換及び／又は付加されたアミノ酸配列を有し、かつ、Ｓ選択的Ｌ－リジン５位水酸化活性を有するポリペプチド；
（Ｃ－１）配列番号２、６、８、１０、１２、１４、１６、１８、２０又は２２に示すアミノ酸配列全長と５５％以上の配列同一性を有するアミノ酸配列を有し、かつ、Ｓ選択的Ｌ－リジン５位水酸化酵素活性を有するポリペプチド；
（Ｆ－１）連続したアミノ酸配列のモチーフである「ＨＧＷＨＷＤＤ」及び「ＨＥＴＭＥＸＬＦ」を有し、かつ、Ｓ選択的Ｌ－リジン５位水酸化活性を保持するポリペプチド（ここで、Ｘは、Ａ、Ｒ、Ｎ、Ｄ、Ｃ、Ｑ、Ｅ、Ｇ、Ｈ、Ｉ、Ｌ、Ｋ、Ｍ、Ｆ、Ｐ、Ｓ、Ｔ、Ｗ、Ｙ又はＶのいずれかのアミノ酸を示す）。
［１５］
　以下の（Ａ－２）、（Ｂ－２）、（Ｄ－２）、（Ｅ－２）又は（Ｆ－２）に示すポリペプチドを含むＲ選択的Ｌ－リジン５位水酸化酵素：
（Ａ－２）配列番号４、２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８、１１０、１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列を有するポリペプチド；
（Ｂ－２）配列番号４、２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８、１１０、１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換及び／又は付加されたアミノ酸配列を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチド；
（Ｄ－２）配列番号２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８又は１１０に示すアミノ酸配列全長と５０％以上の配列同一性を有するアミノ酸配列を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチド；
（Ｅ－２）配列番号１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列全長と５８％以上の配列同一性を有するアミノ酸配列を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチド；（Ｆ－２）連続したアミノ酸配列のモチーフである「ＨＧＷＨＷＤＤ」；と、「ＨＥＴＭＤＸＬＷ」、「ＨＥＴＭＥＸＬＷ」、「ＨＥＴＮＤＸＬＦ」及び「ＨＥＴＮＮＸＬＦ」よりなる群から選ばれるいずれかの連続したアミノ酸配列のモチーフ；を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を保持するポリペプチド（ここで、Ｘは、Ａ、Ｒ、Ｎ、Ｄ、Ｃ、Ｑ、Ｅ、Ｇ、Ｈ、Ｉ、Ｌ、Ｋ、Ｍ、Ｆ、Ｐ、Ｓ、Ｔ、Ｗ、Ｙ又はＶのいずれかのアミノ酸を示す）。
［１６］
　前記のＲ選択的Ｌ－リジン５位水酸化酵素をコードするＤＮＡ。
［１７］
　前記ＤＮＡが、以下の（Ｇ－２）、（Ｈ－２）、（Ｊ－２）、（Ｋ－２）又は（Ｌ－２）である、前記のＤＮＡ：
（Ｇ－２）配列番号３、２３、２５、２７、２９、３１、３３、３５、３７、３９、４１、４３、４５、４７、４９、５１、５５、６７、６９、７１、７３、７５、７９、８１、８３、８５、８７、８９、９１、９３、９５、９７、９９、１０１、１０３、１０５、１０７、１０９、１１１、１１３、１１５、１１９、１２１、１２３、１２５、１２７、１２９、１３１又は１３３に示す塩基配列を含むＤＮＡ；
（Ｈ－２）配列番号３、２３、２５、２７、２９、３１、３３、３５、３７、３９、４１、４３、４５、４７、４９、５１、５５、６７、６９、７１、７３、７５、７９、８１、８３、８５、８７、８９、９１、９３、９５、９７、９９、１０１、１０３、１０５、１０７、１０９、１１１、１１３、１１５、１１９、１２１、１２３、１２５、１２７、１２９、１３１又は１３３に示す塩基配列において、１又は複数個の塩基が置換、欠失、及び／又は付加された塩基配列を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ；
（Ｊ－２）配列番号２３、２５、２７、２９、３１、３３、３５、３７、３９、４１、４３、４５、４７、４９、５１、５５、６７、６９、７１、７３、７５、７９、８１、８３、８５、８７、８９、９１、９３、９５、９７、９９、１０１、１０３、１０５、１０７又は１０９に示す塩基配列がコードするポリペプチドと５０％以上の配列同一性を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ；
（Ｋ－２）配列番号１１１、１１３、１１５、１１９、１２１、１２３、１２５、１２７、１２９、１３１又は１３３に示す塩基配列がコードするポリペプチドと５８％以上の配列同一性を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ；
（Ｌ－２）連続したアミノ酸配列のモチーフである「ＨＧＷＨＷＤＤ」；と、「ＨＥＴＭＤＸＬＷ」、「ＨＥＴＭＥＸＬＷ」、「ＨＥＴＮＤＸＬＦ」及び「ＨＥＴＮＮＸＬＦ」よりなる群から選ばれるいずれかの連続したアミノ酸配列のモチーフ；を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ（ここで、Ｘは、Ａ、Ｒ、Ｎ、Ｄ、Ｃ、Ｑ、Ｅ、Ｇ、Ｈ、Ｉ、Ｌ、Ｋ、Ｍ、Ｆ、Ｐ、Ｓ、Ｔ、Ｗ、Ｙ又はＶのいずれかのアミノ酸を示す）。
［１８］
　Ｌ－リジンに、以下の（Ａ－２）、（Ｂ－２）、（Ｄ－２）、（Ｅ－２）又は（Ｆ－２）に示すポリペプチド又はそれを含む細胞、前記細胞の調製物若しくは前記細胞を培養して得られた培養液を接触させて、（２Ｓ，５Ｒ）－５－ヒドロキシ－Ｌ－リジンを生成させることを特徴とする、（２Ｓ，５Ｒ）－５－ヒドロキシ－Ｌ－リジンの製造方法：
（Ａ－２）配列番号４、２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８、１１０、１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列を有するポリペプチド；
（Ｂ－２）配列番号４、２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８、１１０、１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換及び／又は付加されたアミノ酸配列を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチド；
（Ｄ－２）配列番号２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８又は１１０に示すアミノ酸配列全長と５０％以上の配列同一性を有するアミノ酸配列を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチド；
（Ｅ－２）配列番号１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列全長と５８％以上の配列同一性を有するアミノ酸配列を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチド；（Ｆ－２）連続したアミノ酸配列のモチーフである「ＨＧＷＨＷＤＤ」；と、「ＨＥＴＭＤＸＬＷ」、「ＨＥＴＭＥＸＬＷ」、「ＨＥＴＮＤＸＬＦ」及び「ＨＥＴＮＮＸＬＦ」よりなる群から選ばれるいずれかの連続したアミノ酸配列のモチーフ；を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を保持するポリペプチド（ここで、Ｘは、Ａ、Ｒ、Ｎ、Ｄ、Ｃ、Ｑ、Ｅ、Ｇ、Ｈ、Ｉ、Ｌ、Ｋ、Ｍ、Ｆ、Ｐ、Ｓ、Ｔ、Ｗ、Ｙ又はＶのいずれかのアミノ酸を示す）。
［１９］
　前記細胞が、前記Ｌ－リジン５位水酸化酵素をコードするＤＮＡで形質転換された細胞である、前記の（２Ｓ，５Ｒ）－５－ヒドロキシ－Ｌ－リジンの製造方法。
［２０］
　前記ＤＮＡが、以下の（Ｇ－２）、（Ｈ－２）、（Ｊ－２）、（Ｋ－２）又は（Ｌ－２）である、前記の（２Ｓ，５Ｒ）－５－ヒドロキシ－Ｌ－リジンの製造方法：
（Ｇ－２）配列番号３、２３、２５、２７、２９、３１、３３、３５、３７、３９、４１、４３、４５、４７、４９、５１、５５、６７、６９、７１、７３、７５、７９、８１、８３、８５、８７、８９、９１、９３、９５、９７、９９、１０１、１０３、１０５、１０７、１０９、１１１、１１３、１１５、１１９、１２１、１２３、１２５、１２７、１２９、１３１又は１３３に示す塩基配列を含むＤＮＡ；
（Ｈ－２）配列番号３、２３、２５、２７、２９、３１、３３、３５、３７、３９、４１、４３、４５、４７、４９、５１、５５、６７、６９、７１、７３、７５、７９、８１、８３、８５、８７、８９、９１、９３、９５、９７、９９、１０１、１０３、１０５、１０７、１０９、１１１、１１３、１１５、１１９、１２１、１２３、１２５、１２７、１２９、１３１又は１３３に示す塩基配列において、１又は複数個の塩基が置換、欠失、及び／又は付加された塩基配列を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ；
（Ｊ－２）配列番号２３、２５、２７、２９、３１、３３、３５、３７、３９、４１、４３、４５、４７、４９、５１、５５、６７、６９、７１、７３、７５、７９、８１、８３、８５、８７、８９、９１、９３、９５、９７、９９、１０１、１０３、１０５、１０７又は１０９に示す塩基配列がコードするポリペプチドと５０％以上の配列同一性を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ；
（Ｋ－２）配列番号１１１、１１３、１１５、１１９、１２１、１２３、１２５、１２７、１２９、１３１又は１３３に示す塩基配列がコードするポリペプチドと５８％以上の配列同一性を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ；
（Ｌ－２）連続したアミノ酸配列のモチーフである「ＨＧＷＨＷＤＤ」；と、「ＨＥＴＭＤＸＬＷ」、「ＨＥＴＭＥＸＬＷ」、「ＨＥＴＮＤＸＬＦ」及び「ＨＥＴＮＮＸＬＦ」よりなる群から選ばれるいずれかの連続したアミノ酸配列のモチーフ；を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ（ここで、Ｘは、Ａ、Ｒ、Ｎ、Ｄ、Ｃ、Ｑ、Ｅ、Ｇ、Ｈ、Ｉ、Ｌ、Ｋ、Ｍ、Ｆ、Ｐ、Ｓ、Ｔ、Ｗ、Ｙ又はＶのいずれかのアミノ酸を示す）。
［２１］
　以下の（Ａ－２）、（Ｂ－２）、（Ｄ－２）、（Ｅ－２）又は（Ｆ－２）に示すポリペプチド又はそれを含む細胞、前記細胞の調製物若しくは前記細胞を培養して得られた培養液を含み、Ｒ選択的Ｌ－リジン５位水酸化活性を有する酵素剤組成物：
（Ａ－２）配列番号４、２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８、１１０、１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列を有するポリペプチド；
（Ｂ－２）配列番号４、２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８、１１０、１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換及び／又は付加されたアミノ酸配列を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチド；
（Ｄ－２）配列番号２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８又は１１０に示すアミノ酸配列全長と５０％以上の配列同一性を有するアミノ酸配列を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチド；
（Ｅ－２）配列番号１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列全長と５８％以上の配列同一性を有するアミノ酸配列を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチド；（Ｆ－２）連続したアミノ酸配列のモチーフである「ＨＧＷＨＷＤＤ」と；「ＨＥＴＭＤＸＬＷ」、「ＨＥＴＭＥＸＬＷ」、「ＨＥＴＮＤＸＬＦ」及び「ＨＥＴＮＮＸＬＦ」よりなる群から選ばれるいずれかの連続したアミノ酸配列のモチーフと；を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を保持するポリペプチド（ここで、Ｘは、Ａ、Ｒ、Ｎ、Ｄ、Ｃ、Ｑ、Ｅ、Ｇ、Ｈ、Ｉ、Ｌ、Ｋ、Ｍ、Ｆ、Ｐ、Ｓ、Ｔ、Ｗ、Ｙ又はＶのいずれかのアミノ酸を示す）。 [1]
An L-lysine 5-hydroxylase comprising a polypeptide shown in (A), (B), (C), (D), (E) or (F) below:
(A) a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134;
(B) a polypeptide having an amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134 in which one or more amino acids have been deleted, substituted and/or added, and having L-lysine 5-hydroxylation activity;
(C) a polypeptide having an amino acid sequence having 55% or more sequence identity to the full-length amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20, or 22, and having L-lysine 5-hydroxylase activity;
(D) a polypeptide having an amino acid sequence having 50% or more sequence identity to the full-length amino acid sequence shown in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110, and having L-lysine 5-hydroxylation activity;
(E) a polypeptide having an amino acid sequence having 58% or more sequence identity to the full-length amino acid sequence shown in SEQ ID NO: 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134, and having L-lysine 5-hydroxylation activity;
(F) A polypeptide having a consecutive amino acid sequence motif of "HGWHWDD" and any consecutive amino acid sequence motif selected from the group consisting of "HETMEXLF", "HETMDXLW", "HETMEXLW", "HETNDXLF" and "HETNNXLF", and having L-lysine 5-hydroxylation activity (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V).
[2]
A DNA encoding the L-lysine 5-hydroxylase.
[3]
The DNA is the following (G), (H), (I), (J), (K) or (L):
(G) DNA containing the base sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 57, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133;
(H) a DNA encoding a polypeptide having an L-lysine 5-hydroxylating activity, comprising a nucleotide sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 57, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133 in which one or more nucleotides have been substituted, deleted, and/or added;
(I) a DNA encoding a polypeptide having a sequence identity of 55% or more to a polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, or 21 and having an activity of hydroxylating L-lysine at the 5-position;
(J) a DNA encoding a polypeptide having 50% or more sequence identity to a polypeptide encoded by the base sequence shown in SEQ ID NO: 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107 or 109, and having an activity of hydroxylating L-lysine at position 5;
(K) a DNA encoding a polypeptide having a sequence identity of 58% or more to a polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133 and having L-lysine 5-hydroxylation activity;
(L) A DNA encoding a polypeptide having a consecutive amino acid sequence motif of "HGWHWDD" and any consecutive amino acid sequence motif selected from the group consisting of "HETMEXLF", "HETMDXLW", "HETMEXLW", "HETNDXLF" and "HETNNXLF" and having L-lysine 5-hydroxylation activity (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V).
[4]
A method for producing 5-hydroxy-L-lysine, comprising contacting L-lysine with a polypeptide shown in the following (A), (B), (C), (D), (E) or (F), or a cell containing the polypeptide, a preparation of the cell, or a culture medium obtained by culturing the cell, to produce 5-hydroxy-L-lysine:
(A) a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134;
(B) a polypeptide having an amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134 in which one or more amino acids have been deleted, substituted and/or added, and having L-lysine 5-hydroxylation activity;
(C) a polypeptide having an amino acid sequence having 55% or more sequence identity to the full-length amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20, or 22, and having L-lysine 5-hydroxylase activity;
(D) a polypeptide having an amino acid sequence having 50% or more sequence identity to the full-length amino acid sequence shown in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110, and having L-lysine 5-hydroxylation activity;
(E) a polypeptide having an amino acid sequence having 58% or more identity to the amino acid sequence shown in SEQ ID NO: 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134, and having L-lysine 5-hydroxylation activity;
(F) A polypeptide having a consecutive amino acid sequence motif of "HGWHWDD" and any consecutive amino acid sequence motif selected from the group consisting of "HETMEXLF", "HETMDXLW", "HETMEXLW", "HETNDXLF" and "HETNNXLF", and having L-lysine 5-hydroxylation activity (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V).
[5]
The method for producing 5-hydroxy-L-lysine as described above, wherein the cell is a cell transformed with DNA encoding the L-lysine 5-hydroxylase.
[6]
The method for producing 5-hydroxy-L-lysine as described above, wherein the DNA is the following (G), (H), (I), (J), (K) or (L):
(G) DNA containing the base sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 57, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133;
(H) a DNA encoding a polypeptide having an L-lysine 5-hydroxylating activity, comprising a nucleotide sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 57, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133 in which one or more nucleotides have been substituted, deleted, and/or added;
(I) a DNA encoding a polypeptide having a sequence identity of 55% or more to a polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, or 21 and having an activity of hydroxylating L-lysine at the 5-position;
(J) a DNA encoding a polypeptide having 50% or more sequence identity to a polypeptide encoded by the base sequence shown in SEQ ID NO: 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107 or 109, and having an activity of hydroxylating L-lysine at position 5;
(K) a DNA encoding a polypeptide having a sequence identity of 58% or more to a polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133 and having L-lysine 5-hydroxylation activity;
(L) A DNA encoding a polypeptide having a consecutive amino acid sequence motif "HGWHWDD" and any consecutive amino acid sequence motif selected from the group consisting of "HETMEXLF", "HETMDXLW", "HETMEXLW", "HETNDXLF" and "HETNNXLF", and having L-lysine 5-hydroxylation activity (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V).
[7]
An enzyme composition having L-lysine 5-hydroxylation activity, comprising a polypeptide represented by the following (A), (B), (C), (D), (E) or (F), or a cell containing the same, a preparation of the cell, or a culture medium obtained by culturing the cell:
(A) a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134;
(B) a polypeptide having an amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134 in which one or more amino acids have been deleted, substituted and/or added, and having L-lysine 5-hydroxylation activity;
(C) a polypeptide having an amino acid sequence having 55% or more sequence identity to the full-length amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20, or 22, and having L-lysine 5-hydroxylase activity;
(D) a polypeptide having an amino acid sequence having 50% or more sequence identity to the full-length amino acid sequence shown in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110, and having L-lysine 5-hydroxylation activity;
(E) a polypeptide having an amino acid sequence having 58% or more identity to the amino acid sequence shown in SEQ ID NO: 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134, and having L-lysine 5-hydroxylation activity;
(F) A polypeptide having a consecutive amino acid sequence motif of "HGWHWDD" and any consecutive amino acid sequence motif selected from the group consisting of "HETMEXLF", "HETMDXLW", "HETMEXLW", "HETNDXLF" and "HETNNXLF", and having L-lysine 5-hydroxylation activity (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V).
[8]
An S-selective L-lysine 5-hydroxylase comprising a polypeptide shown in (A-1), (B-1), (C-1) or (F-1) below:
(A-1) a polypeptide having an amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22;
(B-1) a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted and/or added in the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22, and having S-selective L-lysine 5-hydroxylation activity;
(C-1) a polypeptide having an amino acid sequence having 55% or more sequence identity to the full-length amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20, or 22, and having S-selective L-lysine 5-hydroxylase activity;
(F-1) A polypeptide having consecutive amino acid sequence motifs of "HGWHWDD" and "HETMEXLF" and having S-selective L-lysine 5-hydroxylation activity (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V).
[9]
A DNA encoding the S-selective L-lysine 5-hydroxylase.
[10]
The DNA is the following (G-1), (H-1), (I-1) or (L-1):
(G-1) DNA comprising the nucleotide sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, or 21;
(H-1) DNA encoding a polypeptide having a nucleotide sequence in which one or more nucleotides have been substituted, deleted, and/or added in the nucleotide sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, or 21 and having an activity of hydroxylating L-lysine at the 5-position;
(I-1) DNA encoding a polypeptide having 55% or more sequence identity to a polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, or 21 and having S-selective L-lysine 5-hydroxylation activity;
(L-1) A DNA encoding a polypeptide having consecutive amino acid sequence motifs "HGWHWDD" and "HETMEXLF" and having S-selective L-lysine 5-hydroxylation activity (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V).
[11]
A method for producing (2S,5S)-5-hydroxy-L-lysine, comprising contacting L-lysine with a polypeptide shown in the following (A-1), (B-1), (C-1) or (F-1), or a cell containing the polypeptide, a preparation of the cell, or a culture solution obtained by culturing the cell, to produce (2S,5S)-5-hydroxy-L-lysine:
(A-1) a polypeptide having an amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22;
(B-1) a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted and/or added in the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22, and having S-selective L-lysine 5-hydroxylation activity;
(C-1) a polypeptide having an amino acid sequence having 55% or more sequence identity to the full-length amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20, or 22, and having S-selective L-lysine 5-hydroxylase activity;
(F-1) A polypeptide having consecutive amino acid sequence motifs "HGWHWDD" and "HETMEXLF" and having S-selective L-lysine 5-hydroxylation activity (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V).
[12]
The method for producing (2S,5S)-5-hydroxy-L-lysine as described above, wherein the cell is a cell transformed with DNA encoding the L-lysine 5-hydroxylase.
[13]
The method for producing (2S,5S)-5-hydroxy-L-lysine, wherein the DNA is the following (G-1), (H-1), (I-1) or (L-1):
(G-1) DNA comprising the nucleotide sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, or 21;
(H-1) DNA encoding a polypeptide having a nucleotide sequence in which one or more nucleotides have been substituted, deleted, and/or added in the nucleotide sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, or 21 and having an activity of hydroxylating L-lysine at the 5-position;
(I-1) DNA encoding a polypeptide having 55% or more sequence identity to a polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, or 21 and having S-selective L-lysine 5-hydroxylation activity;
(L-1) A DNA encoding a polypeptide having consecutive amino acid sequence motifs "HGWHWDD" and "HETMEXLF" and having S-selective L-lysine 5-hydroxylation activity (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V).
[14]
An enzyme composition comprising a polypeptide shown in (A-1), (B-1), (C-1) or (F-1) below, a cell containing the polypeptide, a preparation of the cell, or a culture medium obtained by culturing the cell, and having S-selective L-lysine 5-hydroxylation activity:
(A-1) a polypeptide having an amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22;
(B-1) a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted and/or added in the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22, and having S-selective L-lysine 5-hydroxylation activity;
(C-1) a polypeptide having an amino acid sequence having 55% or more sequence identity to the full-length amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20, or 22, and having S-selective L-lysine 5-hydroxylase activity;
(F-1) A polypeptide having consecutive amino acid sequence motifs "HGWHWDD" and "HETMEXLF" and having S-selective L-lysine 5-hydroxylation activity (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V).
[15]
An R-selective L-lysine 5-hydroxylase comprising a polypeptide shown in (A-2), (B-2), (D-2), (E-2) or (F-2) below:
(A-2) a polypeptide having an amino acid sequence shown in SEQ ID NO: 4, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134;
(B-2) a polypeptide having an amino acid sequence represented by SEQ ID NO: 4, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134 in which one or more amino acids have been deleted, substituted, and/or added, and having R-selective L-lysine 5-hydroxylation activity;
(D-2) a polypeptide having an amino acid sequence having 50% or more sequence identity to the full length amino acid sequence shown in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110, and having R-selective L-lysine 5-hydroxylation activity;
(E-2) A polypeptide having an amino acid sequence having 58% or more sequence identity with the full length amino acid sequence shown in SEQ ID NO: 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134, and having R-selective L-lysine 5-position hydroxylation activity; (F-2) A polypeptide having a motif of a consecutive amino acid sequence "HGWHWDD"; and a motif of any consecutive amino acid sequence selected from the group consisting of "HETMDXLW", "HETMEXLW", "HETNDXLF" and "HETNNXLF"; and having R-selective L-lysine 5-position hydroxylation activity (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V).
[16]
A DNA encoding the R-selective L-lysine 5-hydroxylase.
[17]
The DNA is the following (G-2), (H-2), (J-2), (K-2) or (L-2):
(G-2) DNA comprising the base sequence shown in SEQ ID NO: 3, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133;
(H-2) DNA encoding a polypeptide having an R-selective L-lysine 5-hydroxylation activity, comprising a nucleotide sequence represented by SEQ ID NO: 3, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133, in which one or more nucleotides have been substituted, deleted and/or added;
(J-2) a DNA encoding a polypeptide having 50% or more sequence identity to a polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107 or 109, and having R-selective L-lysine 5-hydroxylation activity;
(K-2) a DNA having a sequence identity of 58% or more to a polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133, and encoding a polypeptide having R-selective L-lysine 5-hydroxylation activity;
(L-2) A DNA encoding a polypeptide having a consecutive amino acid sequence motif of "HGWHWDD" and any consecutive amino acid sequence motif selected from the group consisting of "HETMDXLW", "HETMEXLW", "HETNDXLF" and "HETNNXLF" and having R-selective L-lysine 5-hydroxylation activity (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V).
[18]
A method for producing (2S,5R)-5-hydroxy-L-lysine, comprising contacting L-lysine with a polypeptide shown in the following (A-2), (B-2), (D-2), (E-2) or (F-2), or a cell containing the same, a preparation of the cell, or a culture solution obtained by culturing the cell, to produce (2S,5R)-5-hydroxy-L-lysine:
(A-2) a polypeptide having an amino acid sequence shown in SEQ ID NO: 4, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134;
(B-2) a polypeptide having an amino acid sequence represented by SEQ ID NO: 4, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134 in which one or more amino acids have been deleted, substituted, and/or added, and having R-selective L-lysine 5-hydroxylation activity;
(D-2) a polypeptide having an amino acid sequence having 50% or more sequence identity to the full length amino acid sequence shown in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110, and having R-selective L-lysine 5-hydroxylation activity;
(E-2) A polypeptide having an amino acid sequence having 58% or more sequence identity with the full length amino acid sequence shown in SEQ ID NO: 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134, and having R-selective L-lysine 5-position hydroxylation activity; (F-2) A polypeptide having a motif of a consecutive amino acid sequence "HGWHWDD"; and a motif of any consecutive amino acid sequence selected from the group consisting of "HETMDXLW", "HETMEXLW", "HETNDXLF" and "HETNNXLF"; and having R-selective L-lysine 5-position hydroxylation activity (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V).
[19]
The method for producing (2S,5R)-5-hydroxy-L-lysine as described above, wherein the cell is a cell transformed with DNA encoding the L-lysine 5-hydroxylase.
[20]
The method for producing (2S,5R)-5-hydroxy-L-lysine, wherein the DNA is the following (G-2), (H-2), (J-2), (K-2) or (L-2):
(G-2) DNA comprising the base sequence shown in SEQ ID NO: 3, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133;
(H-2) DNA encoding a polypeptide having an R-selective L-lysine 5-hydroxylation activity, comprising a nucleotide sequence represented by SEQ ID NO: 3, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133, in which one or more nucleotides have been substituted, deleted and/or added;
(J-2) a DNA encoding a polypeptide having 50% or more sequence identity to a polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107 or 109, and having R-selective L-lysine 5-hydroxylation activity;
(K-2) a DNA having a sequence identity of 58% or more to a polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133, and encoding a polypeptide having R-selective L-lysine 5-hydroxylation activity;
(L-2) A DNA encoding a polypeptide having a consecutive amino acid sequence motif of "HGWHWDD" and any consecutive amino acid sequence motif selected from the group consisting of "HETMDXLW", "HETMEXLW", "HETNDXLF" and "HETNNXLF" and having R-selective L-lysine 5-hydroxylation activity (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V).
[21]
An enzyme composition comprising a polypeptide shown in the following (A-2), (B-2), (D-2), (E-2) or (F-2), or a cell containing the same, a preparation of the cell, or a culture medium obtained by culturing the cell, and having R-selective L-lysine 5-hydroxylation activity:
(A-2) a polypeptide having an amino acid sequence shown in SEQ ID NO: 4, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134;
(B-2) a polypeptide having an amino acid sequence represented by SEQ ID NO: 4, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134 in which one or more amino acids have been deleted, substituted, and/or added, and having R-selective L-lysine 5-hydroxylation activity;
(D-2) a polypeptide having an amino acid sequence having 50% or more sequence identity to the full length amino acid sequence shown in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110, and having R-selective L-lysine 5-hydroxylation activity;
(E-2) A polypeptide having an amino acid sequence having 58% or more sequence identity with the full length amino acid sequence shown in SEQ ID NO: 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134, and having R-selective L-lysine 5-position hydroxylation activity; (F-2) A polypeptide having a consecutive amino acid sequence motif "HGWHWDD" and any consecutive amino acid sequence motif selected from the group consisting of "HETMDXLW", "HETMEXLW", "HETNDXLF" and "HETNNXLF" and having R-selective L-lysine 5-position hydroxylation activity (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V).

　なお、本発明は、以下の構成を採用することも可能である。
［２２］
　Ｌ－リジンに、前記の（Ａ）、（Ｂ）、（Ｃ）、（Ｄ）、（Ｅ）又は（Ｆ）に示すポリペプチド又はそれを含む細胞、前記細胞の調製物若しくは前記細胞を培養して得られた培養液を接触させて、Ｌ－リジンの５位に対して水酸化させることを特徴とする、Ｌ－リジンを水酸化する方法。
［２３］
　前記の（Ａ）、（Ｂ）、（Ｃ）、（Ｄ）、（Ｅ）又は（Ｆ）に示すポリペプチド又はそれを含む細胞、前記細胞の調製物若しくは前記細胞を培養して得られた培養液の、Ｌ－リジン５位水酸化酵素剤組成物としての使用。
［２４］
　前記の（Ａ）、（Ｂ）、（Ｃ）、（Ｄ）、（Ｅ）又は（Ｆ）に示すポリペプチド又はそれを含む細胞、前記細胞の調製物若しくは前記細胞を培養して得られた培養液の、Ｌ－リジン５位水酸化酵素剤組成物の製造における使用。 The present invention can also employ the following configuration.
[22]
A method for hydroxylating L-lysine, comprising contacting L-lysine with a polypeptide selected from (A), (B), (C), (D), (E) or (F) or a cell containing the same, a preparation of the cell, or a culture medium obtained by culturing the cell, to hydroxylate the 5-position of L-lysine.
[23]
Use of a polypeptide as defined in (A), (B), (C), (D), (E) or (F) above, or a cell containing the polypeptide, a preparation of the cell, or a culture medium obtained by culturing the cell, as an L-lysine 5-hydroxylase composition.
[24]
Use of a polypeptide as defined in (A), (B), (C), (D), (E) or (F), or a cell containing the polypeptide, a preparation of the cell, or a culture medium obtained by culturing the cell, in the production of an L-lysine 5-hydroxylase composition.

　本発明によれば、Ｌ－リジン５位水酸化酵素及び５－ヒドロキシ－Ｌ－リジン生成用酵素剤組成物を提供することができる。また、本発明によれば、Ｌ－リジンから高収率かつ低コストで安価に５－ヒドロキシ－Ｌ－リジンを製造する方法を提供することができる。さらに、Ｌ－リジン５位水酸化酵素を使い分けることにより、所望の（２Ｓ，５Ｓ）－５－ヒドロキシ－Ｌ－リジン又は（２Ｓ，５Ｒ）－５－ヒドロキシ－Ｌ－リジンを製造する方法を提供することができる。 According to the present invention, it is possible to provide an L-lysine 5-hydroxylase and an enzyme composition for producing 5-hydroxy-L-lysine. In addition, according to the present invention, it is possible to provide a method for inexpensively producing 5-hydroxy-L-lysine from L-lysine with high yield and at low cost. Furthermore, it is possible to provide a method for producing the desired (2S,5S)-5-hydroxy-L-lysine or (2S,5R)-5-hydroxy-L-lysine by using different L-lysine 5-hydroxylase enzymes.

ａｚｐ遺伝子クラスターを比較した図である。FIG. 1 is a diagram comparing azp gene clusters. ａｚｐＫ２遺伝子及びＰｐ＿ａｚｐＫ２遺伝子のホモログの系統樹分類を示した図である。FIG. 1 shows phylogenetic tree classification of homologues of the azpK2 gene and the Pp_azpK2 gene. 実施例５における、ＡｚｐＫ２を用いた場合の反応生成物のＬＣ－ＭＳ検出結果を示す図である。FIG. 13 shows the results of LC-MS detection of reaction products when AzpK2 was used in Example 5. 実施例７における、５－ヒドロキシリジン及び５－オキソリジンのＦｍｏｃ化体のＨＰＬＣ分析結果を示す図である。FIG. 1 shows the results of HPLC analysis of Fmoc-substituted 5-hydroxylysine and 5-oxolidine in Example 7. 実施例１０における、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ４－１と酸化酵素を組み合わせた５－オキソリジンの検出結果を示す図である。This shows the detection results of 5-oxolidine in Example 10 using a combination of AzpK2 Homolog4-1 and an oxidase. 実施例１３における、遺伝子組換え大腸菌を用いたＳ選択的ＡｚｐＫ２ホモログの活性評価の結果を示す図である。FIG. 13 shows the results of evaluating the activity of an S-selective AzpK2 homolog using recombinant Escherichia coli in Example 13. 実施例１５における、菌体破砕条件の検討結果を示す図である。FIG. 1 shows the results of investigating cell disruption conditions in Example 15. 実施例１５における、反応液のｐＨ検討結果を示す図である。FIG. 13 shows the results of investigating the pH of the reaction solution in Example 15. 実施例１５における、基質濃度の検討結果を示す図である。FIG. 15 shows the results of investigating the substrate concentration in Example 15. 実施例１５における、基質濃度の検討結果を示す図である。FIG. 15 shows the results of investigating the substrate concentration in Example 15. 実施例１７における、Ｌ－リジン５位水酸化酵素のアミノ酸配列の同一性比較結果を示す図である。FIG. 13 shows the results of identity comparison of amino acid sequences of L-lysine 5-hydroxylase in Example 17. 実施例１７における、Ｌ－リジン５位水酸化酵素のアミノ酸配列の同一性比較結果を示す図である。FIG. 13 shows the results of identity comparison of amino acid sequences of L-lysine 5-hydroxylase in Example 17. 実施例１８における、ＡｚｐＫ２タンパク質結晶のＸ線構造解析によるモデル構築結果を示す図である。FIG. 13 shows the results of model construction by X-ray structure analysis of the AzpK2 protein crystal in Example 18. 実施例１９における、ＡｚｐＫ２とＢｅｓＤのアミノ酸同一性比較結果を示す図である。FIG. 13 shows the results of amino acid identity comparison between AzpK2 and BesD in Example 19. 実施例２０における、ＡｚｐＫ２及びＢｅｓＤのＡｌｐｈａＦｏｌｄ２による立体構造予測結果を示す図である。FIG. 13 shows the results of prediction of the three-dimensional structures of AzpK2 and BesD by AlphaFold2 in Example 20. 実施例２１における、ＡｚｐＫ２アミノ酸置換体の活性評価の結果を示す図である。FIG. 13 shows the results of activity evaluation of amino acid substituted AzpK2 mutants in Example 21. 実施例２２における、ＡｚｐＫ２とＡｚｐＫ２ホモログのアミノ酸１３１～１４５番目に該当するロゴプロットを示す図である。FIG. 23 shows logo plots corresponding to amino acids 131 to 145 of AzpK2 and AzpK2 homologs in Example 22. 実施例２２における、ＡｚｐＫ２とＡｚｐＫ２ホモログのアミノ酸２３１～２４５番目に該当するロゴプロットを示す図である。FIG. 23 shows a logo plot corresponding to amino acids 231 to 245 of AzpK2 and AzpK2 homologs in Example 22.

　以下、本明細書において用いられる用語等について説明する。
　本明細書において「Ｌ－リジン」とはＣＡＳレジストリ番号５６－８７－１のＬ－リジンを意味する。 The terms used in this specification will be explained below.
As used herein, "L-lysine" refers to L-lysine having CAS Registry Number 56-87-1.

　本明細書において、「３位」、「４位」、「５位」とは、Ｌ－リジンの「３位」、「４位」、「５位」の炭素原子の位置のことであり、下記式（ＩＶ）中に示す番号の炭素原子の位置を指す。 In this specification, "position 3", "position 4", and "position 5" refer to the positions of the carbon atoms at positions 3, 4, and 5 of L-lysine, respectively, and refer to the positions of the carbon atoms numbered as shown in formula (IV) below.

　本明細書において、「Ｌ－リジンの５位」とは、前記式（ＩＶ）の番号「５」に位置する炭素原子を表す。 In this specification, "position 5 of L-lysine" refers to the carbon atom numbered "5" in formula (IV) above.

　本明細書において、「５－ヒドロキシ－Ｌ－リジン」とは、「（２Ｓ，５Ｓ）－５－ヒドロキシ－Ｌ－リジン」又は「（２Ｓ，５Ｒ）－５－ヒドロキシ－Ｌ－リジン」のうちのいずれか１つ、又は両方を意味する。 In this specification, "5-hydroxy-L-lysine" means either one or both of "(2S,5S)-5-hydroxy-L-lysine" and "(2S,5R)-5-hydroxy-L-lysine."

　本明細書において、「５－ヒドロキシリジン」とは、「（２Ｓ，５Ｓ）－５－ヒドロキシ－Ｌ－リジン」、「（２Ｓ，５Ｒ）－５－ヒドロキシ－Ｌ－リジン」、「（２Ｒ，５Ｓ）－５－ヒドロキシ－Ｄ－リジン」及び「（２Ｒ，５Ｒ）－５－ヒドロキシ－Ｄ－リジン」よりなる群から選ばれるいずれか１つ、又は２つから４つを意味する。 In this specification, "5-hydroxylysine" means any one or two to four selected from the group consisting of "(2S,5S)-5-hydroxy-L-lysine", "(2S,5R)-5-hydroxy-L-lysine", "(2R,5S)-5-hydroxy-D-lysine" and "(2R,5R)-5-hydroxy-D-lysine".

　本明細書において、「水酸化」とは、化合物中の特定のＣ－Ｈ結合をＣ－ＯＨ結合に変換する反応（化合物中の特定の炭素原子に水酸基を付加する反応）を意味する。 In this specification, "hydroxylation" refers to a reaction that converts a specific C-H bond in a compound to a C-OH bond (a reaction that adds a hydroxyl group to a specific carbon atom in a compound).

　本明細書において、「水酸化活性」とは、水酸化における、タンパク質の単位質量（例えばミリグラム）、乾燥細胞質量、又は固定化触媒質量あたりの触媒活性を意味する。 In this specification, "hydroxylation activity" refers to the catalytic activity in hydroxylation per unit mass of protein (e.g., milligram), dry cell mass, or immobilized catalyst mass.

　本明細書において、「Ｌ－リジン５位水酸化活性」とは、Ｌ－リジンの５位の炭素原子に水酸基を付加する能力を意味する。 In this specification, "L-lysine 5-hydroxylation activity" means the ability to add a hydroxyl group to the carbon atom at position 5 of L-lysine.

　このような活性は、例えば、Ｌ－リジンを基質として含有し、さらに２－オキソグルタル酸を含有する反応系において、触媒として、Ｌ－リジン５位水酸化酵素、又はそれを含む細胞、当該細胞の調製物若しくは当該細胞を培養して得られた培養液を用いて、後述する実施例のように５－ヒドロキシ－Ｌ－リジンの生成を直接的又は間接的に確認することで、確認することができる。 Such activity can be confirmed, for example, by directly or indirectly confirming the production of 5-hydroxy-L-lysine in a reaction system containing L-lysine as a substrate and further containing 2-oxoglutarate, using L-lysine 5-hydroxylase as a catalyst, or cells containing it, a preparation of said cells, or a culture medium obtained by culturing said cells, as in the Examples described below.

　５－ヒドロキシ－Ｌ－リジンを直接的に検出する方法としては、５－ヒドロキシ－Ｌ－リジンをＨＰＬＣに供して分離し、ＵＶ吸収による検出を行う方法が挙げられるが、５－ヒドロキシ－Ｌ－リジンはＵＶ吸収率が低く検出が難しい。また、５－ヒドロキシ－Ｌ－リジンを間接的に検出する方法としては、５－ヒドロキシ－Ｌ－リジンと銅イオンを含む移動相を使用して５－ヒドロキシ－Ｌ－リジンの銅錯体を形成し、ＨＰＬＣに供して銅錯体のＵＶ吸収スペクトルを検出する方法、又は５－ヒドロキシ－Ｌ－リジンのアミノ基に９－フルオレニルメチルオキシカルボニル（以下「Ｆｍｏｃ」と称することがある。）基等のＵＶ吸収のある誘導基を導入し、その誘導体をＨＰＬＣに供して分離し、分離した誘導体をＵＶ吸収法又は質量分析法（ＭＳ）を使用して検出及び定量する方法が挙げられる。 　A method for directly detecting 5-hydroxy-L-lysine includes subjecting 5-hydroxy-L-lysine to HPLC for separation and detection by UV absorption, but 5-hydroxy-L-lysine has low UV absorption and is difficult to detect. Methods for indirectly detecting 5-hydroxy-L-lysine include a method for forming a copper complex of 5-hydroxy-L-lysine using a mobile phase containing 5-hydroxy-L-lysine and copper ions, subjecting the complex to HPLC to detect the UV absorption spectrum of the copper complex, or a method for introducing a UV-absorbing derivative such as a 9-fluorenylmethyloxycarbonyl (hereinafter sometimes referred to as "Fmoc") group into the amino group of 5-hydroxy-L-lysine, subjecting the derivative to HPLC for separation, and detecting and quantifying the separated derivative using UV absorption or mass spectrometry (MS).

　また、５－ヒドロキシ－Ｌ－リジンの５位の水酸基の立体を判別する方法としては、下記式（Ｖ）： The method for determining the configuration of the hydroxyl group at the 5th position of 5-hydroxy-L-lysine is shown below (V):

　に示す反応のように、５－ヒドロキシ－Ｌ－リジン酸化酵素を用いて、（２Ｓ,５Ｓ）－５－ヒドロキシ－Ｌ－リジン又は（２Ｓ,５Ｒ）－５－ヒドロキシ－Ｌ－リジンの水酸基を立体選択的に酸化して５－オキソ－Ｌ－リジンを生成し、その生成の際に５－オキソ－Ｌ－リジンと等モル量生じるＮＡＤＨを検出する方法が挙げられる。この方法によれば、５－ヒドロキシ－Ｌ－リジンの検出及び５－ヒドロキシ－Ｌ－リジンの水酸基の立体の判別が可能である。 As shown in the reaction diagram, 5-hydroxy-L-lysine oxidase is used to stereoselectively oxidize the hydroxyl group of (2S,5S)-5-hydroxy-L-lysine or (2S,5R)-5-hydroxy-L-lysine to produce 5-oxo-L-lysine, and NADH, which is produced in an equimolar amount to 5-oxo-L-lysine during the production, is detected. This method makes it possible to detect 5-hydroxy-L-lysine and distinguish the stereochemistry of the hydroxyl group of 5-hydroxy-L-lysine.

　また、下記式（ＶＩ）： Also, the following formula (VI):

　に示す反応のように、５－ヒドロキシ－Ｌ－リジンから５－ヒドロキシ－Ｌ－ピペコリン酸を生成し、５－ヒドロキシ－Ｌ－ピペコリン酸の５位の水酸基を判別することにより５－ヒドロキシ－Ｌ－リジンの水酸基を決定する方法も挙げられる。５－ヒドロキシ－Ｌ－リジンから５－ヒドロキシ－Ｌ－ピペコリン酸を生成する方法としては、Ｌ－リジン／Ｌ－オルニチンシクロデアミナーゼを用いる方法の他、アミノ酸脱水素酵素とイミン還元酵素等の複数の酸化還元酵素を組み合わせる方法が挙げられる。 As shown in the reaction diagram below, another method is to generate 5-hydroxy-L-pipecolic acid from 5-hydroxy-L-lysine and determine the hydroxyl group at the 5-position of 5-hydroxy-L-pipecolic acid to determine the hydroxyl group of 5-hydroxy-L-lysine. Methods for generating 5-hydroxy-L-pipecolic acid from 5-hydroxy-L-lysine include a method using L-lysine/L-ornithine cyclodeaminase, as well as a method using a combination of multiple oxidoreductases, such as amino acid dehydrogenase and imine reductase.

　本明細書において、「酵素活性の１単位」、「活性の１単位」又は「Ｕ」とは、特定の温度における、１分あたり１μｍｏｌの５－ヒドロキシリジンを生成するのに必要な水酸化活性を意味する。 As used herein, "one unit of enzyme activity," "one unit of activity," or "U" refers to the hydroxylation activity required to produce 1 μmol of 5-hydroxylysine per minute at a particular temperature.

　本明細書で用いるアミノ酸の１文字表記及び３文字表記は、「塩基配列又はアミノ酸配列を含む明細書等の作成のためのガイドライン（ST.26対応）」（特許庁）に従う。具体的には以下のとおりである。 The one-letter and three-letter symbols for amino acids used in this specification follow the "Guidelines for the preparation of specifications, etc., including nucleotide or amino acid sequences (ST.26 compatible)" (Japan Patent Office). Specifically, they are as follows:

　本明細書においては、タンパク質（ポリペプチド）中のアミノ酸残基を表す際に、アミノ酸の３文字表記と数字とを組み合わせた場合はタンパク質中のアミノ酸残基とその残基番号を表す。例えば、「Ｈｉｓ１３９」はタンパク質中の１３９番目のアミノ酸残基であるヒスチジンを表す。また、数字の前後にアミノ酸の１文字表記を記載した場合は、タンパク質中のアミノ酸残基の置換を表すものであり、数字（残基番号）の前に元のアミノ酸残基を、数字（残基番号）の後に置換したアミノ酸残基を記載している。例えば、「Ｒ７６Ａ」はタンパク質中の７６番目のアミノ酸残基であるアルギニンがアラニンに置換していることを表す。 In this specification, when expressing an amino acid residue in a protein (polypeptide), a combination of the three-letter code of an amino acid and a number represents the amino acid residue in the protein and its residue number. For example, "His139" represents histidine, the 139th amino acid residue in a protein. Furthermore, when a one-letter code of an amino acid is written before and after a number, it represents a substitution of an amino acid residue in a protein, with the original amino acid residue written before the number (residue number) and the substituted amino acid residue written after the number (residue number). For example, "R76A" represents the substitution of alanine for arginine, the 76th amino acid residue in a protein.

　本明細書において、「シグネチャーモチーフ」、「アミノ酸モチーフ」及び「モチーフ」は、特定の活性を有する酵素グループ（酵素ファミリー）が有する、共通の保存された構造を意味する。これらのシグネチャーモチーフ等により、特定の基質群に対して類似する酵素活性を有する、構造的に関連する酵素ファミリーを、特徴付けたり同定したりすることができる。これらのシグネチャーモチーフ等は、通常、単一の連続したアミノ酸配列や、隣接しない保存されたモチーフのコレクションとして存在する。当該保存されたモチーフもアミノ酸配列で表すことができる。 As used herein, "signature motif," "amino acid motif," and "motif" refer to a common conserved structure shared by a group of enzymes (enzyme families) with a particular activity. These signature motifs, etc., can be used to characterize or identify a family of structurally related enzymes that have similar enzymatic activity against a particular set of substrates. These signature motifs, etc., typically exist as a single contiguous amino acid sequence or a collection of non-contiguous conserved motifs. The conserved motifs can also be expressed as amino acid sequences.

　本明細書において、「遺伝子クラスター」とは、複数の遺伝子配列が物理的に近接しており、これらの遺伝子配列が、通常、協調して機能することにより、特定の生物学的プロセスや生理学的機能に寄与することができる、遺伝子の集合体を意味する。 As used herein, the term "gene cluster" refers to a collection of genes in which multiple gene sequences are in close physical proximity and typically function in a coordinated manner to contribute to a specific biological process or physiological function.

　この集合体は、通常、遺伝子発現の調節、転写、翻訳等の生物学的プロセスにおいて、相互に作用し、特定の細胞機能や生物学的経路に影響を与えることがあるが、遺伝子クラスターを構成する遺伝子の数、種類、順序等は生物間で異なる場合がある。 These collections typically interact with each other in biological processes such as gene expression regulation, transcription, and translation, and can affect specific cellular functions or biological pathways, but the number, types, and order of genes that make up a gene cluster can vary between organisms.

　本明細書において、「同一性（％）」とは、２種若しくはそれ以上のポリペプチド配列間、又は２種若しくはそれ以上のポリヌクレオチド配列間の関係を示す指標であり、例えば、２種若しくはそれ以上のポリペプチド配列を比較することによって求めることができる。配列同一性とは、ポリペプチド配列またはポリヌクレオチド配列において、２つの配列を最適の態様で整列させた場合に、２つの配列間で共有する一致したアミノ酸又はヌクレオチドの個数の百分率を意味する。すなわち、同一性＝（一致した位置の数／位置の全数）×１００で算出でき、「同一性」は、例えば、以下に挙げたような公知の方法によって容易に計算することができるが、これらに限定されるものではない。 As used herein, "identity (%)" is an index showing the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, and can be determined, for example, by comparing two or more polypeptide sequences. Sequence identity means the percentage of the number of matching amino acids or nucleotides shared between two polypeptide or polynucleotide sequences when the two sequences are aligned in an optimal manner. In other words, identity can be calculated as follows: identity = (number of matching positions/total number of positions) x 100. "Identity" can be easily calculated, for example, by known methods such as those listed below, but is not limited to these.

　Ｃｏｍｐｕｔａｔｉｏｎａｌ Ｍｏｌｅｃｕｌａｒ Ｂｉｏｌｏｇｙ （Ｌｅｓｋ，Ａ．Ｍ．編集） オックスフォード・ユニバーシティ・プレス（Ｏｘｆｏｒｄ Ｕｎｉｖｅｒｓｉｔｙ Ｐｒｅｓｓ），ニューヨーク州 （１９８８）；　Ｂｉｏｃｏｍｐｕｔｉｎｇ：Ｉｎｆｏｒｍａｔｉｃｓ ａｎｄ Ｇｅｎｏｍｅ Ｐｒｏｊｅｃｔｓ （Ｓｍｉｔｈ，Ｄ．Ｗ．編集） アカデミック・プレス（Ａｃａｄｅｍｉｃ Ｐｒｅｓｓ），ニューヨーク州 （１９９３）；　Ｃｏｍｐｕｔｅｒ Ａｎａｌｙｓｉｓ ｏｆ Ｓｅｑｕｅｎｃｅ Ｄａｔａ，Ｐａｒｔ Ｉ （Ｇｒｉｆｆｉｎ，Ａ．Ｍ．，ａｎｄ Ｇｒｉｆｆｉｎ，Ｈ．Ｇ．編集） ヒューマナ・プレス，ニュージャージー州（１９９４）；　Ｓｅｑｕｅｎｃｅ Ａｎａｌｙｓｉｓ ｉｎ Ｍｏｌｅｃｕｌａｒ Ｂｉｏｌｏｇｙ （ｖｏｎ Ｈｅｉｎｊｅ，Ｇ．編集） アカデミック・プレス（１９８７）；　及びＳｅｑｕｅｎｃｅ Ａｎａｌｙｓｉｓ Ｐｒｉｍｅｒ （Ｇｒｉｂｓｋｏｖ，Ｍ．ａｎｄ Ｄｅｖｅｒｅｕｘ，Ｊ．編集） ストックトン・プレス（Ｓｔｏｃｋｔｏｎ Ｐｒｅｓｓ），ニューヨーク州（１９９１）。 Computational Molecular Biology (ed. Lesk, A.M.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (ed. Smith, D.W.) Academic Press, New York (1993); Computer Analysis of Sequence Data Sequence Analysis in Molecular Biology, Part I (eds. Griffin, A.M., and Griffin, H.G.), Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (eds. von Heinje, G.), Academic Press (1987); and Sequence Analysis Primer (eds. Gribskov, M. and Devereux, J.), Stockton Press, NY (1991).

　また、同一性（％）は、公に利用可能なコンピュータープログラムによって計算することができる。例えば、レーザージーン　バイオインフォマティクス　コンピューティング　スィート （ＬＡＳＥＲＧＥＮＥ ｂｉｏｉｎｆｏｒｍａｔｉｃｓ ｃｏｍｐｕｔｉｎｇｓｕｉｔｅ）のメガライン（Ｍｅｇａｌｉｇｎ）プログラム （ＤＮＡＳＴＡＲ社（ＤＮＡＳＴＡＲ Ｉｎｃ．），ウィスコンシン州マディソン;　又はエンボス・オープン（ＥＭＢＯＳＳ Ｏｐｅｎ） ソフトウェアスィート （ＥＭＢＬ－ＥＢＩ；　Ｒｉｃｅ等，Ｔｒｅｎｄｓ ｉｎ Ｇｅｎｅｔｉｃｓ １６（６） ｐｐ２７６－２７７（２０００））等を用いて容易に計算することができるが、これらに限定されるものではない。 Percent identity can also be calculated using publicly available computer programs, such as, but not limited to, the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wisconsin; or the EMBOSS Open software suite (EMBL-EBI; Rice et al., Trends in Genetics 16(6) pp. 276-277 (2000)).

　この場合、配列のマルチプルアライメントを用いて、アライメントのクラスタル（Ｃｌｕｓｔａｌ）法（すなわち、ＣＬＵＳＴＡＬＷ；例えばバージョン２．４）（Ｈｉｇｇｉｎｓ及びＳｈａｒｐ，ＣＡＢＩＯＳ，５：１５１－１５３（１９８９）；Ｈｉｇｇｉｎｓ等，Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓ．２２：４６７３－４６８０（１９９４）；及びＣｈｅｎｎａ等，Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓ ３１（１３）：３４９７－５００（２００３）、これは、欧州バイオインフォマティクス研究所（Ｅｕｒｏｐｅａｎ Ｂｉｏｉｎｆｏｒｍａｔｉｃｓ Ｉｎｓｔｉｔｕｔｅ）を介して欧州分子生物学研究所（Ｅｕｒｏｐｅａｎ Ｍｏｌｅｃｕｌａｒ Ｂｉｏｌｏｇｙ Ｌａｂｏｒａｔｏｒｙ）より入手可能）をデフォルトパラメーターを用いて行うことができる。 In this case, multiple alignment of sequences can be performed using the Clustal method of alignment (i.e., CLUSTALW; e.g. version 2.4) (Higgins and Sharp, CABIOS, 5:151-153 (1989); Higgins et al., Nucleic Acids Res. 22:4673-4680 (1994); and Chenna et al., Nucleic Acids Res 31(13):3497-500 (2003), available from the European Molecular Biology Laboratory via the European Bioinformatics Institute) using default parameters.

　また、ＣＬＵＳＴＡＬＷタンパク質アライメントにとって適切なパラメーターは、ＧＡＰ存在ペナルティー＝１５、ＧＡＰ伸長＝０．２、マトリックス＝Ｇｏｎｎｅｔ（例えば、Ｇｏｎｎｅｔ２５０）、タンパク質ＥＮＤＧＡＰ＝－１、タンパク質ＧＡＰＤＩＳＴ＝４、及びＫＴＵＰＬＥ＝１を含む。一つの態様においては、迅速又は遅いアライメントがデフォルト設定で用いられ、ここにおいて好ましくは遅いアライメントである。あるいは、ＫＴＵＰＬＥ＝１、ＧＡＰペナルティー＝１０、ＧＡＰ伸長＝１、マトリックス＝ＢＬＯＳＵＭ（例えばＢＬＯＳＵＭ６４）、ウィンドウ＝５、及び保存された上の対角（ＴＯＰ ＤＩＡＧＯＮＡＬＳ ＳＡＶＥＤ）＝５も使用できるように、ＣＬＵＳＴＡＬＷ法（バージョン１．８３）を用いるパラメーターを改変してもよい。 Also, suitable parameters for CLUSTALW protein alignment include GAP presence penalty=15, GAP extension=0.2, matrix=Gonnet (e.g., Gonnet250), protein ENDGAP=-1, protein GAP DIST=4, and KTUPLE=1. In one embodiment, either fast or slow alignment is used with default settings, with slow alignment being preferred. Alternatively, parameters using the CLUSTALW method (version 1.83) may be modified to also use KTUPLE=1, GAP penalty=10, GAP extension=1, matrix=BLOSUM (e.g., BLOSUM64), window=5, and TOP DIAGONALS SAVED=5.

　本明細書において、「配列解析ソフトウェア」とは、アミノ酸配列又はヌクレオチドの解析に有用なコンピューターアルゴリズム又はソフトウェアプログラムを意味する。配列解析ソフトウェアは、市販のものでもよいし、又は個別に開発されたものでもよい。このような配列解析ソフトウェアとしては、例えば、以下のものが挙げられるが、これらに限定されるものではない。 As used herein, "sequence analysis software" refers to a computer algorithm or software program useful for analyzing amino acid sequences or nucleotides. Sequence analysis software may be commercially available or independently developed. Examples of such sequence analysis software include, but are not limited to, the following:

　ＧＣＧプログラム （ウィスコンシン・パッケージ・バージョン（ＷｉｓｃｏｎｓｉｎＰａｃｋａｇｅ Ｖｅｒｓｉｏｎ）９．０、ジェネティックス・コンピューター・グループ（Ｇｅｎｅｔｉｃｓ Ｃｏｍｐｕｔｅｒ Ｇｒｏｕｐ）（ＧＣＧ），ウィスコンシン州マディソン）；ＢＬＡＳＴＰ、ＢＬＡＳＴＮ、ＢＬＡＳＴＸ （Ａｌｔｓｃｈｕｌ等，Ｊ．Ｍｏｌ．Ｂｉｏｌ．２１５：４０３－４１０（１９９０）；ＤＮＡＳＴＡＲ （ＤＮＡＳＴＡＲ，Ｉｎｃ．１２２８ Ｓ．Ｐａｒｋ Ｓｔ．マディソン，ウィスコンシン州５３７１５米国）；ＣＬＵＳＴＡＬＷ （例えば、バージョン２．４；Ｔｈｏｍｐｓｏｎ等，Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓｅａｒｃｈ，２２（２２）：４６７３－４６８０（１９９４）；スミス－ウォーターマン（Ｓｍｉｔｈ－Ｗａｔｅｒｍａｎ）アルゴリズムを内包したＦＡＳＴＡプログラム （Ｗ．Ｒ．Ｐｅａｒｓｏｎ，Ｃｏｍｐｕｔ．Ｍｅｔｈｏｄｓ Ｇｅｎｏｍｅ Ｒｅｓ．，［Ｐｒｏｃ．Ｉｎｔ．Ｓｙｍｐ．］（１９９４），Ｍｅｅｔｉｎｇ Ｄａｔｅ １９９２，１１１－２０．編集者：Ｓｕｈａｉ，Ｓａｎｄｏｒ．Ｐｕｂｌｉｓｈｅｒ：プレナム（Ｐｌｅｎｕｍ），ニューヨーク州ニューヨーク）；シーケンチャー（Ｓｅｑｕｅｎｃｈｅｒ）ｖ．４．０５ GCG programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, WI); BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215:403-410 (1990); DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison, WI 53715 USA); CLUSTALW (e.g., Version 2.4; Th ompson et al., Nucleic Acids Research, 22(22):4673-4680 (1994); the FASTA program, which incorporates the Smith-Waterman algorithm (W.R. Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor: Suhai, Sandor. Publisher: Plenum, New York, NY); Sequencher v. 4.05

　本明細書において、アミノ酸配列又はヌクレオチドの解析に配列解析ソフトウェアを用いる場合、原則として、解析結果は当該プログラムの「デフォルト値」に基づくものである。 In this specification, when sequence analysis software is used to analyze amino acid sequences or nucleotides, the analysis results are, in principle, based on the "default values" of the program.

　即ち、本明細書において「デフォルト値」とは、ソフトウェアの製造元によって設定された値又はパラメーターのセットを意味する。デフォルト値は、最初に初期設定した際にソフトウェアでもともとローディングされるものである。 In other words, in this specification, "default values" refers to a set of values or parameters established by the software manufacturer. Default values are originally loaded into the software when it is first initialized.

　本明細書において、「Ｘ線構造解析」とは、タンパク質又はその他の化合物の分子構造を明らかにするための手法を包括するものであり、タンパク質の調製、精製、結晶化条件の最適化、結晶へのＸ線照射、そして得られたデータの解析から成り立つ。以下に、一つの手法を示すが、これに限定されるものではない。タンパク質の調製は、生細胞又は無細胞を利用してタンパク質合成を行うプロセスであり、タンパク質の性質によって様々な手段が適用される。タンパク質の精製では、イオン交換クロマトグラフィーを用いる手法が広く使用され、タンパク質をカラムに充填したイオン交換樹脂に供し、イオン交換樹脂と溶液中のイオンとの相互作用により、タンパク質の分離及び精製が行われる。結晶化条件の最適化では、まず初期スクリーニングによって結晶化の条件を特定した後、最適な結晶化条件を見つけるために結晶化用バッファーのｐＨと沈殿剤の量を調整する。ｐＨの調整は、タンパク質の帯電状態や安定性に影響を与える重要な要素であり、沈殿剤は結晶成長を制御し、理想的な結晶の形成を促進する。最適な条件で結晶を得ることにより、Ｘ線構造解析の精度が向上し、解析結果の品質が高まる。得られた結晶を一定の環境に保たれた状態でＸ線を照射することにより、Ｘ線の回折像が得られ、ここで得られた回折像をＸＤＳソフトウェアを用いて処理することにより、位相情報が抽出できる。さらに抽出された位相情報を、例えばＡｕｔｏＢｕｉｌｄのような構造モデル構築ソフトを用いることにより、タンパク質の三次元構造モデルを構築できる。結晶化の際、解析対象のタンパク質以外にも、反応基質、基質の類似化合物又は補酵素等をタンパク質と共に結晶化させることによって、基質との相互作用や反応機構のようなタンパク質の機能にとって重要な情報を明らかにできる場合がある。 In this specification, "X-ray structural analysis" encompasses a method for clarifying the molecular structure of a protein or other compound, and consists of protein preparation, purification, optimization of crystallization conditions, X-ray irradiation of crystals, and analysis of the obtained data. One method is shown below, but it is not limited to this. Protein preparation is a process of protein synthesis using living cells or acellular organisms, and various means are applied depending on the properties of the protein. In protein purification, a method using ion exchange chromatography is widely used, in which the protein is subjected to an ion exchange resin packed in a column, and the protein is separated and purified by the interaction between the ion exchange resin and the ions in the solution. In optimizing the crystallization conditions, the crystallization conditions are first identified by initial screening, and then the pH of the crystallization buffer and the amount of precipitant are adjusted to find the optimal crystallization conditions. The adjustment of pH is an important factor that affects the charged state and stability of the protein, and the precipitant controls the crystal growth and promotes the formation of ideal crystals. By obtaining crystals under optimal conditions, the accuracy of X-ray structural analysis is improved and the quality of the analysis results is improved. By irradiating the obtained crystals with X-rays while maintaining a constant environment, an X-ray diffraction image is obtained, and the obtained diffraction image is processed using XDS software to extract phase information. Furthermore, by using the extracted phase information with structural model construction software such as AutoBuild, a three-dimensional structural model of the protein can be constructed. During crystallization, in addition to the protein to be analyzed, reactive substrates, substrate analogues, coenzymes, etc. are crystallized together with the protein, which may reveal information important to the function of the protein, such as interactions with the substrate and reaction mechanisms.

　本明細書において、「タンパク質構造予測ソフト」とは、タンパク質の配列情報を元に、そのタンパク質の３次元構造を予測するためのソフトウェアを意味する。タンパク質の構造は、その機能や性質を理解する上で極めて重要であるが、タンパク質の構造は非常に複雑であり、さまざまなモデリング手法とそれを支援するソフトウェアが開発されている。モデリング手法としては、例えば、比較モデリングとｄｅ　ｎｏｖｏモデリングがある。比較モデリングでは、既に構造が決定されたタンパク質を鋳型として、構造を予測したいタンパク質（ターゲット）を比較することによって、ターゲットの構造を予測する。鋳型とターゲットが類似した構造を持つ場合、鋳型の構造をもとにターゲットの構造を推定できる。特に、ターゲットと鋳型が類似したタンパク質である場合、この手法は非常に有用である。ｄｅ　ｎｏｖｏモデリングでは、既存の構造情報を必要とせず、タンパク質のアミノ酸配列情報を元に構造を予測する。タンパク質の構造はアミノ酸配列のパターンによって決定されると考えられており、ｄｅ　ｎｏｖｏモデリングではこのパターンを解析し、構造を予測する。本発明で使用したＡｌｐｈａＦｏｌｄ２は、タンパク質構造予測ソフトウェアの一つであり、比較モデリングとｄｅ　ｎｏｖｏモデリングを組み合わせたハイブリッド型の構造予測ソフトウェアである。これは、最初に既知の構造を持つタンパク質をテンプレートとして使用して初期のモデルを構築し、その後、ｄｅ　ｎｏｖｏモデリングによりモデルを修正し、より高精度な構造予測を行う。ＡｌｐｈａＦｏｌｄ２はオープンソースで提供されており、ＧｉｔＨｕｂ上でソースコードが公開されている。このソースコードをダウンロードし、適切なパーソナルコンピューター上でビルドすることにより、ＡｌｐｈａＦｏｌｄ２を実行できるようになる。ＡｌｐｈａＦｏｌｄ２を利用する際は、タンパク質の配列情報をＰＤＢファイルやＦＡＳＴＡファイル等の形式で入力し、目的の構造予測情報を得ることが可能となる。 In this specification, "protein structure prediction software" means software for predicting the three-dimensional structure of a protein based on the protein sequence information. The structure of a protein is extremely important in understanding its function and properties, but the structure of a protein is very complex, and various modeling methods and software to support it have been developed. Modeling methods include, for example, comparative modeling and de novo modeling. In comparative modeling, a protein whose structure has already been determined is used as a template to compare the protein (target) whose structure is to be predicted, thereby predicting the target structure. If the template and target have similar structures, the target structure can be estimated based on the structure of the template. This method is particularly useful when the target and template are similar proteins. In de novo modeling, existing structural information is not required, and the structure is predicted based on the amino acid sequence information of the protein. It is believed that the structure of a protein is determined by the pattern of the amino acid sequence, and in de novo modeling, this pattern is analyzed to predict the structure. AlphaFold2 used in the present invention is one of the protein structure prediction software, and is a hybrid type structure prediction software that combines comparative modeling and de novo modeling. This software first uses a protein with a known structure as a template to build an initial model, and then modifies the model by de novo modeling to perform more accurate structure prediction. AlphaFold2 is provided as open source, and the source code is published on GitHub. AlphaFold2 can be executed by downloading this source code and building it on an appropriate personal computer. When using AlphaFold2, protein sequence information can be input in the format of a PDB file, FASTA file, or the like, and the desired structure prediction information can be obtained.

　本明細書において、「ｍＭ」はｍｍｏｌ／Ｌを、「μＭ」はμｍｏｌ／Ｌを表す。
　本明細書において、「Ｗ／Ｖ％」は、重量（weight）／体積（volume）％を表す。
　本明細書において、「ＨＥＰＥＳ」は、４－２－ヒドロキシエチル-1-ピペラジンエタンスルホン酸を表す。
　本明細書において、「ＭＥＳ」は、２－モルホリノエタンスルホン酸を表す。
　本明細書において、「ＶＣ」はＬ－アスコルビン酸を表す。
　本明細書において、「５ＨＬ」は５－ヒドロキシ－Ｌ－リジンを表す。 In this specification, "mM" represents mmol/L and "μM" represents μmol/L.
In this specification, "W/V %" stands for weight/volume %.
As used herein, "HEPES" refers to 4-2-hydroxyethyl-1-piperazineethanesulfonic acid.
As used herein, "MES" stands for 2-morpholinoethanesulfonic acid.
As used herein, "VC" stands for L-ascorbic acid.
As used herein, "5HL" refers to 5-hydroxy-L-lysine.

　本明細書における、取得したＬ－リジン５位水酸化酵素のアミノ酸配列の配列番号と配列略称等の一覧は表１のとおりである。また、前記アミノ酸配列をコードするＤＮＡの塩基配列の配列番号は表２のとおりである。 In this specification, the sequence numbers and sequence abbreviations of the amino acid sequences of the obtained L-lysine 5-hydroxylase are listed in Table 1. The sequence numbers of the base sequences of the DNAs encoding the amino acid sequences are listed in Table 2.

　以下、本発明を詳細に説明する。
１．本発明のＬ－リジン５位水酸化酵素の探索
　本発明のＬ－リジン５位水酸化酵素は、以下のようにして見出されたものである。
　非特許文献７の情報を基に、Ｌ－リジン５位水酸化酵素活性を有する遺伝子の探索を行った。具体的には、非特許文献７に記載されているアラゾペプチン生合成遺伝子クラスターを有するＳｔｒｅｐｔａｃｉｄｉｐｈｉｌｕｓ　ｇｒｉｓｅｏｐｌａｎｕｓ株（以下、「Ｓ．ｇｒｉｓｅｏｐｌａｎｕｓ株」と称することがある。）及びＫｉｔａｓａｔｏｓｐｏｒａ　ａｚａｔｉｃａ株（以下、「Ｋ．ａｚａｔｉｃａ株」と称することがある。）の遺伝子情報を基にａｚｐ遺伝子クラスターを有する微生物を探索した。 The present invention will be described in detail below.
1. Search for the L-lysine 5-hydroxylase of the present invention The L-lysine 5-hydroxylase of the present invention was discovered as follows.
A search was conducted for a gene having L-lysine 5-hydroxylase activity based on the information in Non-Patent Document 7. Specifically, a search was conducted for a microorganism having an azp gene cluster based on the genetic information of a Streptacidiphilus griseoplanus strain (hereinafter sometimes referred to as "S. griseoplanus strain") and a Kitasatospora azatica strain (hereinafter sometimes referred to as "K. azatica strain") having the arazopeptin biosynthetic gene cluster described in Non-Patent Document 7.

　その結果、図１に示すようにＡｃｔｉｎｏｓｙｎｎｅｍａ　ｐｒｅｔｉｏｓｕｍ株（以下、「Ａ．ｐｒｅｔｉｏｓｕｍ株」と称することがある。）およびＡｃｔｉｎｏｓｙｎｎｅｍａ　ｍｉｒｕｍ　ＤＳＭ　４３８２７株（以下、Ａ．ｍｉｒｕｍ株」と称することがある。）のａｚｐ遺伝子クラスターのホモログクラスターにおいて、ａｚｐＫ遺伝子を持たないが、代わりに機能未知の２－オキソグルタル酸依存型水酸化酵素遺伝子が存在することを見出した（図１中、★で示す）。この２－オキソグルタル酸依存型水酸化酵素をＡｚｐＫ２、また当該ＡｚｐＫ２タンパク質をコードするＤＮＡをａｚｐＫ２遺伝子と命名した。 As a result, as shown in Figure 1, it was found that the homologue cluster of the azp gene cluster in Actinosynnema pretiosum strain (hereinafter sometimes referred to as "A. pretiosum strain") and Actinosynnema mirum DSM 43827 strain (hereinafter sometimes referred to as "A. mirum strain") does not have the azpK gene, but instead contains a 2-oxoglutarate-dependent hydroxylase gene with unknown function (indicated by * in Figure 1). This 2-oxoglutarate-dependent hydroxylase was named AzpK2, and the DNA encoding the AzpK2 protein was named the azpK2 gene.

　ａｚｐＫ２遺伝子を用いて形質転換体を作製して、ＡｚｐＫ２タンパク質のＬ－リジン５位水酸化活性を測定したところ、Ｌ－リジンの５位に対してＳ選択的に水酸化する活性を有することを見出した。 A transformant was created using the azpK2 gene, and the L-lysine 5-hydroxylation activity of the AzpK2 protein was measured, revealing that it has the activity to selectively hydroxylate the S-5 position of L-lysine.

　さらに、ａｚｐ遺伝子クラスター内に存在する６－ｄｉａｚｏ－５－ｏｘｏ－Ｌ－ｎｏｒｌｅｕｃｉｎｅの生合成遺伝子群（ａｚｐＪ，ａｚｐＫ，ａｚｐＬ）について同様に探索したところ、Ｐｓｅｕｄｏｍｏｎａｓ　ｐｓｙｃｈｒｏｔｏｌｅｒａｎｓ　ＮＳ３８３株（以下、「Ｐ．ｐｓｙｃｈｒｏｔｏｌｅｒａｎｓ株」と称することがある。）において、ａｚｐＪ遺伝子とａｚｐＬ遺伝子の間に機能未知のａｚｐＫ２遺伝子ホモログがあることを見出した（図１中、☆で示す）。これをＰｐ＿ａｚｐＫ２遺伝子、Ｐｐ＿ａｚｐＫ２遺伝子がコードするタンパク質をＰｐ＿ＡｚｐＫ２と命名した。 Furthermore, a similar search was conducted for the biosynthetic genes (azpJ, azpK, azpL) of 6-diazo-5-oxo-L-norleucine present in the azp gene cluster, and it was found that in Pseudomonas psychotolerans NS383 strain (hereinafter sometimes referred to as "P. psychotolerans strain"), there is an azpK2 gene homologue with unknown function between the azpJ gene and the azpL gene (indicated by a star in Figure 1). This gene was named Pp_azpK2 gene, and the protein encoded by the Pp_azpK2 gene was named Pp_AzpK2.

　図1は、ａｚｐ遺伝子クラスターに関する図であり、図１中の各記号の意味は以下のとおりである。 Figure 1 shows the azp gene cluster, and the symbols in Figure 1 have the following meanings:

　また、Ｐｐ＿ａｚｐＫ２遺伝子を用いて形質転換体を作製して、Ｐｐ＿ＡｚｐＫ２タンパク質のＬ－リジン５位水酸化活性を測定したところ、Ｌ－リジンの５位に対してＲ選択的に水酸化する活性を有することを見出した。 In addition, a transformant was created using the Pp_azpK2 gene, and the L-lysine 5-hydroxylation activity of the Pp_AzpK2 protein was measured, revealing that it has the activity of R-selectively hydroxylating the 5-position of L-lysine.

　さらに、ＡｚｐＫ２タンパク質をコードするＤＮＡ（ａｚｐＫ２遺伝子）及びＰｐ＿ＡｚｐＫ２タンパク質をコードするＤＮＡ（Ｐｐ＿ａｚｐＫ２遺伝子）について、それぞれの形質転換体を用いてＬ－リジンから５－ヒドロキシ－Ｌ－リジンを合成したところ、高い蓄積濃度で５－ヒドロキシ－Ｌ－リジンを合成することを見出した。 Furthermore, when 5-hydroxy-L-lysine was synthesized from L-lysine using transformants of DNA encoding the AzpK2 protein (azpK2 gene) and DNA encoding the Pp_AzpK2 protein (Pp_azpK2 gene), it was found that 5-hydroxy-L-lysine was synthesized at high accumulated concentrations.

　また、ａｚｐＫ２遺伝子及びＰｐ＿ａｚｐＫ２遺伝子について、それぞれＤＮＡ　Ｄａｔａｂａｎｋ　ｏｆ　ＪＡＰＡＮ　（ＤＤＢＪ）等のデータベースを用いてホモロジー検索を行って、ａｚｐＫ２遺伝子及びＰｐ＿ａｚｐＫ２遺伝子のそれぞれのホモログ遺伝子１０００種の遺伝子配列情報を取得した。 Furthermore, a homology search was performed for the azpK2 gene and the Pp_azpK2 gene using databases such as the DNA Databank of JAPAN (DDBJ), and gene sequence information for 1,000 homologous genes of each of the azpK2 gene and the Pp_azpK2 gene was obtained.

　次いで、ａｚｐＫ２遺伝子のホモログ遺伝子とＰｐ＿ａｚｐＫ２遺伝子のホモログ遺伝子の重複を排除して、ａｚｐＫ２遺伝子およびＰｐ＿ａｚｐＫ２遺伝子を含めたホモログ遺伝子１１１５種の遺伝子配列情報を取得した。 Next, overlapping homologous genes of the azpK2 gene and the Pp_azpK2 gene were eliminated, and gene sequence information for 1,115 homologous genes, including the azpK2 gene and the Pp_azpK2 gene, was obtained.

　これらのホモログ遺伝子がコードするアミノ酸配列をＮｅｉｇｈｂｏｒ－ｊｏｉｎｉｎｇ法を用いて作成した分子系統樹を元に分類したところ、１０個のＣｌａｄｅに分類された。分類された各ＣｌａｄｅをそれぞれＣｌａｄｅ１～１０と命名した。１０個のＣｌａｄｅに分類されたホモログ遺伝子１１１５種（ａｚｐＫ２およびＰｐ＿ａｚｐＫ２遺伝子を含む）がコードするタンパク質のうち、代表的なものを図２に示す。 The amino acid sequences encoded by these homolog genes were classified based on a molecular phylogenetic tree created using the Neighbor-joining method, and were classified into 10 Clades. Each classified Clade was named Clades 1 to 10. Representative proteins encoded by the 1,115 homolog genes (including the azpK2 and Pp_azpK2 genes) classified into the 10 Clades are shown in Figure 2.

　この１０個のＣｌａｄｅに属するタンパク質について、Ｌ－リジン５位水酸化酵素活性を確認したところ、Ｃｌａｄｅ１～４に属するタンパク質がＬ－リジン５位水酸化酵素活性を有することを見出した。さらに、Ｃｌａｄｅ１に属するタンパク質はＬ－リジンの５位に対してＳ選択的に水酸化する活性を有すること及びＣｌａｄｅ２～４に属するタンパク質はＬ－リジンの５位に対してＲ選択的に水酸化する活性を有することを見出した。この１０個のＣｌａｄｅのうち、Ｃｌａｄｅ１にはＡｚｐＫ２タンパク質、Ｃｌａｄｅ１０にはＰｐ＿ＡｚｐＫ２タンパク質、Ｃｌａｄｅ５には非特許文献８で報告されているＬ－リジンハロゲン化酵素が含まれていた。 When the L-lysine 5-hydroxylase activity of proteins belonging to these 10 Clades was confirmed, it was found that proteins belonging to Clades 1 to 4 have L-lysine 5-hydroxylase activity. Furthermore, it was found that proteins belonging to Clades 1 have the activity of selectively hydroxylating the 5th position of L-lysine in an S-selective manner, and proteins belonging to Clades 2 to 4 have the activity of selectively hydroxylating the 5th position of L-lysine in an R-selective manner. Of these 10 Clades, Clade 1 contained the AzpK2 protein, Clade 10 contained the Pp_AzpK2 protein, and Clade 5 contained the L-lysine halogenase reported in Non-Patent Document 8.

　また、ＡｚｐＫ２タンパク質について、Ｘ線構造解析、立体構造予測、Ｃｌａｄｅ５に属するＬ－リジンハロゲン化酵素との比較解析、及びＣｌａｄｅ１～５のアミノ酸配列との同一性解析を行った結果、ＡｚｐＫ２タンパク質の１３６～１４２番目のアミノ酸残基からなるアミノ酸モチーフ及び２３５～２４２番目のアミノ酸残からなるアミノ酸モチーフがＬ－リジン５位水酸化活性の発現に重要であることを見出した。 Furthermore, X-ray structural analysis, three-dimensional structure prediction, comparative analysis with L-lysine halogenase belonging to Clade 5, and identity analysis with the amino acid sequences of Clades 1 to 5 were performed on the AzpK2 protein, and it was found that an amino acid motif consisting of amino acid residues 136 to 142 and an amino acid motif consisting of amino acid residues 235 to 242 of the AzpK2 protein are important for expressing L-lysine 5-hydroxylation activity.

　ここで、２－オキソグルタル酸依存性ジオキシゲナーゼのうち、主に水酸化反応を触媒する酵素に関連するシグネチャーモチーフは、以下の２つの保存されたモチーフを含むことが知られている。なお、アミノ酸残基の位置は、ＡｚｐＫ２タンパク質のアミノ酸配列（配列番号２）に基づいて番号付けをしたものである。
１）Ｈｉｓ１３９－Ｘ１４０－Ａｓｐ１４１：「ＨＸＤ」モチーフ
２）Ｔｙｒ１９７－Ａｒｇ２１６：「Ｙ－Ｒ」モチーフ
（ここで本明細書において、特に断らない限り、Ｘは、Ａ、Ｒ、Ｎ、Ｄ、Ｃ、Ｑ、Ｅ、Ｇ、Ｈ、Ｉ、Ｌ、Ｋ、Ｍ、Ｆ、Ｐ、Ｓ、Ｔ、Ｗ、Ｙ又はＶのいずれかのアミノ酸を示す。） Here, among 2-oxoglutarate-dependent dioxygenases, the signature motif associated with enzymes that mainly catalyze hydroxylation reactions is known to contain the following two conserved motifs: The positions of amino acid residues are numbered based on the amino acid sequence of the AzpK2 protein (SEQ ID NO: 2).
1) His139-X140-Asp141: "HXD"motif; 2) Tyr197-Arg216: "Y-R" motif (unless otherwise specified herein, X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V).

　本発明のＬ－リジン５位水酸化酵素は、前記シグネチャーモチーフを有するものであって、Ｅ．Ｃ．番号（Ｅｎｚｙｍｅ Ｃｏｍｍｉｓｓｉｏｎ ｎｕｍｂｅｒｓ）１．１４．１１に属する２－オキソグルタル酸依存性ジオキシゲナーゼに分類される。 The L-lysine 5-hydroxylase of the present invention has the above-mentioned signature motif and is classified as a 2-oxoglutarate-dependent dioxygenase that belongs to the E.C. number (Enzyme Commission numbers) 1.14.11.

　従来公知のグローバルアライメントのアルゴリズム（すなわち、配列解析ソフトウェア）を用いて、Ｌ－リジン５位水酸化活性を有する酵素を示す２種又はそれ以上のアミノ酸配列をアライメントし、比較することにより、共通するシグネチャーモチーフを有するかどうかを決定することができる。 By using a conventionally known global alignment algorithm (i.e., sequence analysis software), two or more amino acid sequences representing enzymes having L-lysine 5-hydroxylation activity can be aligned and compared to determine whether they share a common signature motif.

　そこで、Ｌ－リジン５位水酸化活性を有することが判明したタンパク質のアミノ酸配列について、配列番号２を参照配列として、ＣＬＵＳＴＡＬアライメント（ＣＬＵＳＴＡＬＷ）を用いて解析したところ、本発明のＬ－リジン５位水酸化酵素は、前記シグネチャーモチーフに加えて、以下の３）及び４）の連続したアミノ酸配列のモチーフを含むものであることを見出した。
３）Ｈｉｓ１３６－Ｇｌｙ１３７－Ｔｒｐ１３８－Ｈｉｓ１３９－Ｔｒｐ１４０－Ａｓｐ１４１－Ａｓｐ１４２：「ＨＧＷＨＷＤＤ」（配列番号１４４）モチーフ（１３６～１４２番目のアミノ酸のモチーフ；Ｃｌａｄｅ1～４に共通）
４）Ｈｉｓ２３５－Ｇｌｎ２３６－Ｔｈｒ２３７－Ｍｅｔ２３８－Ｇｌｎ２３９－Ｘ２４０－Ｌｅｕ２４１－Ｐｈｅ２４２：「ＨＥＴＭＥＸＬＦ」（配列番号１４５）モチーフ（２３５～２４２番目のアミノ酸のモチーフ１；Ｃｌａｄｅ1に共通）、
　Ｈｉｓ２３５－Ｇｌｎ２３６－Ｔｈｒ２３７－Ｍｅｔ２３８－Ａｓｐ２３９－Ｘ２４０－Ｌｅｕ２４１－Ｔｒｐ２４２：「ＨＥＴＭＤＸＬＷ」（配列番号１４６）モチーフ（２３５～２４２番目のアミノ酸のモチーフ２；Ｃｌａｄｅ２及び３に共通する第１配列）、　Ｈｉｓ２３５－Ｇｌｎ２３６－Ｔｈｒ２３７－Ｍｅｔ２３８－Ｇｌｎ２３９－Ｘ２４０－Ｌｅｕ２４１－Ｔｒｐ２４２：「ＨＥＴＭＥＸＬＷ」（配列番号１４７）モチーフ（２３５～２４２番目のアミノ酸のモチーフ３；Ｃｌａｄｅ２及び３に共通する第２配列）、　Ｈｉｓ２３５－Ｇｌｎ２３６－Ｔｈｒ２３７－Ａｓｎ２３８－Ａｓｐ２３９－Ｘ２４０－Ｌｅｕ２４１－Ｐｈｅ２４２：「ＨＥＴＮＤＸＬＦ」（配列番号１４８）モチーフ（２３５～２４２番目のアミノ酸のモチーフ４；Ｃｌａｄｅ４に共通する第１配列）、
　又はＨｉｓ２３５－Ｇｌｎ２３６－Ｔｈｒ２３７－Ａｓｎ２３８－Ａｓｎ２３９－Ｘ２４０－Ｌｅｕ２４１－Ｐｈｅ２４２：「ＨＥＴＮＮＸＬＦ」（配列番号１４９）モチーフ（２３５～２４２番目のアミノ酸のモチーフ５；Ｃｌａｄｅ４に共通する第２配列） Thus, the amino acid sequences of proteins found to have L-lysine 5-hydroxylation activity were analyzed using CLUSTAL alignment (CLUSTALW) with SEQ ID NO: 2 as a reference sequence, and it was found that the L-lysine 5-hydroxylase of the present invention contains, in addition to the signature motif, the following consecutive amino acid sequence motifs 3) and 4):
3) His136-Gly137-Trp138-His139-Trp140-Asp141-Asp142: "HGWHWDD" (SEQ ID NO: 144) motif (motif of amino acids 136 to 142; common to Clade 1 to 4)
4) His235-Gln236-Thr237-Met238-Gln239-X240-Leu241-Phe242: "HETMEXLF" (SEQ ID NO: 145) motif (motif 1 of amino acids 235 to 242; common to Clade 1);
His235-Gln236-Thr237-Met238-Asp239-X240-Leu241-Trp242: "HETMDXLW" (SEQ ID NO: 146) motif (motif 2 of amino acids 235 to 242; first sequence common to Clades 2 and 3); His235-Gln236-Thr237-Met238-Gln239-X240-Leu241-Trp242: "HETMEXLW" (SEQ ID NO: 147) motif (motif 3 of amino acids 235 to 242; second sequence common to Clades 2 and 3); His235-Gln236-Thr237-Asn238-Asp239-X240-Leu241-Phe242: "HETNDXLF" (SEQ ID NO: 148) motif (motif 4 at amino acids 235 to 242; first sequence common to Clade 4);
Or His235-Gln236-Thr237-Asn238-Asn239-X240-Leu241-Phe242: "HETNNXLF" (SEQ ID NO: 149) motif (motif 5 at amino acids 235 to 242; second sequence common to Clade 4)

　なお、アミノ酸配列には、アミノ酸残基のわずかな（例えば、５個又はそれ未満）の挿入又は欠失がある可能性があることから、前記のアミノ酸残基の位置は、参照配列である配列番号２に基づいて番号付けをしたものである。 Note that since there may be a small number of insertions or deletions (e.g., 5 or fewer) of amino acid residues in the amino acid sequence, the positions of the amino acid residues are numbered based on the reference sequence, SEQ ID NO:2.

　本発明は、上記のような知見に基づき、成されたものである。以下、本発明について、さらに詳細に説明する。 The present invention was made based on the above findings. The present invention will be explained in more detail below.

２．本発明のＬ－リジン５位水酸化酵素等
　本発明のＬ－リジン５位水酸化酵素は、配列番号２、４、６、８、１０、１２、１４、１６、１８、２０、２２、２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８、１１０、１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２若しくは１３４に示すアミノ酸配列を有するポリペプチド、又は当該アミノ酸配列のホモログを有するポリペプチドであって、Ｌ－リジン５位水酸化活性を有するものである。 2. L-lysine 5-hydroxylase of the present invention, etc. The L-lysine 5-hydroxylase of the present invention is a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134, or a polypeptide having a homologue of the amino acid sequence, and has an activity of hydroxylating L-lysine 5-position.

　本発明のＬ－リジン５位水酸化酵素は、例えば、前記Ｃｌａｄｅ１、２、３、４及び１０に属するポリペプチドであって、Ｌ－リジン５位水酸化活性を有するものである。 The L-lysine 5-hydroxylase of the present invention is, for example, a polypeptide belonging to Clade 1, 2, 3, 4, and 10, and has L-lysine 5-hydroxylation activity.

　具体的には、本発明のＬ－リジン５位水酸化酵素は、以下の（Ａ）、（Ｂ）、（Ｃ）、（Ｄ）、（Ｅ）又は（Ｆ）に示すポリペプチドを有するものであることが好ましい。
（Ａ）配列番号２、４、６、８、１０、１２、１４、１６、１８、２０、２２、２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８、１１０、１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列を有するポリペプチド
（Ｂ）配列番号２、４、６、８、１０、１２、１４、１６、１８、２０、２２、２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８、１１０、１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換及び／又は付加されたアミノ酸配列を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチド
（Ｃ）配列番号２、６、８、１０、１２、１４、１６、１８、２０又は２２に示すアミノ酸配列と５５％以上の同一性を有するアミノ酸配列を有し、かつ、Ｌ－リジン５位水酸化酵素活性を有するポリペプチド
（Ｄ）配列番号２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８又は１１０に示すアミノ酸配列と５０％以上の同一性を有するアミノ酸配列を有し、かつ、Ｌ－リジン５位水酸化酵素活性を有するポリペプチド
（Ｅ）配列番号１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列と５８％以上の同一性を有するアミノ酸配列を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチド
（Ｆ）連続したアミノ酸配列のモチーフである「ＨＧＷＨＷＤＤ」；と、「ＨＥＴＭＥＸＬＦ」、「ＨＥＴＭＤＸＬＷ」「ＨＥＴＭＥＸＬＷ」、「ＨＥＴＮＤＸＬＦ」及び「ＨＥＴＮＮＸＬＦ」よりなる群から選ばれるいずれかの連続したアミノ酸配列のモチーフ；とを有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチド Specifically, the L-lysine 5-hydroxylase of the present invention preferably has a polypeptide shown in (A), (B), (C), (D), (E) or (F) below.
(A) A polypeptide having an amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134. (B) A polypeptide having an amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 2 A polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted and/or added in the amino acid sequence shown in SEQ ID NO: 2, 6, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134, and having L-lysine 5-hydroxylation activity (C) (D) a polypeptide having an amino acid sequence having 50% or more identity with the amino acid sequence shown in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108 or 110, and having L-lysine 5-hydroxylase activity; E) A polypeptide having an amino acid sequence having 58% or more identity to the amino acid sequence shown in SEQ ID NO: 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134 and having L-lysine 5-hydroxylation activity. (F) A polypeptide having a consecutive amino acid sequence motif "HGWHWDD"; and any consecutive amino acid sequence motif selected from the group consisting of "HETMEXLF", "HETMDXLW", "HETMEXLW", "HETNDXLF" and "HETNNXLF", and having L-lysine 5-hydroxylation activity.

　ポリペプチド（Ａ）は、配列番号２、４、６、８、１０、１２、１４、１６、１８、２０、２２、２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８、１１０、１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列（以下、「アミノ酸配列（ａ）群」と称することがある。）を有するものである。アミノ酸配列（ａ）群を有するポリペプチドは、Ｌ－リジン５位水酸化活性を有しており、Ｌ－リジンの５位を水酸化して５－ヒドロキシ－Ｌ－リジンを生成することができる。 Polypeptide (A) has the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134 (hereinafter sometimes referred to as "amino acid sequence (a) group"). A polypeptide having the amino acid sequence (a) group has L-lysine 5-position hydroxylation activity and can hydroxylate the 5-position of L-lysine to produce 5-hydroxy-L-lysine.

　ポリペプチド（Ａ）は、Ｃｌａｄｅ１、２、３、４又は１０に属するポリペプチドであって、Ｌ－リジン５位水酸化活性を有するものである。 Polypeptide (A) is a polypeptide belonging to Class 1, 2, 3, 4 or 10 and has L-lysine 5-hydroxylation activity.

　アミノ酸配列（ａ）群を有するポリペプチドの中では、Ｌ－リジン５位水酸化活性が高く、５－ヒドロキシ－Ｌ－リジンの収率が高いことから、配列番号２、４、６、８、１０、１２、１４、１６、２２、２６、２８、３０、３４、３６、４０、４４、４６、５２、７４、８２、８４、９０、９８、１００又は１０４に示すアミノ酸配列を有するポリペプチドが好ましく、配列番号８、１０、１６、９０及び１０４に示すアミノ酸配列を有するポリペプチドがより好ましい。 Among the polypeptides having the amino acid sequence (a) group, those having the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 22, 26, 28, 30, 34, 36, 40, 44, 46, 52, 74, 82, 84, 90, 98, 100 or 104 are preferred, as they have high L-lysine 5-hydroxylation activity and high yield of 5-hydroxy-L-lysine, and those having the amino acid sequences shown in SEQ ID NO: 8, 10, 16, 90 and 104 are more preferred.

　また、アミノ酸配列（ａ）群を有するポリペプチドのうち、配列番号２、６、８、１０、１２、１４、１６、１８、２０又は２２に示すアミノ酸配列を有するポリペプチドは、Ｌ－リジンの５位をＳ選択的に水酸化することができるものであり、（２Ｓ，５Ｓ）－５－ヒドロキシ－Ｌ－リジンの製造に好適に用いることができる。これらのポリペプチドは、Ｃｌａｄｅ１に属するものである。これらの中では、Ｌ－リジン５位水酸化活性が高く、（２Ｓ，５Ｓ）－５－ヒドロキシ－Ｌ－リジンの収率が高いことから、配列番号２、６、８、１０、１２、１４、１６又は２２に示すアミノ酸配列を有するポリペプチドが好ましく、配列番号８、１０又は１６に示すアミノ酸配列を有するポリペプチドがより好ましい。 In addition, among the polypeptides having the amino acid sequence of group (a), the polypeptides having the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22 are capable of selectively S-hydroxylating the 5-position of L-lysine and can be suitably used for producing (2S,5S)-5-hydroxy-L-lysine. These polypeptides belong to Class 1. Among these, the polypeptides having the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16 or 22 are preferred, and the polypeptides having the amino acid sequence shown in SEQ ID NO: 8, 10 or 16 are more preferred, because they have high L-lysine 5-position hydroxylation activity and high yield of (2S,5S)-5-hydroxy-L-lysine.

　また、アミノ酸配列（ａ）群を有するポリペプチドのうち、配列番号４、２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８、１１０、１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列を有するポリペプチドは、Ｌ－リジンの５位をＲ選択的に水酸化することができるものであり、（２Ｓ、５Ｒ）－５－ヒドロキシ－Ｌ－リジンの製造に好適に用いることができる。これらのポリペプチドは、Ｃｌａｄｅ２、３、４又は１０に属するものである。これらの中では、Ｌ－リジン５位水酸化活性が高く、（２Ｓ、５Ｒ）－５－ヒドロキシ－Ｌ－リジンの収率が高いことから、配列番号２６、２８、３０、３４、３６、４０、４４、４６、５２、７４、８２、８４、９０、９８、１００又は１０４に示すアミノ酸配列を有するポリペプチドが好ましく、配列番号９０又は１０４に示すアミノ酸配列を有するポリペプチドがより好ましい。 Furthermore, among the polypeptides having the amino acid sequence (a) group, the polypeptides having the amino acid sequence shown in SEQ ID NO: 4, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134 can R-selectively hydroxylate the 5-position of L-lysine, and can be suitably used for producing (2S, 5R)-5-hydroxy-L-lysine. These polypeptides belong to Clade 2, 3, 4, or 10. Among these, the polypeptides having the amino acid sequence shown in SEQ ID NO: 26, 28, 30, 34, 36, 40, 44, 46, 52, 74, 82, 84, 90, 98, 100, or 104 are preferred, as they have a high L-lysine 5-hydroxylation activity and a high yield of (2S,5R)-5-hydroxy-L-lysine, and the polypeptides having the amino acid sequence shown in SEQ ID NO: 90 or 104 are more preferred.

　ポリペプチド（Ｂ）、（Ｃ）、（Ｄ）、（Ｅ）又は（Ｆ）は、アミノ酸配列（ａ）群のホモログを有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチドである。 Polypeptide (B), (C), (D), (E) or (F) is a polypeptide that has a homologue of the amino acid sequence (a) group and has L-lysine 5-hydroxylation activity.

　ポリペプチド（Ｂ）は、アミノ酸配列（ａ）群において、１個又は複数個のアミノ酸が欠失、置換、若しくは付加されたアミノ酸配列を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチドである。置換又は付加の場合は、１又は複数個のアミノ酸が保守的に置換又は付加された、保守的に変異されたものが好ましい。ここで「１個又は複数個のアミノ酸」とは、通常、１個～１００個、好ましくは１個～５０個、より好ましくは１個～２０個、さらに好ましくは１個～１０個、なお一層好ましくは１個～５個、特に好ましくは１個～２個、最も好ましくは１個のアミノ酸である。 Polypeptide (B) is a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence group (a), and having L-lysine 5-hydroxylation activity. In the case of substitution or addition, a conservative mutation in which one or more amino acids are conservatively substituted or added is preferred. Here, "one or more amino acids" generally means 1 to 100, preferably 1 to 50, more preferably 1 to 20, even more preferably 1 to 10, still more preferably 1 to 5, particularly preferably 1 to 2, and most preferably 1 amino acid.

　ポリペプチド（Ｂ）は、一態様では、Ｃｌａｄｅ１、２、３、４又は１０に属するポリペプチドであって、Ｌ－リジン５位水酸化活性を有するものである。 In one embodiment, the polypeptide (B) is a polypeptide belonging to Clade 1, 2, 3, 4 or 10 and has L-lysine 5-hydroxylation activity.

　ポリペプチド（Ｃ）は、アミノ酸配列（ａ）群のホモログのうち、配列番号２、６、８、１０、１２、１４、１６、１８、２０又は２２に示すアミノ酸配列のホモログを有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチドである。ポリペプチド（Ｃ）は、一態様では、Ｃｌａｄｅ１に属するものである。 Polypeptide (C) is a polypeptide that has a homologue of the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20, or 22 among the homologues of the amino acid sequence (a) group, and has L-lysine 5-hydroxylation activity. In one embodiment, polypeptide (C) belongs to Clade 1.

　当該アミノ酸配列のホモログは、それぞれ、配列番号２、６、８、１０、１２、１４、１６、１８、２０又は２２に示すアミノ酸配列全長と５５％以上の配列同一性を有するものである。これらのうち、Ｌ－リジン５位水酸化活性の観点から、それぞれ、好ましくは７０％以上、より好ましくは８０％以上、さらに好ましくは８５％以上、なお一層好ましくは９０％以上、特に好ましくは９５％以上、より特に好ましくは９７％以上、さらに特に好ましくは９８％以上、最も好ましくは９９％以上の配列同一性を有するものが好ましい。 The homologues of the amino acid sequence have a sequence identity of 55% or more with the full length amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22, respectively. Among these, from the viewpoint of L-lysine 5-hydroxylation activity, those having a sequence identity of preferably 70% or more, more preferably 80% or more, even more preferably 85% or more, still more preferably 90% or more, particularly preferably 95% or more, even more particularly preferably 97% or more, even more particularly preferably 98% or more, and most preferably 99% or more are preferred.

　ポリペプチド（Ｃ）は、Ｌ－リジンの５位をＳ選択的に水酸化することができるものであり、（２Ｓ，５Ｓ）－５－ヒドロキシ－Ｌ－リジンの製造に好適に用いることができる。 Polypeptide (C) is capable of selectively S-hydroxylating the 5-position of L-lysine and can be suitably used to produce (2S,5S)-5-hydroxy-L-lysine.

　ポリペプチド（Ｄ）は、アミノ酸配列（ａ）群のホモログのうち、配列番号２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８又は１１０に示すアミノ酸配列のホモログを有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチドである。ポリペプチド（Ｄ）は、一態様では、Ｃｌａｄｅ２又は３に属するものである。 Polypeptide (D) is a polypeptide having a homologue of the amino acid sequence shown in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110 among the homologues of amino acid sequence (a) group, and having L-lysine 5-position hydroxylation activity. In one embodiment, polypeptide (D) belongs to Clade 2 or 3.

　当該アミノ酸配列のホモログは、それぞれ、配列番号２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８又は１１０に示すアミノ酸配列全長と５０％以上の配列同一性を有するものである。これらのうち、Ｌ－リジン５位水酸化活性の観点から、それぞれ、好ましくは７０％以上、より好ましくは８０％以上、さらに好ましくは８５％以上、なお一層好ましくは９０％以上、特に好ましくは９５％以上、より特に好ましくは９７％以上、さらに特に好ましくは９８％以上、最も好ましくは９９％以上の配列同一性を有するものが好ましい。 The homologues of the amino acid sequence have a sequence identity of 50% or more with the full length amino acid sequence shown in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108 or 110. Among these, from the viewpoint of L-lysine 5-hydroxylation activity, those having a sequence identity of preferably 70% or more, more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, particularly preferably 95% or more, even more particularly preferably 97% or more, even more particularly preferably 98% or more, and most preferably 99% or more are preferred.

　ポリペプチド（Ｄ）は、Ｌ－リジンの５位をＲ選択的に水酸化することができるものであり、（２Ｓ，５Ｒ）－５－ヒドロキシ－Ｌ－リジンの製造に好適に用いることができる。 Polypeptide (D) is capable of R-selectively hydroxylating the 5-position of L-lysine and can be suitably used to produce (2S,5R)-5-hydroxy-L-lysine.

　ポリペプチド（Ｅ）は、アミノ酸配列（ａ）群のホモログのうち、配列番号１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４のホモログを有し、かつ、Ｌ－リジン５位水酸化活性を保持するポリペプチドである。ポリペプチド（Ｅ）は、一態様では、Ｃｌａｄｅ４に属するものである。 Polypeptide (E) is a polypeptide that has a homolog of SEQ ID NO: 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134 among the homologs of amino acid sequence (a) group, and retains L-lysine 5-hydroxylation activity. In one embodiment, polypeptide (E) belongs to Clade 4.

　当該アミノ酸配列のホモログは、それぞれ、配列番号１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列全長と５８％以上の配列同一性を有するものである。これらのうち、Ｌ－リジン５位水酸化活性の観点から、それぞれ、好ましくは７０％以上、より好ましくは８０％以上、さらに好ましくは８５％以上、なお一層好ましくは９０％以上、特に好ましくは９５％以上、より特に好ましくは９７％以上、さらに特に好ましくは９８％以上、最も好ましくは９９％以上の配列同一性を有するものが好ましい。 Homologs of the amino acid sequence have a sequence identity of 58% or more with the full length amino acid sequence shown in SEQ ID NO: 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134, respectively. Among these, from the viewpoint of L-lysine 5-hydroxylation activity, those having a sequence identity of preferably 70% or more, more preferably 80% or more, even more preferably 85% or more, still more preferably 90% or more, particularly preferably 95% or more, even more particularly preferably 97% or more, even more particularly preferably 98% or more, and most preferably 99% or more are preferred.

　ポリペプチド（Ｅ）は、Ｌ－リジンの５位をＲ選択的に水酸化することができるものであり、（２Ｓ，５Ｒ）－５－ヒドロキシ－Ｌ－リジンの製造に好適に用いることができる。 Polypeptide (E) is capable of R-selectively hydroxylating the 5-position of L-lysine and can be suitably used to produce (2S,5R)-5-hydroxy-L-lysine.

　ポリペプチド（Ｆ）は、アミノ酸配列（ａ）群のホモログのうち、連続したアミノ酸配列のモチーフである「ＨＧＷＨＷＤＤ」；と、「ＨＥＴＭＥＸＬＦ」、「ＨＥＴＭＤＸＬＷ」、「ＨＥＴＭＥＸＬＷ」、「ＨＥＴＮＤＸＬＦ」及び「ＨＥＴＮＮＸＬＦ」よりなる群から選ばれるいずれかの連続したアミノ酸配列のモチーフ；とを有し、かつ、Ｌ－リジン５位水酸化活性を保持するポリペプチドである。より具体的には、ポリペプチド（Ｆ）は、連続したアミノ酸配列のモチーフである「ＨＧＷＨＷＤＤ」；と、「ＨＥＴＭＥＸＬＦ」、「ＨＥＴＭＤＸＬＷ」「ＨＥＴＭＥＸＬＷ」、「ＨＥＴＮＤＸＬＦ」及び「ＨＥＴＮＮＸＬＦ」よりなる群から選ばれるいずれか１つの連続したアミノ酸配列のモチーフを有するものである。 Polypeptide (F) is a polypeptide that has a consecutive amino acid sequence motif "HGWHWDD" among homologs of the amino acid sequence (a) group; and any consecutive amino acid sequence motif selected from the group consisting of "HETMEXLF", "HETMDXLW", "HETMEXLW", "HETNDXLF" and "HETNNXLF"; and retains L-lysine 5-hydroxylation activity. More specifically, polypeptide (F) has a consecutive amino acid sequence motif "HGWHWDD"; and any one consecutive amino acid sequence motif selected from the group consisting of "HETMEXLF", "HETMDXLW", "HETMEXLW", "HETNDXLF" and "HETNNXLF".

　上記連続したアミノ酸配列のモチーフは、具体的には以下のとおりである。アミノ酸配列には、アミノ酸残基のわずかな（例えば、５個又はそれ未満）の挿入又は欠失がある可能性があることから、アミノ酸残基の位置は、参照配列である配列番号２に基づいて番号付けをしたものである。
　「ＨＧＷＨＷＤＤ」モチーフ：Ｈｉｓ１３６－Ｇｌｙ１３７－Ｔｒｐ１３８－Ｈｉｓ１３９－Ｔｒｐ１４０－Ａｓｐ１４１－Ａｓｐ１４２
　「ＨＥＴＭＥＸＬＦ」モチーフ：Ｈｉｓ２３５－Ｇｌｎ２３６－Ｔｈｒ２３７－Ｍｅｔ２３８－Ｇｌｎ２３９－Ｘ２４０－Ｌｅｕ２４１－Ｐｈｅ２４２
　「ＨＥＴＭＤＸＬＷ」モチーフ：Ｈｉｓ２３５－Ｇｌｎ２３６－Ｔｈｒ２３７－Ｍｅｔ２３８－Ａｓｐ２３９－Ｘ２４０－Ｌｅｕ２４１－Ｔｒｐ２４２
　「ＨＥＴＭＥＸＬＷ」モチーフ：Ｈｉｓ２３５－Ｇｌｎ２３６－Ｔｈｒ２３７－Ｍｅｔ２３８－Ｇｌｎ２３９－Ｘ２４０－Ｌｅｕ２４１－Ｔｒｐ２４２
　「ＨＥＴＮＤＸＬＦ」モチーフ：Ｈｉｓ２３５－Ｇｌｎ２３６－Ｔｈｒ２３７－Ａｓｎ２３８－Ａｓｐ２３９－Ｘ２４０－Ｌｅｕ２４１－Ｐｈｅ２４２
　「ＨＥＴＮＮＸＬＦ」モチーフ：Ｈｉｓ２３５－Ｇｌｎ２３６－Ｔｈｒ２３７－Ａｓｎ２３８－Ａｓｎ２３９－Ｘ２４０－Ｌｅｕ２４１－Ｐｈｅ２４２ Specifically, the motif of the consecutive amino acid sequence is as follows: Since the amino acid sequence may have a small (e.g., 5 or less) insertion or deletion of amino acid residues, the positions of the amino acid residues are numbered based on the reference sequence, SEQ ID NO: 2.
"HGWHWDD" motif: His136-Gly137-Trp138-His139-Trp140-Asp141-Asp142
"HETMEXLF" motif: His235-Gln236-Thr237-Met238-Gln239-X240-Leu241-Phe242
"HETMDXLW" motif: His235-Gln236-Thr237-Met238-Asp239-X240-Leu241-Trp242
"HETMEXLW" motif: His235-Gln236-Thr237-Met238-Gln239-X240-Leu241-Trp242
"HETNDXLF" motif: His235-Gln236-Thr237-Asn238-Asp239-X240-Leu241-Phe242
"HETNNXLF" motif: His235-Gln236-Thr237-Asn238-Asn239-X240-Leu241-Phe242

　これらの連続したアミノ酸配列のモチーフ「ＨＧＷＨＷＤＤ」、「ＨＥＴＭＥＸＬＦ」、「ＨＥＴＭＤＸＬＷ」、「ＨＥＴＭＥＸＬＷ」、「ＨＥＴＮＤＸＬＦ」及び「ＨＥＴＮＮＸＬＦ」は、Ｌ－リジン５位水酸化活性の発現に重要な役割を果たすものである。また、「ＨＥＴＭＥＸＬＦ」、「ＨＥＴＭＤＸＬＷ」、「ＨＥＴＭＥＸＬＷ」、「ＨＥＴＮＤＸＬＦ」及び「ＨＥＴＮＮＸＬＦ」は、Ｌ－リジン５位水酸化活性の光学選択性の決定に重要な役割を果たすものである。特に、「ＨＥＴＭＥＸＬＦ」はＳ選択的なＬ－リジン５位水酸化活性の発現に重要な役割を果たすものであり、「ＨＥＴＭＤＸＬＷ」、「ＨＥＴＭＥＸＬＷ」、「ＨＥＴＮＤＸＬＦ」及び「ＨＥＴＮＮＸＬＦ」はＲ選択的なＬ－リジン５位水酸化活性の発現に重要な役割を果たすものである。 These consecutive amino acid sequence motifs "HGWHWDD", "HETMEXLF", "HETMDXLW", "HETMEXLW", "HETNDXLF" and "HETNNXLF" play an important role in expressing L-lysine 5-hydroxylation activity. Furthermore, "HETMEXLF", "HETMDXLW", "HETMEXLW", "HETNDXLF" and "HETNNXLF" play an important role in determining the optical selectivity of L-lysine 5-hydroxylation activity. In particular, "HETMEXLF" plays an important role in expressing S-selective L-lysine 5-hydroxylation activity, and "HETMDXLW", "HETMEXLW", "HETNDXLF" and "HETNNXLF" play an important role in expressing R-selective L-lysine 5-hydroxylation activity.

　ポリペプチド（Ｆ）の中では、以下のポリペプチド（Ｆ－１）～（Ｆ－３）が好ましい。 Among the polypeptides (F), the following polypeptides (F-1) to (F-3) are preferred.

　ポリペプチド（Ｆ－１）は、アミノ酸配列（ａ）群のホモログのうち、配列番号２、６、８、１０、１２、１４、１６、１８、２０又は２２に示すアミノ酸配列のホモログであって、連続したアミノ酸配列のモチーフ「ＨＧＷＨＷＤＤ」及び「ＨＥＴＭＥＸＬＦ」を有するアミノ酸配列を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチドである。 Polypeptide (F-1) is a polypeptide that is a homolog of the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20, or 22 among the homologs of the amino acid sequence (a) group, has an amino acid sequence having the consecutive amino acid sequence motifs "HGWHWDD" and "HETMEXLF", and has L-lysine 5-position hydroxylation activity.

　ポリペプチド（Ｆ－１）は、Ｌ－リジンの５位をＳ選択的に水酸化することができるものであり、（２Ｓ，５Ｓ）－５－ヒドロキシ－Ｌ－リジンの製造に好適に用いることができる。また、ポリペプチド（Ｆ－１）は、一態様では、Ｃｌａｄｅ１に属するものである。 Polypeptide (F-1) is capable of selectively S-hydroxylating the 5-position of L-lysine and can be suitably used for producing (2S,5S)-5-hydroxy-L-lysine. In one embodiment, polypeptide (F-1) belongs to Clade 1.

　ポリペプチド（Ｆ－２－１）は、アミノ酸配列（ａ）群のホモログのうち、配列番号２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８又は１１０に示すアミノ酸配列のホモログであって、連続したアミノ酸配列のモチーフ「ＨＧＷＨＷＤＤ」、及び「ＨＥＴＭＤＸＬＷ」又は「ＨＥＴＭＥＸＬＷ」のいずれか１つを有するアミノ酸配列を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチドである。 Polypeptide (F-2-1) is a polypeptide that is a homolog of the amino acid sequence shown in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110 among the homologs of amino acid sequence (a), has an amino acid sequence having consecutive amino acid sequence motifs "HGWHWDD" and either "HETMDXLW" or "HETMEXLW", and has L-lysine 5-position hydroxylation activity.

　ポリペプチド（Ｆ－２－１）は、Ｌ－リジンの５位をＲ選択的に水酸化することができるものであり、（２Ｓ，５Ｒ）－５－ヒドロキシ－Ｌ－リジンの製造に好適に用いることができる。また、ポリペプチド（Ｆ－２－１）は、一態様では、Ｃｌａｄｅ２又は３に属するものである。 Polypeptide (F-2-1) is capable of R-selectively hydroxylating the 5-position of L-lysine, and can be suitably used for producing (2S,5R)-5-hydroxy-L-lysine. In one embodiment, polypeptide (F-2-1) belongs to Class 2 or 3.

　ポリペプチド（Ｆ－２－２）は、アミノ酸配列（ａ）群のホモログのうち、配列番号１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列のホモログであって、連続したアミノ酸配列のモチーフ「ＨＧＷＨＷＤＤ」、及び「ＨＥＴＮＤＸＬＦ」又は「ＨＥＴＮＮＸＬＦ」のいずれか１つを有するアミノ酸配列を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチドである。 Polypeptide (F-2-2) is a polypeptide that is a homolog of the amino acid sequence shown in SEQ ID NO: 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134 among the homologs of the amino acid sequence (a) group, has an amino acid sequence having the consecutive amino acid sequence motifs "HGWHWDD" and either "HETNDXLF" or "HETNNXLF", and has L-lysine 5-position hydroxylation activity.

　ポリペプチド（Ｆ－２－２）は、Ｌ－リジンの５位をＲ選択的に水酸化することができるものであり、（２Ｓ，５Ｒ）－５－ヒドロキシ－Ｌ－リジンの製造に好適に用いることができる。また、ポリペプチド（Ｆ－２－２）は、一態様では、Ｃｌａｄｅ４に属するものである。 Polypeptide (F-2-2) is capable of R-selectively hydroxylating the 5-position of L-lysine and can be suitably used for producing (2S,5R)-5-hydroxy-L-lysine. In one embodiment, polypeptide (F-2-2) belongs to Class 4.

　また、本明細書において例示されたＬ－リジン５位水酸化酵素間での全体の同一性（％）の比較から、シグネチャーモチーフを保持しているものであって、互いに同一性が低い酵素（例えば、配列番号２に対してわずか３１％の同一性しか有さない酵素）であっても、有意なＬ－リジン５位水酸化活性を有することが示された。 Furthermore, a comparison of the overall identity (%) between the L-lysine 5-hydroxylases exemplified in this specification showed that even enzymes that share the signature motif but have low identity to each other (for example, an enzyme with only 31% identity to SEQ ID NO:2) have significant L-lysine 5-hydroxylation activity.

　なお、アミノ酸配列（ａ）群のアミノ酸配列は、いずれもタンパク質として単離する等、実際に存在が確認された報告例は無く、また、タンパク質としての機能についても全く不明であったものである。 Furthermore, there have been no reports of the actual existence of any of the amino acid sequences in group (a) being confirmed, such as by isolating them as proteins, and their function as proteins is completely unknown.

　また、本発明のＬ－リジン５位水酸化酵素は、２－オキソグルタル酸依存性ジオキシゲナーゼのうち、主に水酸化反応を触媒する酵素に関連するシグネチャーモチーフである、以下の１）及び２）の２つの保存されたモチーフを有するものであって、Ｅ．Ｃ．番号１．１４．１１に属する２－オキソグルタル酸依存性ジオキシゲナーゼに分類されると考えられる。以下の１）及び２）において、アミノ酸残基の位置は、ＡｚｐＫ２タンパク質のアミノ酸配列（配列番号２）に基づいて番号付けをしたものである。
１）Ｈｉｓ１３９－Ｘ１４０－Ａｓｐ１４１：「ＨＸＤ」モチーフ
２）Ｔｙｒ１９７－Ａｒｇ２１６：「Ｙ－Ｒ」モチーフ Furthermore, the L-lysine 5-hydroxylase of the present invention has the following two conserved motifs 1) and 2), which are signature motifs associated with enzymes that mainly catalyze hydroxylation reactions among 2-oxoglutarate-dependent dioxygenases, and is considered to be classified as a 2-oxoglutarate-dependent dioxygenase belonging to the E.C. number 1.14.11. In the following 1) and 2), the positions of amino acid residues are numbered based on the amino acid sequence of the AzpK2 protein (SEQ ID NO: 2).
1) His139-X140-Asp141: "HXD" motif 2) Tyr197-Arg216: "Y-R" motif

　本発明のＬ－リジン５位水酸化酵素は、例えば、前述の表１に示す由来生物から精製して得ることができる。また、Ｌ－リジン５位水酸化酵素をコードするＤＮＡをＰＣＲ、ハイブリダイゼーション等の公知の方法により又は化学合成核酸を用いてクローン化し、それを適当な宿主で発現させることによって得ることもできる。 The L-lysine 5-hydroxylase of the present invention can be obtained, for example, by purification from the origin organisms shown in Table 1 above. It can also be obtained by cloning the DNA encoding the L-lysine 5-hydroxylase by known methods such as PCR or hybridization, or by using chemically synthesized nucleic acid, and expressing it in an appropriate host.

　アミノ酸配列（ａ）群のアミノ酸配列を有するＬ－リジン５位水酸化酵素をコードするＤＮＡとしては、例えば、前述の表２に示す塩基配列を有するＤＮＡが挙げられる。また、本発明のＬ－リジン５位水酸化酵素をコードする限り、表２に示す塩基配列を有するＤＮＡのホモログでもよい。 As an example of a DNA encoding an L-lysine 5-hydroxylase having an amino acid sequence in the amino acid sequence (a) group, there can be mentioned a DNA having the base sequence shown in Table 2 above. In addition, as long as it encodes the L-lysine 5-hydroxylase of the present invention, it may be a homologue of the DNA having the base sequence shown in Table 2.

　本発明のＬ－リジン５位水酸化酵素をコードするＤＮＡとしては、以下の（Ｇ）、（Ｈ）、（Ｉ）、（Ｊ）、（Ｋ）又は（Ｌ）に示すＤＮＡが挙げられる。 The DNA encoding the L-lysine 5-hydroxylase of the present invention includes the DNA shown in (G), (H), (I), (J), (K) or (L) below.

　（Ｇ）は、アミノ酸配列（ａ）群を有するＬ－リジン５位水酸化酵素をコードするＤＮＡであって、配列番号１、３、５、７、９、１１、１３、１５、１７、１９、２１、２３、２５、２７、２９、３１、３３、３５、３７、３９、４１、４３、４５、４７、４９、５１、５５、５７、６７、６９、７１、７３、７５、７９、８１、８３、８５、８７、８９、９１、９３、９５、９７、９９、１０１、１０３、１０５、１０７、１０９、１１１、１１３、１１５、１１９、１２１、１２３、１２５、１２７、１２９、１３１又は１３３に示す塩基配列（以下、「塩基配列（ｇ）群」と称することがある。）を有するＤＮＡである。 (G) is a DNA encoding an L-lysine 5-hydroxylase having the amino acid sequence (a) group, and has the base sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 57, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133 (hereinafter, sometimes referred to as "base sequence (g) group").

　（Ｈ）～（Ｌ）は、塩基配列（ｇ）群を有するＤＮＡのホモログであって、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡである。 (H) to (L) are DNAs that are homologs of DNAs having the base sequence group (g) and encode polypeptides having L-lysine 5-hydroxylation activity.

　（Ｈ）は、塩基配列（ｇ）群において、１又は複数個の塩基が置換、欠失、及び／又は付加された塩基配列を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡである。置換又は付加の場合は、１又は複数個の塩基が保守的に置換又は付加された、保守的に変異されたものが好ましい。ここでいう１又は複数個の塩基とは、通常、１個～１５０個、好ましくは１個～６０個、より好ましくは１個～３０個、さらに好ましくは１個～１５個、特に好ましくは１個～５個の塩基、より特に好ましくは１個～３個、さらに特に好ましくは１個～２個、最も好ましくは１個である。 (H) is a DNA having a base sequence in the base sequence group (g) in which one or more bases have been substituted, deleted, and/or added, and encoding a polypeptide having L-lysine 5-hydroxylation activity. In the case of substitution or addition, a conservative mutation in which one or more bases have been conservatively substituted or added is preferred. The one or more bases referred to here are usually 1 to 150, preferably 1 to 60, more preferably 1 to 30, even more preferably 1 to 15, particularly preferably 1 to 5 bases, even more particularly preferably 1 to 3, even more particularly preferably 1 to 2, and most preferably 1.

　（Ｉ）は、塩基配列（ｇ）群を有するＤＮＡのホモログのうち、配列番号１、５、７、９、１１、１３、１５、１７、１９又は２１に示す塩基配列がコードするポリペプチドと５５％以上の同一性を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡである。ここでいう５５％以上の同一性とは、通常、５５％以上、好ましくは７０％以上、より好ましくは８０％以上、さらに好ましくは８５％以上、なお一層好ましくは９０％以上、特に好ましくは９５％以上、より特に好ましくは９７％以上、さらに特に好ましくは９８％以上、最も好ましくは９９％以上の同一性である。 (I) is a DNA that has 55% or more identity with a polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19 or 21 among homologues of DNA having the nucleotide sequence (g) group, and encodes a polypeptide having L-lysine 5-hydroxylation activity. Here, 55% or more identity means usually 55% or more, preferably 70% or more, more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, particularly preferably 95% or more, even more particularly preferably 97% or more, even more particularly preferably 98% or more, and most preferably 99% or more.

　（Ｊ）は、塩基配列（ｇ）群を有するＤＮＡのホモログのうち、配列番号２３、２５、２７、２９、３１、３３、３５、３７、３９、４１、４３、４５、４７、４９、５１、５５、６７、６９、７１、７３、７５、７９、８１、８３、８５、８７、８９、９１、９３、９５、９７、９９、１０１、１０３、１０５、１０７又は１０９に示す塩基配列がコードするポリペプチドと５０％以上の同一性を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡである。ここでいう５０％以上の同一性とは、通常、５０％以上、好ましくは７０％以上、より好ましくは８０％以上、さらに好ましくは８５％以上、なお一層好ましくは９０％以上、特に好ましくは９５％以上、より特に好ましくは９７％以上、さらに特に好ましくは９８％以上、最も好ましくは９９％以上の同一性である。 (J) is a DNA that has 50% or more identity to a polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, or 109 among homologues of DNA having the nucleotide sequence (g) group, and encodes a polypeptide having L-lysine 5-position hydroxylation activity. Here, 50% or more identity means usually 50% or more, preferably 70% or more, more preferably 80% or more, even more preferably 85% or more, still more preferably 90% or more, particularly preferably 95% or more, even more particularly preferably 97% or more, even more particularly preferably 98% or more, and most preferably 99% or more.

　（Ｋ）は、塩基配列（ｇ）群を有するＤＮＡのホモログのうち、配列番号１１１、１１３、１１５、１１９、１２１、１２３、１２５、１２７、１２９、１３１又は１３３に示す塩基配列を有するＤＮＡのいずれかがコードするアミノ酸配列と５８％以上の同一性を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡである。ここでいう５８％以上の同一性とは、通常、５８％以上、好ましくは７０％以上、より好ましくは８０％以上、さらに好ましくは８５％以上、なお一層好ましくは９０％以上、特に好ましくは９５％以上、より特に好ましくは９７％以上、さらに特に好ましくは９８％以上、最も好ましくは９９％以上の同一性である。 (K) is a DNA that has 58% or more identity with an amino acid sequence encoded by any of DNAs having the base sequence shown in SEQ ID NO: 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133 among homologs of DNAs having the base sequence (g) group, and encodes a polypeptide having L-lysine 5-hydroxylation activity. Here, 58% or more identity means usually 58% or more, preferably 70% or more, more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, particularly preferably 95% or more, even more particularly preferably 97% or more, even more particularly preferably 98% or more, and most preferably 99% or more.

　（Ｌ）は、塩基配列（ｇ）群を有するＤＮＡのホモログのうち、連続したアミノ酸配列のモチーフである「ＨＧＷＨＷＤＤ」；と、「ＨＥＴＭＥＸＬＦ」、「ＨＥＴＭＤＸＬＷ」、「ＨＥＴＭＥＸＬＷ」、「ＨＥＴＮＤＸＬＦ」及び「ＨＥＴＮＮＸＬＦ」よりなる群から選ばれるいずれかの連続したアミノ酸配列のモチーフ；を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡである。 (L) is a DNA that is a homologue of DNA having the base sequence (g) group, and has a consecutive amino acid sequence motif of "HGWHWDD" and any consecutive amino acid sequence motif selected from the group consisting of "HETMEXLF", "HETMDXLW", "HETMEXLW", "HETNDXLF" and "HETNNXLF", and encodes a polypeptide having L-lysine 5-hydroxylation activity.

　当業者であれば、塩基配列（ｇ）群のＤＮＡに、部位特異的変異導入法（Ｎｕｃｌｅｉｃ　Ａｃｉｄｓ　Ｒｅｓ．　１０，ｐｐ．　６４８７　（１９８２），Ｍｅｔｈｏｄｓ　ｉｎ　Ｅｎｚｙｍｏｌ．　１００，ｐｐ．　４４８　（１９８３），Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ，ＰＣＲ　Ａ　Ｐｒａｃｔｉｃａｌ　Ａｐｐｒｏａｃｈ　ＩＲＬ　Ｐｒｅｓｓ　ｐｐ．　２００　（１９９１）等を用いて、適宜、置換、欠失、挿入、及び／又は付加変異を導入することにより、前記のようなＤＮＡのホモログを得ることが可能である。 Those skilled in the art can obtain a homologue of the above DNA by introducing substitution, deletion, insertion, and/or additional mutations into the DNA of the base sequence (g) group using site-directed mutagenesis (Nucleic Acids Res. 10, pp. 6487 (1982), Methods in Enzymol. 100, pp. 448 (1983), Molecular Cloning, PCR A Practical Approach IRL Press pp. 200 (1991), etc.

　また、アミノ酸配列（ａ）群のアミノ酸配列またはその一部や、塩基配列（ｇ）群の塩基配列またはその一部をもとに、例えばＤＮＡ Ｄａｔａｂａｎｋ ｏｆ ＪＡＰＡＮ（ＤＤＢＪ）等のデータベースに対してホモロジー検索を行って、Ｌ－リジン５位水酸化酵素のアミノ酸情報又はそれをコードするＤＮＡの塩基配列情報を入手することも可能である。 In addition, it is also possible to perform a homology search in a database such as the DNA Databank of JAPAN (DDBJ) based on the amino acid sequence of the amino acid sequence (a) group or a part thereof, or the base sequence of the base sequence (g) group or a part thereof, to obtain amino acid information of L-lysine 5-hydroxylase or base sequence information of the DNA encoding it.

　後述する本発明の５－ヒドロキシ－Ｌ－リジンの製造方法においては、Ｌ－リジン５位水酸化酵素を直接反応に使用してもよいが、Ｌ－リジン５位水酸化酵素を含む細胞、当該細胞の調製物、又は当該細胞を培養して得られた培養液を用いてもよい。 In the method for producing 5-hydroxy-L-lysine of the present invention described below, L-lysine 5-hydroxylase may be used directly in the reaction, but cells containing L-lysine 5-hydroxylase, a preparation of said cells, or a culture medium obtained by culturing said cells may also be used.

　Ｌ－リジン５位水酸化酵素を含む細胞としては、もともとＬ－リジン５位水酸化酵素を有する微生物等の細胞を用いてもよいが、入手しやすさ等から、Ｌ－リジン５位水酸化酵素をコードするＤＮＡで形質転換した微生物等の細胞を用いることが好ましい。ここで、細胞としては、その生死は問わず、例えば、入手しやすさ等から休止菌体等を好適に用いることができる。 As cells containing L-lysine 5-hydroxylase, cells of a microorganism or the like that originally have L-lysine 5-hydroxylase may be used, but in terms of ease of availability, it is preferable to use cells of a microorganism or the like that have been transformed with DNA that codes for L-lysine 5-hydroxylase. Here, the cells may be either live or dead, and in terms of ease of availability, for example, resting cells can be preferably used.

　また、Ｌ－リジン５位水酸化酵素を含む細胞の調製物としては、Ｌ－リジン５位水酸化酵素を含み、当該酵素活性を有する調製物であって、例えば、当該細胞をアセトン、ジメチルスルホキシド、トルエン等の有機溶媒や界面活性剤により処理したもの、凍結乾燥処理したもの、物理的又は酵素的に破砕したもの等の細胞処理物、細胞中の酵素画分を粗製物又は精製物として取得したもの、当該細胞処理物又は取得物をポリアクリルアミドゲル、カラギーナンゲル等に代表される担体に固定化したもの等を用いることができる。 Furthermore, examples of cell preparations containing L-lysine 5-hydroxylase include preparations that contain L-lysine 5-hydroxylase and have the enzyme activity, such as cells treated with an organic solvent such as acetone, dimethyl sulfoxide, or toluene, or a surfactant, freeze-dried cells, or cells that have been physically or enzymatically disrupted, cell treatments in which the enzyme fraction in the cells has been obtained as a crude or purified product, and cell treatments or preparations immobilized on a carrier such as polyacrylamide gel or carrageenan gel.

　Ｌ－リジン５位水酸化酵素を含む細胞を培養して得られた培養液としては、Ｌ－リジン５位水酸化酵素を含み、当該酵素活性を有する培養液であって、例えば、当該細胞と液体培地の懸濁液や、当該細胞が分泌発現型細胞である場合は当該細胞を遠心分離等で除去した上清又はその濃縮物を用いることができる。 The culture medium obtained by culturing cells containing L-lysine 5-hydroxylase is a culture medium that contains L-lysine 5-hydroxylase and has the enzyme activity, and may be, for example, a suspension of the cells and a liquid medium, or, if the cells are secretory expression cells, the supernatant obtained by removing the cells by centrifugation or the like, or a concentrate thereof.

　Ｌ－リジン５位水酸化酵素をコードするＤＮＡで形質転換した微生物等の細胞（形質転換体）の作製方法としては、例えば、微生物等の宿主細胞において安定に存在するプラスミドベクター、ファージベクター又はウイルスベクター中に、Ｌ－リジン５位水酸化酵素をコードするＤＮＡを導入し、構築された発現ベクターを当該宿主細胞中に導入する方法、直接宿主ゲノム中に当該ＤＮＡを導入し、その遺伝情報を転写・翻訳させる方法等が挙げられる。このとき、宿主において適当なプロモーターをＤＮＡの５’－側上流に連結させることは好ましく、さらに、ターミネーターを３’－側下流に連結させることがより好ましい。このようなプロモーター及びターミネーターとしては、宿主として利用する細胞中において機能することが知られているプロモーター及びターミネーターであれば特に限定されず、例えば、「微生物学基礎講座８遺伝子工学・共立出版」等に記載されている、宿主細胞において利用可能なベクター、プロモーター及びターミネーターを用いることができる。 Methods for producing cells (transformants) of microorganisms or the like transformed with DNA encoding L-lysine 5-hydroxylase include, for example, a method of introducing DNA encoding L-lysine 5-hydroxylase into a plasmid vector, phage vector, or virus vector that is stable in a host cell of a microorganism, and introducing the constructed expression vector into the host cell, and a method of directly introducing the DNA into the host genome and transcribing and translating the genetic information. In this case, it is preferable to link a suitable promoter to the 5'-side upstream of the DNA in the host, and more preferably to link a terminator to the 3'-side downstream. Such promoters and terminators are not particularly limited as long as they are promoters and terminators known to function in the cells used as the host, and for example, vectors, promoters, and terminators that can be used in host cells, as described in "Basic Microbiology Lectures 8: Genetic Engineering, Kyoritsu Shuppan" and the like, can be used.

　具体的には、Ｌ－リジン５位水酸化酵素をコードするＤＮＡを公知の発現ベクターに発現可能に挿入することにより得られたＬ－リジン水酸化酵素発現ベクターで宿主細胞を形質転換することにより、Ｌ－リジン５位水酸化酵素をコードするＤＮＡが導入された形質転換体を得ることができる。また、宿主細胞の染色体ＤＮＡにＬ－リジン５位水酸化酵素をコードするＤＮＡを相同組み換え等の手法によって発現可能に組み込むことによって形質転換体を得ることができる。 Specifically, a transformant having DNA encoding L-lysine 5-hydroxylase introduced therein can be obtained by transforming a host cell with an L-lysine hydroxylase expression vector obtained by expressibly inserting DNA encoding L-lysine 5-hydroxylase into a known expression vector. Alternatively, a transformant can be obtained by expressibly incorporating DNA encoding L-lysine 5-hydroxylase into the chromosomal DNA of a host cell by a method such as homologous recombination.

　Ｌ－リジン５位水酸化酵素を発現させるための形質転換の対象となる宿主細胞（微生物）としては、それ自体がＬ－リジンの水酸化反応に悪影響を与えない限り特に限定されることはなく、具体的には以下に示すような微生物を挙げることができる。
　Ｅｓｃｈｅｒｉｃｈｉａ属、Ｂａｃｉｌｌｕｓ属、Ｐｓｅｕｄｏｍｏｎａｓ属、Ｓｅｒｒａｔｉａ属、Ｂｒｅｖｉｂａｃｔｅｒｉｕｍ属、Ｃｏｒｙｎｅｂａｃｔｅｒｉｕｍ属、Ｓｔｒｅｐｔｏｃｏｃｃｕｓ属、Ｌａｃｔｏｂａｃｉｌｌｕｓ属等に属する宿主ベクター系の確立されている細菌。
　Ｒｈｏｄｏｃｏｃｃｕｓ属、Ｓｔｒｅｐｔｏｍｙｃｅｓ属等に属する宿主ベクター系の確立されている放線菌。
　Ｓａｃｃｈａｒｏｍｙｃｅｓ属、Ｋｌｕｙｖｅｒｏｍｙｃｅｓ属、Ｓｃｈｉｚｏｓａｃｃｈａｒｏｍｙｃｅｓ属、Ｚｙｇｏｓａｃｃｈａｒｏｍｙｃｅｓ属、Ｙａｒｒｏｗｉａ属、Ｔｒｉｃｈｏｓｐｏｒｏｎ属、Ｒｈｏｄｏｓｐｏｒｉｄｉｕｍ属、Ｈａｎｓｅｎｕｌａ属、Ｐｉｃｈｉａ属、Ｃａｎｄｉｄａ属等に属する宿主ベクター系の確立されている酵母。
　Ｎｅｕｒｏｓｐｏｒａ属、Ａｓｐｅｒｇｉｌｌｕｓ属、Ｃｅｐｈａｌｏｓｐｏｒｉｕｍ属、Ｔｒｉｃｈｏｄｅｒｍａ属等に属する宿主ベクター系の確立されているカビ。 The host cell (microorganism) to be transformed to express L-lysine 5-hydroxylase is not particularly limited as long as it does not adversely affect the hydroxylation reaction of L-lysine. Specific examples of the host cell include the following microorganisms:
Bacteria belonging to the genera Escherichia, Bacillus, Pseudomonas, Serratia, Brevibacterium, Corynebacterium, Streptococcus, Lactobacillus, etc., for which a host-vector system has been established.
Actinomycetes belonging to the genera Rhodococcus and Streptomyces, etc., for which host-vector systems have been established.
Yeasts belonging to the genera Saccharomyces, Kluyveromyces, Schizosaccharomyces, Zygosaccharomyces, Yarrowia, Trichosporon, Rhodosporidium, Hansenula, Pichia, Candida, etc. for which host-vector systems have been established.
Fungi of the genera Neurospora, Aspergillus, Cephalosporium, Trichoderma, etc., for which host-vector systems have been established.

　形質転換体作製のための手順、宿主に適合した組換えベクターの構築及び宿主の培養方法は、分子生物学、生物工学、遺伝子工学の分野において慣用されている技術に準じて行うことができる（例えば、Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇに記載の方法）。 The procedures for producing transformants, the construction of recombinant vectors suitable for the host, and the method for culturing the host can be carried out in accordance with techniques commonly used in the fields of molecular biology, biotechnology, and genetic engineering (for example, the methods described in Molecular Cloning).

　以下、具体的に、好ましい宿主微生物、各微生物における好ましい形質転換の手法、ベクター、プロモーター、ターミネーター等の例を挙げるが、本発明はこれらの例に限定されない。 Below are specific examples of preferred host microorganisms, preferred transformation methods for each microorganism, vectors, promoters, terminators, etc., but the present invention is not limited to these examples.

　Ｅｓｃｈｅｒｉｃｈｉａ属、特にＥｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉにおいては、プラスミドベクターとしては、ｐＢＲ、ｐＵＣ系プラスミド等が挙げられ、ｌａｃ（β－ガラクトシダーゼ）、ｔｒｐ（トリプトファンオペロン）、ｔａｃ、ｔｒｃ（ｌａｃ、ｔｒｐの融合）、λファージＰＬ、ＰＲ等に由来するプロモーター等が挙げられる。また、ターミネーターとしては、ｔｒｐＡ由来、ファージ由来、ｒｒｎＢリボソーマルＲＮＡ由来のターミネーター等が挙げられる。 In the genus Escherichia, particularly Escherichia coli, examples of plasmid vectors include pBR and pUC-based plasmids, and examples of promoters include those derived from lac (β-galactosidase), trp (tryptophan operon), tac, trc (lac and trp fusion), and λ phages PL and PR. Examples of terminators include those derived from trpA, phages, and rrnB ribosomal RNA.

　Ｂａｃｉｌｌｕｓ属においては、ベクターとしては、ｐＵＢ１１０系プラスミド、ｐＣ１９４系プラスミド等を挙げることができ、また、染色体にインテグレートすることもできる。プロモーター及びターミネーターとしては、アルカリプロテアーゼ、中性プロテアーゼ、α－アミラーゼ等の酵素遺伝子のプロモーターやターミネーター等が利用できる。 In the genus Bacillus, examples of vectors include pUB110-based plasmids and pC194-based plasmids, and they can also be integrated into chromosomes. Examples of promoters and terminators that can be used include promoters and terminators of enzyme genes such as alkaline protease, neutral protease, and α-amylase.

　Ｐｓｅｕｄｏｍｏｎａｓ属においては、ベクターとしては、Ｐｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉｄａ、Ｐｓｅｕｄｏｍｏｎａｓ　ｃｅｐａｃｉａ等で確立されている一般的な宿主ベクター系や、トルエン化合物の分解に関与するプラスミド、ＴＯＬプラスミドを基本にした広宿主域ベクター（ＲＳＦ１０１０等に由来する自律的複製に必要な遺伝子を含む）ｐＫＴ２４０（Ｇｅｎｅ　２６，２７３－２８２（１９８３））等を挙げることができる。 In the genus Pseudomonas, examples of vectors include general host vector systems established for Pseudomonas putida, Pseudomonas cepacia, etc., plasmids involved in the decomposition of toluene compounds, and the broad host range vector pKT240 (Gene 26, 273-282 (1983)) based on the TOL plasmid (containing genes necessary for autonomous replication derived from RSF1010, etc.).

　また、前記以外でも、各種微生物に応じた宿主ベクター系が確立されており、それらを適宜使用することができる。 In addition to the above, host-vector systems have been established for various microorganisms, and these can be used as appropriate.

　また、微生物以外でも、植物、動物において様々な宿主・ベクター系が確立されており、例えば、昆虫（例えば、蚕）等の動物中（Ｎａｔｕｒｅ　３１５，　５９２－５９４　（１９８５））や、菜種、トウモロコシ、ジャガイモ等の植物中に大量に異種タンパク質を発現させる系、及び大腸菌無細胞抽出液や小麦胚芽等の無細胞タンパク質合成系等を好適に使用することができる。 In addition to microorganisms, various host-vector systems have been established in plants and animals. For example, systems that express large amounts of heterologous proteins in animals such as insects (e.g., silkworms) (Nature 315, 592-594 (1985)) and plants such as rapeseed, corn, and potato, as well as cell-free protein synthesis systems such as cell-free extracts of E. coli and wheat germ can be suitably used.

　本発明のＬ－リジン５位水酸化酵素のうち、以下の（Ａ－１）、（Ｂ－１）、（Ｃ－１）又は（Ｆ－１）に示すポリペプチドを含むものは、Ｓ選択的Ｌ－リジン５位水酸化酵素である。
（Ａ－１）配列番号２、６、８、１０、１２、１４、１６、１８、２０又は２２に示すアミノ酸配列を有するポリペプチド
（Ｂ－１）配列番号２、６、８、１０、１２、１４、１６、１８、２０又は２２に示すアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換及び／又は付加されたアミノ酸配列を有し、かつ、Ｓ選択的Ｌ－リジン５位水酸化活性を有するポリペプチド
（Ｃ－１）配列番号２、６、８、１０、１２、１４、１６、１８、２０又は２２に示すアミノ酸配列全長と５５％以上の配列同一性を有するアミノ酸配列を有し、かつ、Ｓ選択的Ｌ－リジン５位水酸化酵素活性を有するポリペプチド
（Ｆ－１）連続したアミノ酸配列のモチーフである「ＨＧＷＨＷＤＤ」及び「ＨＥＴＭＥＸＬＦ」を有し、かつ、Ｓ選択的Ｌ－リジン５位水酸化活性を保持するポリペプチド Among the L-lysine 5-hydroxylases of the present invention, those containing the polypeptide shown in (A-1), (B-1), (C-1) or (F-1) below are S-selective L-lysine 5-hydroxylases.
(A-1) A polypeptide having an amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22. (B-1) A polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted and/or added in the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22, and having S-selective L-lysine 5-hydroxylation activity. (C-1) A polypeptide having an amino acid sequence having 55% or more sequence identity with the full length amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22, and having S-selective L-lysine 5-hydroxylation activity. (F-1) A polypeptide having consecutive amino acid sequence motifs "HGWHWDD" and "HETMEXLF", and having S-selective L-lysine 5-hydroxylation activity.

　これらのＳ選択的Ｌ－リジン５位水酸化酵素は、例えば、以下の（Ｇ－１）、（Ｈ－１）、（Ｉ－１）又は（Ｌ－１）に示すＤＮＡをＰＣＲ、ハイブリダイゼーション等の公知の方法により又は化学合成核酸を用いてクローン化し、それを適当な宿主で発現させることによって得ることができる。
（Ｇ－１）配列番号１、５、７、９、１１、１３、１５、１７、１９又は２１に示す塩基配列を含むＤＮＡ
（Ｈ－１）配列番号１、５、７、９、１１、１３、１５、１７、１９又は２１に示す塩基配列において、１又は複数個の塩基が置換、欠失、及び／又は付加された塩基配列を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ
（Ｉ－１）配列番号１、５、７、９、１１、１３、１５、１７、１９又は２１に示す塩基配列がコードするポリペプチドと５５％以上の配列同一性を有し、かつ、Ｓ選択的Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ
（Ｌ－１）連続したアミノ酸配列のモチーフである「ＨＧＷＨＷＤＤ」及び「ＨＥＴＭＥＸＬＦ」を有し、かつ、Ｓ選択的Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ These S-selective L-lysine 5-hydroxylases can be obtained, for example, by cloning the DNA shown in (G-1), (H-1), (I-1) or (L-1) below by a known method such as PCR or hybridization or by using a chemically synthesized nucleic acid, and expressing the clone in an appropriate host.
(G-1) DNA containing the base sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19 or 21
(H-1) A DNA encoding a polypeptide having a nucleotide sequence in which one or more nucleotides are substituted, deleted, and/or added in the nucleotide sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, or 21, and having an activity of hydroxylating the 5-position of L-lysine.
(I-1) A DNA encoding a polypeptide having 55% or more sequence identity with a polypeptide encoded by the base sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19 or 21 and having S-selective L-lysine 5-hydroxylation activity.
(L-1) DNA encoding a polypeptide having the consecutive amino acid sequence motifs "HGWHWDD" and "HETMEXLF" and having S-selective L-lysine 5-hydroxylation activity.

　また、本発明のＬ－リジン５位水酸化酵素のうち、以下の（Ａ－２）、（Ｂ－２）、（Ｄ－２）、（Ｅ－２）又は（Ｆ－２）に示すポリペプチドを含むものは、Ｒ選択的Ｌ－リジン５位水酸化酵素である。
（Ａ－２）配列番号４、２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８、１１０、１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列を有するポリペプチド
（Ｂ－２）配列番号４、２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８、１１０、１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換及び／又は付加されたアミノ酸配列を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチド
（Ｄ－２）配列番号２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８又は１１０に示すアミノ酸配列全長と５０％以上の配列同一性を有するアミノ酸配列を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチド
（Ｅ－２）配列番号１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列全長と５８％以上の配列同一性を有するアミノ酸配列を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチド
（Ｆ－２）連続したアミノ酸配列のモチーフである「ＨＧＷＨＷＤＤ」；と、「ＨＥＴＭＤＸＬＷ」、「ＨＥＴＭＥＸＬＷ」、「ＨＥＴＮＤＸＬＦ」及び「ＨＥＴＮＮＸＬＦ」よりなる群から選ばれるいずれかの連続したアミノ酸配列のモチーフ；とを有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を保持するポリペプチド Furthermore, among the L-lysine 5-hydroxylases of the present invention, those containing the polypeptide shown in (A-2), (B-2), (D-2), (E-2) or (F-2) below are R-selective L-lysine 5-hydroxylases.
(A-2) A polypeptide having an amino acid sequence shown in SEQ ID NO: 4, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134 (B-2) SEQ ID NO: 4, 24, 26, 28, 30, 32, 34, 36, 38 , 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134, in which one or more amino acids are deleted, substituted and/or added, and which has R-selective L-lysine 5-hydroxylation activity. A polypeptide (E-2) having an amino acid sequence having 50% or more sequence identity with the full length amino acid sequence shown in No. 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108 or 110 and having R-selective L-lysine 5-position hydroxylation activity. SEQ ID NO: 112, 114, 116, 120, 122, 124, 126, 128 130, 132 or 134, and having R-selective L-lysine 5-hydroxylation activity (F-2) A polypeptide having a contiguous amino acid sequence motif "HGWHWDD" and any contiguous amino acid sequence motif selected from the group consisting of "HETMDXLW", "HETMEXLW", "HETNDXLF" and "HETNNXLF", and having R-selective L-lysine 5-hydroxylation activity.

　これらのＲ選択的Ｌ－リジン５位水酸化酵素は、例えば、以下の（Ｇ－２）、（Ｈ－２）、（Ｊ－２）、（Ｋ－２）又は（Ｌ－２）に示すＤＮＡをＰＣＲ、ハイブリダイゼーション等の公知の方法により又は化学合成核酸を用いてクローン化し、それを適当な宿主で発現させることによって得ることができる。
（Ｇ－２）配列番号３、２３、２５、２７、２９、３１、３３、３５、３７、３９、４１、４３、４５、４７、４９、５１、５５、６７、６９、７１、７３、７５、７９、８１、８３、８５、８７、８９、９１、９３、９５、９７、９９、１０１、１０３、１０５、１０７、１０９、１１１、１１３、１１５、１１９、１２１、１２３、１２５、１２７、１２９、１３１又は１３３に示す塩基配列を含むＤＮＡ
（Ｈ－２）配列番号３、２３、２５、２７、２９、３１、３３、３５、３７、３９、４１、４３、４５、４７、４９、５１、５５、６７、６９、７１、７３、７５、７９、８１、８３、８５、８７、８９、９１、９３、９５、９７、９９、１０１、１０３、１０５、１０７、１０９、１１１、１１３、１１５、１１９、１２１、１２３、１２５、１２７、１２９、１３１又は１３３に示す塩基配列において、１又は複数個の塩基が置換、欠失、及び／又は付加された塩基配列を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ
（Ｊ－２）配列番号２３、２５、２７、２９、３１、３３、３５、３７、３９、４１、４３、４５、４７、４９、５１、５５、６７、６９、７１、７３、７５、７９、８１、８３、８５、８７、８９、９１、９３、９５、９７、９９、１０１、１０３、１０５、１０７又は１０９に示す塩基配列がコードするポリペプチドと５０％以上の配列同一性を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ
（Ｋ－２）配列番号１１１、１１３、１１５、１１９、１２１、１２３、１２５、１２７、１２９、１３１又は１３３に示す塩基配列がコードするポリペプチドと５８％以上の配列同一性を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ
（Ｌ－２）連続したアミノ酸配列のモチーフである「ＨＧＷＨＷＤＤ」；と、「ＨＥＴＭＤＸＬＷ」、「ＨＥＴＭＥＸＬＷ」、「ＨＥＴＮＤＸＬＦ」及び「ＨＥＴＮＮＸＬＦ」よりなる群から選ばれるいずれかの連続したアミノ酸配列のモチーフ；を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ These R-selective L-lysine 5-hydroxylases can be obtained, for example, by cloning the DNA shown in (G-2), (H-2), (J-2), (K-2) or (L-2) below by a known method such as PCR or hybridization or by using a chemically synthesized nucleic acid, and expressing the clone in an appropriate host.
(G-2) DNA containing the base sequence shown in SEQ ID NO: 3, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133
(H-2) A DNA encoding a polypeptide having an R-selective L-lysine 5-hydroxylation activity, comprising a base sequence in which one or more bases are substituted, deleted, and/or added in the base sequence shown in SEQ ID NO: 3, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131, or 133.
(J-2) A DNA encoding a polypeptide having 50% or more sequence identity to a polypeptide encoded by the base sequence shown in SEQ ID NO: 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107 or 109 and having R-selective L-lysine 5-hydroxylation activity.
(K-2) DNA encoding a polypeptide having 58% or more sequence identity with a polypeptide encoded by the base sequence shown in SEQ ID NO: 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133 and having R-selective L-lysine 5-hydroxylation activity.
(L-2) A DNA encoding a polypeptide having a consecutive amino acid sequence motif of "HGWHWDD" and any consecutive amino acid sequence motif selected from the group consisting of "HETMDXLW", "HETMEXLW", "HETNDXLF" and "HETNNXLF", and having R-selective L-lysine 5-hydroxylation activity.

３．酵素剤組成物
　本発明の酵素剤組成物は、本発明のＬ－リジン５位水酸化酵素、又はそれを含む細胞、当該細胞の調製物、又は当該細胞を培養して得られた培養液（以下、合わせて「本発明のＬ－リジン５位水酸化酵素等」と称することがある。）を含み、前記Ｌ－リジン５位水酸化酵素活性を有するものである。本発明の酵素剤組成物は、触媒として使用することにより、Ｌ－リジンから高収率かつ低コストで安価に５－ヒドロキシ－Ｌ－リジンを製造することができる。また、本発明の酵素剤組成物は、Ｌ－リジンを水酸化する際の位置選択性及び立体選択性が高いため、効率よく、目的とする光学異性体の５－ヒドロキシ－Ｌ－リジンを得ることができる。 3. Enzyme Composition The enzyme composition of the present invention contains the L-lysine 5-hydroxylase of the present invention, or a cell containing it, a preparation of the cell, or a culture solution obtained by culturing the cell (hereinafter, these may be collectively referred to as "the L-lysine 5-hydroxylase of the present invention, etc."), and has the L-lysine 5-hydroxylase activity. When used as a catalyst, the enzyme composition of the present invention can inexpensively produce 5-hydroxy-L-lysine from L-lysine with high yield and low cost. In addition, the enzyme composition of the present invention has high regioselectivity and stereoselectivity when hydroxylating L-lysine, and therefore can efficiently obtain the desired optical isomer of 5-hydroxy-L-lysine.

　本発明の酵素剤組成物は、有効成分である本発明のＬ－リジン５位水酸化酵素等の他、賦形剤、緩衝剤、懸濁剤、安定剤、保存剤、防腐剤、生理食塩水等を含有していてもよい。賦形剤としては乳糖、ソルビトール、Ｄ－マンニトール、白糖等を用いることができる。緩衝剤としてはリン酸塩、クエン酸塩、酢酸塩等を用いることができる。安定剤としてはプロピレングリコール、アスコルビン酸等を用いることができる。保存剤としてはフェノール、塩化ベンザルコニウム、ベンジルアルコール、クロロブタノール、メチルパラベン等を用いることができる。防腐剤としては塩化ベンザルコニウム、パラオキシ安息香酸、クロロブタノール等と用いることができる。本発明の酵素剤組成物におけるＬ－リジン５位水酸化酵素等の含有量としては、Ｌ－リジン５位水酸化酵素の効果が発揮される範囲で適宜設定される。例えば、後述の本発明の５－ヒドロキシ－Ｌ－リジンの製造方法に使用する量を達成し得るように適宜設定される。例えば、酵素剤組成物全体の０．０１～１００重量％、０．１～１０重量％、又は１～５０重量％等で配合され得る。本発明の酵素剤組成物は、通常行われている製剤化方法で製造することができる。 The enzyme composition of the present invention may contain, in addition to the active ingredient L-lysine 5-hydroxylase of the present invention, excipients, buffers, suspending agents, stabilizers, preservatives, antiseptics, physiological saline, etc. Examples of excipients that can be used include lactose, sorbitol, D-mannitol, sucrose, etc. Examples of buffers that can be used include phosphates, citrates, acetates, etc. Examples of stabilizers that can be used include propylene glycol, ascorbic acid, etc. Examples of preservatives that can be used include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, etc. Examples of preservatives that can be used include benzalkonium chloride, paraoxybenzoic acid, chlorobutanol, etc. The content of L-lysine 5-hydroxylase, etc. in the enzyme composition of the present invention is appropriately set within a range in which the effect of L-lysine 5-hydroxylase is exerted. For example, the content is appropriately set so as to achieve the amount used in the production method of 5-hydroxy-L-lysine of the present invention described below. For example, it can be blended at 0.01 to 100% by weight, 0.1 to 10% by weight, or 1 to 50% by weight of the total enzyme composition. The enzyme composition of the present invention can be manufactured using a commonly used formulation method.

４．Ｌ－リジン５位水酸化酵素を用いた５－ヒドロキシ－Ｌ－リジンの製造方法
　本発明の５－ヒドロキシ－Ｌ－リジンの製造方法は、Ｌ－リジンに、本発明のＬ－リジン５位水酸化酵素、又はそれを含む細胞、当該細胞の調製物若しくは当該細胞を培養して得られた培養液を接触させることにより、下記式（Ｉ）： 4. Method for Producing 5-hydroxy-L-lysine Using L-lysine 5-Hydroxylase The method for producing 5-hydroxy-L-lysine of the present invention comprises contacting L-lysine with the L-lysine 5-hydroxylase of the present invention, or a cell containing it, a preparation of the cell, or a culture solution obtained by culturing the cell, thereby obtaining a compound represented by the following formula (I):

　で表される５－ヒドロキシ－Ｌ－リジンを製造することを特徴とする。 The method is characterized by producing 5-hydroxy-L-lysine represented by the formula:

　前記式（Ｉ）としては、５位のヒドロキシル基がＳ体である下記式（II）： The above formula (I) includes the following formula (II) in which the hydroxyl group at the 5-position is S-form:

　又は、５位のヒドロキシル基がＲ体である下記式（III）： Or the following formula (III) in which the hydroxyl group at the 5-position is the R-form:

　のどちらか一方であることが好ましい。 It is preferable to have one of the two.

　式（II）で表される化合物は（２Ｓ，５Ｓ）－５－ヒドロキシ－Ｌ－リジンであり、式（III）で表される化合物は（２Ｓ，５Ｒ）－５－ヒドロキシ－Ｌ－リジンである。 The compound represented by formula (II) is (2S,5S)-5-hydroxy-L-lysine, and the compound represented by formula (III) is (2S,5R)-5-hydroxy-L-lysine.

　本発明の製造方法において、本発明のＬ－リジン５位水酸化酵素等が前記ポリペプチド（Ａ）及び／又は（Ｂ）を含有する場合は、式（Ｉ）で表される５－ヒドロキシ－Ｌ－リジンを製造することができる。
　本発明のＬ－リジン５位水酸化酵素等が前記ポリペプチド（Ｃ）を含有する場合は式（II）で表される（２Ｓ，５Ｓ）－５－ヒドロキシ－Ｌ－リジンを製造することができる。
　本発明のＬ－リジン５位水酸化酵素等が前記ポリペプチド（Ｄ）又は（Ｅ）を含有する場合は、式（III）で表される（２Ｓ，５Ｒ）－５－ヒドロキシ－Ｌ－リジンを製造することができる。
　本発明のＬ－リジン５位水酸化酵素等が前記ポリペプチド（Ｆ）を含有する場合は、式（Ｉ）で表される５－ヒドロキシ－Ｌ－リジン、本発明のＬ－リジン５位水酸化酵素等が前記ポリペプチド（Ｆ－１）を含有する場合は、式（II）で表される（２Ｓ，５Ｓ）－５－ヒドロキシ－Ｌ－リジン、本発明のＬ－リジン５位水酸化酵素等が前記ポリペプチド（Ｆ－２）及び／又は（Ｆ－３）を含有する場合は、式（III）で表される（２Ｓ，５Ｒ）－５－ヒドロキシ－Ｌ－リジンを製造することができる。 In the production method of the present invention, when the L-lysine 5-hydroxylase or the like of the present invention contains the above-mentioned polypeptides (A) and/or (B), 5-hydroxy-L-lysine represented by formula (I) can be produced.
When the L-lysine 5-hydroxylase of the present invention contains the above polypeptide (C), it is possible to produce (2S,5S)-5-hydroxy-L-lysine represented by the formula (II).
When the L-lysine 5-hydroxylase or the like of the present invention contains the above polypeptide (D) or (E), it is possible to produce (2S,5R)-5-hydroxy-L-lysine represented by formula (III).
When the L-lysine 5-hydroxylase or the like of the present invention contains the polypeptide (F), 5-hydroxy-L-lysine represented by formula (I) can be produced; when the L-lysine 5-hydroxylase or the like of the present invention contains the polypeptide (F-1), (2S,5S)-5-hydroxy-L-lysine represented by formula (II) can be produced; and when the L-lysine 5-hydroxylase or the like of the present invention contains the polypeptides (F-2) and/or (F-3), (2S,5R)-5-hydroxy-L-lysine represented by formula (III) can be produced.

　本発明の製造方法においては、複数の種類のＬ－リジン５位水酸化酵素等を併用してもよい。この場合、同じ立体選択性を有するＬ－リジン５位水酸化酵素等を併用することが好ましい。 In the production method of the present invention, multiple types of L-lysine 5-hydroxylases may be used in combination. In this case, it is preferable to use L-lysine 5-hydroxylases having the same stereoselectivity in combination.

　Ｌ－リジンに本発明のＬ－リジン５位水酸化酵素等を接触させる方法は、両者を接触させることができれば特に限定されないが、通常、水性媒体中、又は水性媒体と有機溶媒との混合物中等の液体中で行うことが好ましい。また、水性媒体と有機溶媒との混合物の場合は、水性媒体の比率が大きい方が好ましい。水性媒体としては、例えば、水又はグッド緩衝液、リン酸緩衝液、トリス緩衝液、ホウ酸緩衝液等の公知の緩衝液（バッファー）が挙げられる。有機溶媒としては、メタノール、エタノール、１－プロパノール、２－プロパノール、１－ブタノール、ｔｅｒｔ－ブタノール、アセトン、ジメチルスルホキシド等、反応基質であるＬ-リジンの溶解度が高いものを使用することができる。また、有機溶媒としては、反応副産物の除去等に効果のある、酢酸エチル、酢酸ブチル、トルエン、クロロホルム、ｎ－ヘキサン、メチルｔｅｒｔ－ブチルエーテル等を使用することもできる。 The method of contacting L-lysine with the L-lysine 5-position hydroxylase of the present invention is not particularly limited as long as it allows the two to come into contact, but it is usually preferable to carry out the method in a liquid such as an aqueous medium or a mixture of an aqueous medium and an organic solvent. In the case of a mixture of an aqueous medium and an organic solvent, it is preferable that the ratio of the aqueous medium is large. Examples of the aqueous medium include water and well-known buffer solutions (buffers) such as Good's buffer, phosphate buffer, Tris buffer, and borate buffer. As the organic solvent, those that have high solubility of L-lysine as a reaction substrate, such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, tert-butanol, acetone, and dimethyl sulfoxide, can be used. As the organic solvent, ethyl acetate, butyl acetate, toluene, chloroform, n-hexane, methyl tert-butyl ether, and the like, which are effective in removing reaction by-products, can also be used.

　また、Ｌ－リジンに本発明のＬ－リジン５位水酸化酵素等を接触させて反応させる方法としては、例えば、本発明のＬ－リジン５位水酸化酵素等を含む液体に反応基質であるＬ－リジンを添加する、Ｌ－リジンを含む液体に本発明のＬ－リジン５位水酸化酵素を添加する等が挙げられる。酵素への阻害作用があった場合の影響を減らす、目的生成物の蓄積濃度を向上させる、副生物を低減する等の観点から、本発明のＬ－リジン５位水酸化酵素等を含む液体に反応基質であるＬ－リジンを添加することが好ましい。 In addition, examples of methods for contacting L-lysine with the L-lysine 5-hydroxylase or the like of the present invention to cause a reaction include adding L-lysine, which is a reaction substrate, to a liquid containing the L-lysine 5-hydroxylase or the like of the present invention, and adding the L-lysine 5-hydroxylase or the like of the present invention to a liquid containing L-lysine. From the viewpoints of reducing the influence of any inhibitory action on the enzyme, increasing the accumulated concentration of the target product, and reducing by-products, it is preferable to add L-lysine, which is a reaction substrate, to a liquid containing the L-lysine 5-hydroxylase or the like of the present invention.

　Ｌ－リジンと本発明のＬ－リジン５位水酸化酵素等が接触し反応することにより、Ｌ－リジンが水酸化されて５－ヒドロキシ－Ｌ－リジンが生成する。 When L-lysine comes into contact with and reacts with the L-lysine 5-hydroxylase of the present invention, L-lysine is hydroxylated to produce 5-hydroxy-L-lysine.

　使用する本発明のＬ－リジン５位水酸化酵素等の量は、Ｌ－リジンを水酸化して５－ヒドロキシ－Ｌ－リジンを生成できる量であればよく、特に限定されない。例えば、Ｌ－リジン５位水酸化酵素含む細胞を使用する場合は、反応系中（例えば反応液中）の当該細胞の濃度が、通常、湿菌体重で０．１ｇ／Ｌ～５０ｇ／Ｌ、好ましくは１ｇ／Ｌ～２０ｇ／Ｌとなるように使用することができる。また、当該細胞の調製物や当該細胞を培養して得られた培養液を使用する場合は、使用するＬ－リジン５位水酸化酵素の比活性を求め、反応系中（例えば反応液中）の当該細胞の濃度が、通常、湿菌体重で０．１ｇ／Ｌ～５０ｇ／Ｌ、好ましくは１ｇ／Ｌ～２０ｇ／Ｌとなるような量を使用することができる。 The amount of the L-lysine 5-hydroxylase of the present invention used is not particularly limited as long as it is an amount capable of hydroxylating L-lysine to produce 5-hydroxy-L-lysine. For example, when cells containing L-lysine 5-hydroxylase are used, they can be used so that the concentration of the cells in the reaction system (e.g., in the reaction solution) is usually 0.1 g/L to 50 g/L, preferably 1 g/L to 20 g/L, based on wet cell weight. When a preparation of the cells or a culture solution obtained by culturing the cells is used, the specific activity of the L-lysine 5-hydroxylase used can be determined, and an amount can be used so that the concentration of the cells in the reaction system (e.g., in the reaction solution) is usually 0.1 g/L to 50 g/L, preferably 1 g/L to 20 g/L, based on wet cell weight.

　反応基質であるＬ－リジンは、反応系中（例えば反応液中）の基質濃度が、通常、０．０１ｍＭ～２００ｍＭ、好ましくは１０ｍＭ～２００ｍＭ、より好ましくは２０ｍＭ～２００ｍＭ、特に好ましくは５０ｍＭ～２００ｍＭの範囲となるように使用することができる。 The reaction substrate L-lysine can be used so that the substrate concentration in the reaction system (e.g., in the reaction solution) is usually in the range of 0.01 mM to 200 mM, preferably 10 mM to 200 mM, more preferably 20 mM to 200 mM, and particularly preferably 50 mM to 200 mM.

　また、基質であるＬ－リジンとしては、市販のリジンを使用することができるが、酵素・菌体等を用いて生物学的な方法により製造されたものを使用することもできる。 In addition, as the substrate L-lysine, commercially available lysine can be used, but lysine produced by biological methods using enzymes, bacteria, etc. can also be used.

　また、反応基質であるＬ－リジンとしては、Ｌ－リジンそのものを用いることが好ましいが、反応系中（例えば反応液中）でＬ－リジンとなるＬ－リジン塩酸塩等の化合物を用いることもできる。 As the reaction substrate, L-lysine, it is preferable to use L-lysine itself, but it is also possible to use a compound such as L-lysine hydrochloride that becomes L-lysine in the reaction system (e.g., in the reaction solution).

　本発明のＬ－リジン５位水酸化酵素等を含む液体に反応基質であるＬ－リジンを添加する場合、Ｌ－リジンは反応開始時に一括して添加してもよいが、酵素への阻害作用があった場合の影響を減らす、目的生成物の蓄積濃度を向上させる、副生物を低減する等の観点から、連続的又は間欠的に添加することが望ましい。 When adding L-lysine as a reaction substrate to a liquid containing the L-lysine 5-hydroxylase of the present invention, etc., L-lysine may be added all at once at the start of the reaction, but it is preferable to add it continuously or intermittently from the standpoint of reducing the effect of any inhibitory action on the enzyme, increasing the accumulated concentration of the target product, reducing by-products, etc.

　また、Ｌ－リジンと本発明のＬ－リジン５位水酸化酵素等の接触（反応）は、２－オキソグルタル酸及び２価の鉄イオンの存在下で好気的に行なうことが好ましい。 In addition, the contact (reaction) of L-lysine with the L-lysine 5-hydroxylase of the present invention is preferably carried out aerobically in the presence of 2-oxoglutaric acid and divalent iron ions.

　２－オキソグルタル酸は、通常、反応基質であるＬ－リジンと等モル又はそれ以上、好ましくは等モル～２倍モル、さらに好ましくは等モル～１．８倍モル、より好ましくは等モル～１．５倍モル、特に好ましくは等モル～１．２倍モルの範囲で使用することができる。 2-oxoglutaric acid can usually be used in an equimolar or greater amount relative to the reaction substrate L-lysine, preferably equimolar to 2-fold molar amount, more preferably equimolar to 1.8-fold molar amount, even more preferably equimolar to 1.5-fold molar amount, and particularly preferably equimolar to 1.2-fold molar amount.

　２－オキソグルタル酸は、反応開始時に一括して添加してもよいが、酵素への阻害作用があった場合の影響を減らす、目的生成物の蓄積濃度を向上させる、副生物を低減する等の観点から、連続的若しくは間欠的に添加することが望ましい。 2-Oxoglutaric acid may be added all at once at the start of the reaction, but it is preferable to add it continuously or intermittently in order to reduce the effects of any inhibitory effects on the enzyme, to increase the accumulated concentration of the target product, and to reduce by-products.

　なお、２－オキソグルタル酸の代わりに、宿主が代謝可能なグルコース等の安価な化合物を用いて宿主に代謝させ、その代謝過程で生じる２－オキソグルタル酸を反応に使用させることも可能である。 In addition, instead of 2-oxoglutaric acid, it is also possible to use an inexpensive compound that the host can metabolize, such as glucose, and have the host metabolize it, and use the 2-oxoglutaric acid produced during the metabolic process in the reaction.

　また、２－オキソグルタル酸の代わりに、安価な化合物から複数の酵素によって２－オキソグルタル酸を生成させて、その２－オキソグルタル酸を反応に使用させることも可能である。 Instead of 2-oxoglutaric acid, it is also possible to produce 2-oxoglutaric acid from inexpensive compounds using multiple enzymes and use that 2-oxoglutaric acid in the reaction.

　２価の鉄イオンは、反応系中（例えば反応液中）の濃度が、通常０．０１ｍＭ～１００ｍＭ、好ましくは０．１ｍＭ～１０ｍＭの範囲で使用することができる。２価の鉄イオンは、硫酸鉄等として、反応開始時に一括して添加することが好ましいが、反応中に添加した２価の鉄イオンが３価に酸化されたり、沈殿を形成して減少してしまったりする場合には追添加することも効果的である。 Divalent iron ions can be used at a concentration in the reaction system (e.g., in the reaction solution) of usually 0.01 mM to 100 mM, preferably 0.1 mM to 10 mM. It is preferable to add the divalent iron ions as ferrous sulfate or the like all at once at the start of the reaction, but if the divalent iron ions added during the reaction are oxidized to trivalent iron ions or decrease due to the formation of a precipitate, it is also effective to add more.

　また、本発明のＬ－リジン水酸化酵素等に既に充分な量の２価の鉄イオンが含まれている場合は、必ずしも２価の鉄イオンを添加しなくてもよい。 In addition, if the L-lysine hydroxylase or the like of the present invention already contains a sufficient amount of divalent iron ions, it is not necessarily necessary to add divalent iron ions.

　Ｌ－リジンと本発明のＬ－リジン５位水酸化酵素等の接触（反応）は、通常、反応に悪影響を及ぼさない所望の圧力下で、通常、４℃～６０℃、好ましくは１０℃～４５℃、より好ましくは１５℃～３０℃の反応温度で行うことができる。また、当該接触（反応）は、通常、ｐＨ３～１１、好ましくはｐＨ５～８の条件下で行うことができる。当該接触（反応）時間は、Ｌ－リジンが水酸化されて所望の５－ヒドロキシ－Ｌ－リジンが生成する時間であればよく、特に限定されないが、通常、３０分～７２時間、好ましくは１時間～２４時間である。 The contact (reaction) of L-lysine with the L-lysine 5-hydroxylase or the like of the present invention can usually be carried out under a desired pressure that does not adversely affect the reaction, and at a reaction temperature of usually 4°C to 60°C, preferably 10°C to 45°C, and more preferably 15°C to 30°C. The contact (reaction) can usually be carried out under conditions of pH 3 to 11, and preferably pH 5 to 8. The contact (reaction) time is not particularly limited as long as it is a time during which L-lysine is hydroxylated to produce the desired 5-hydroxy-L-lysine, and is usually 30 minutes to 72 hours, preferably 1 hour to 24 hours.

　生成した５－ヒドロキシ－Ｌ－リジンは、必要に応じて、反応液中の細胞やタンパク質等を遠心分離や膜処理等、当業者に公知の分離又は精製方法により分離した後に、１－ブタノール、ｔｅｒｔ－ブタノール等の有機溶媒による抽出、蒸留、イオン交換樹脂やシリカゲル等を用いたカラムクロマトグラフィー、又は等電点における晶析や一塩酸塩、二塩酸塩、カルシウム塩等での晶析等、当業者に公知の方法を適宜組み合わせることにより精製することができる。 The produced 5-hydroxy-L-lysine can be purified, if necessary, by separating the cells and proteins in the reaction solution using a separation or purification method known to those skilled in the art, such as centrifugation or membrane processing, and then by an appropriate combination of methods known to those skilled in the art, such as extraction with an organic solvent such as 1-butanol or tert-butanol, distillation, column chromatography using ion exchange resins or silica gel, or crystallization at the isoelectric point or crystallization with monohydrochloride, dihydrochloride, calcium salts, etc.

　また、生成した５－ヒドロキシ－Ｌ－リジンのアミノ基に非水溶性の誘導基／保護基を導入する反応を行った後、有機溶媒で抽出して晶析することにより精製することもできる。当該有機溶媒としては、メタノール、エタノール、１－プロパノール、２－プロパノール、１－ブタノール、ｔｅｒｔ－ブタノール、アセトン、ジメチルスルホキシド、酢酸エチル、酢酸ブチル、トルエン、クロロホルム、ｎ－ヘキサン、メチルｔｅｒｔ－ブチルエーテル等を使用することができる。 Furthermore, after carrying out a reaction to introduce a water-insoluble derivative/protecting group into the amino group of the produced 5-hydroxy-L-lysine, it can be purified by extraction with an organic solvent and crystallization. Examples of the organic solvent that can be used include methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, tert-butanol, acetone, dimethyl sulfoxide, ethyl acetate, butyl acetate, toluene, chloroform, n-hexane, and methyl tert-butyl ether.

　本発明では、Ｌ－リジンに、以下の（Ａ－１）、（Ｂ－１）、（Ｃ－１）又は（Ｆ－１）に示すポリペプチド又はそれを含む細胞、前記細胞の調製物若しくは前記細胞を培養して得られた培養液を接触させることにより、（２Ｓ，５Ｓ）－５－ヒドロキシ－Ｌ－リジンを製造することができる。
（Ａ－１）配列番号２、６、８、１０、１２、１４、１６、１８、２０又は２２に示すアミノ酸配列を有するポリペプチド
（Ｂ－１）配列番号２、６、８、１０、１２、１４、１６、１８、２０又は２２に示すアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換及び／又は付加されたアミノ酸配列を有し、かつ、Ｓ選択的Ｌ－リジン５位水酸化活性を有するポリペプチド
（Ｃ－１）配列番号２、６、８、１０、１２、１４、１６、１８、２０又は２２に示すアミノ酸配列全長と５５％以上の配列同一性を有するアミノ酸配列を有し、かつ、Ｓ選択的Ｌ－リジン５位水酸化酵素活性を有するポリペプチド
（Ｆ－１）連続したアミノ酸配列のモチーフである「ＨＧＷＨＷＤＤ」及び「ＨＥＴＭＥＸＬＦ」を有し、かつ、Ｓ選択的Ｌ－リジン５位水酸化活性を保持するポリペプチド In the present invention, (2S,5S)-5-hydroxy-L-lysine can be produced by contacting L-lysine with a polypeptide shown in the following (A-1), (B-1), (C-1), or (F-1), or a cell containing the polypeptide, a preparation of the cells, or a culture medium obtained by culturing the cells.
(A-1) A polypeptide having an amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22. (B-1) A polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted and/or added in the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22, and having S-selective L-lysine 5-hydroxylation activity. (C-1) A polypeptide having an amino acid sequence having 55% or more sequence identity with the full length amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22, and having S-selective L-lysine 5-hydroxylation activity. (F-1) A polypeptide having consecutive amino acid sequence motifs "HGWHWDD" and "HETMEXLF", and having S-selective L-lysine 5-hydroxylation activity.

　また、本発明では、Ｌ－リジンに、以下の（Ａ－２）、（Ｂ－２）、（Ｄ－２）、（Ｅ－２）又は（Ｆ－２）に示すポリペプチド又はそれを含む細胞、前記細胞の調製物若しくは前記細胞を培養して得られた培養液を接触させることにより、（２Ｓ，５Ｒ）－５－ヒドロキシ－Ｌ－リジンを製造することができる。
（Ａ－２）配列番号４、２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８、１１０、１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列を有するポリペプチド
（Ｂ－２）配列番号４、２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８、１１０、１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換及び／又は付加されたアミノ酸配列を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチド
（Ｄ－２）配列番号２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８又は１１０に示すアミノ酸配列全長と５０％以上の配列同一性を有するアミノ酸配列を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチド
（Ｅ－２）配列番号１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列全長と５８％以上の配列同一性を有するアミノ酸配列を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチド
（Ｆ－２）連続したアミノ酸配列のモチーフである「ＨＧＷＨＷＤＤ」；と、「ＨＥＴＭＤＸＬＷ」、「ＨＥＴＭＥＸＬＷ」、「ＨＥＴＮＤＸＬＦ」及び「ＨＥＴＮＮＸＬＦ」よりなる群から選ばれるいずれかの連続したアミノ酸配列のモチーフ；を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を保持するポリペプチド Furthermore, in the present invention, (2S,5R)-5-hydroxy-L-lysine can be produced by contacting L-lysine with a polypeptide shown in the following (A-2), (B-2), (D-2), (E-2), or (F-2), or a cell containing the same, a preparation of the cells, or a culture medium obtained by culturing the cells.
(A-2) A polypeptide having an amino acid sequence shown in SEQ ID NO: 4, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134 (B-2) A polypeptide having an amino acid sequence shown in SEQ ID NO: 4, 24, 26, 28, 30, 32, 34, 36, 3 8, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134, in which one or more amino acids are deleted, substituted and/or added, and which has R-selective L-lysine 5-hydroxylation activity. A polypeptide (E-2) having an amino acid sequence having 50% or more sequence identity with the full length amino acid sequence shown in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108 or 110 and having R-selective L-lysine 5-hydroxylation activity. 8, 130, 132 or 134, and having R-selective L-lysine 5-hydroxylation activity (F-2) A polypeptide having a consecutive amino acid sequence motif of "HGWHWDD" and any consecutive amino acid sequence motif selected from the group consisting of "HETMDXLW", "HETMEXLW", "HETNDXLF" and "HETNNXLF", and retaining R-selective L-lysine 5-hydroxylation activity.

　このように、本発明の５－ヒドロキシ－Ｌ－リジンの製造方法は、Ｌ－リジン５位水酸化酵素を使い分けることにより、所望の（２Ｓ，５Ｓ）－５－ヒドロキシ－Ｌ－リジン又は（２Ｓ，５Ｒ）－５－ヒドロキシ－Ｌ－リジンを製造することができる。 In this way, the method for producing 5-hydroxy-L-lysine of the present invention can produce the desired (2S,5S)-5-hydroxy-L-lysine or (2S,5R)-5-hydroxy-L-lysine by using the appropriate L-lysine 5-hydroxylase.

　以下、実施例により本発明を更に詳しく説明するが、本発明はこれに限定されるものではない。 The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these.

＜実施例１＞（ａｚｐＫ２遺伝子のクローニング）
　理化学研究所バイオリソース研究センター（ＲＩＫＥＮ　ＢＲＣ）微生物材料開発室（以下「ＪＣＭ」と称する。）より購入した放線菌Ａｃｔｉｎｏｓｙｎｎｅｍａ　ｍｉｒｕｍ　ＪＣＭ３２２５を使用し、ｌｙｓｏｚｙｍｅ、ＳＤＳ、クロロホルム、及びイソプロパノール沈殿法によるゲノムＤＮＡ抽出法の常法に従ってゲノムＤＮＡを得た。この抽出したゲノムＤＮＡをテンプレートとして、配列番号１５０及び１５１に示すプライマーを使用したＰＣＲ反応を行い、ａｚｐＫ２遺伝子を増幅した。ＰＣＲ反応には、ＰｒｉｍｅＳｔａｒ　ＨＳ　ＤＮＡ　ｐｏｌｙｍｅｒａｓｅ（タカラバイオ社製）を使用し、目的のａｚｐＫ２遺伝子の増幅産物を得た。反応溶液の組成及び反応サイクルは下記の通りである。 Example 1 (Cloning of azpK2 gene)
Actinomycete Actinosynnema mirum JCM3225 purchased from the Microorganism Materials Development Laboratory of the RIKEN BioResource Research Center (RIKEN BRC) (hereinafter referred to as "JCM") was used to obtain genomic DNA according to the conventional method of genomic DNA extraction using lysozyme, SDS, chloroform, and isopropanol precipitation. Using the extracted genomic DNA as a template, a PCR reaction was performed using the primers shown in SEQ ID NOs: 150 and 151 to amplify the azpK2 gene. PrimeStar HS DNA polymerase (manufactured by Takara Bio Inc.) was used in the PCR reaction to obtain an amplification product of the target azpK2 gene. The composition of the reaction solution and the reaction cycle are as follows.

ＰＣＲ　反応溶液組成
　滅菌水：８．６μＬ
　５ｍｏｌ／Ｌ　ｂｅｔａｉｎｅ：４．０μＬ
　５×ＰｒｉｍｅＳＴＡＲ（登録商標）　Ｂｕｆｆｅｒ：４．０μＬ
　２．５ｍＭ　ｄＮＴＰ　ｍｉｘ：１．６μＬ
　ＡｚｐＫ２_Ｆ（１００μＭ）：０．６μＬ
　ＡｚｐＫ２_Ｒ（１００μＭ）：０．６μＬ
　ゲノムＤＮＡ（２．５～２５μｇ／μＬ）：０．４μＬ
　ＰｒｉｍｅＳｔａｒ（登録商標）　ＨＳ　ＤＮＡ　ｐｏｌｙｍｅｒａｓｅ：０．２μＬ PCR reaction solution composition Sterile water: 8.6 μL
5mol/L betaine: 4.0μL
5x PrimeSTAR (registered trademark) Buffer: 4.0 μL
2.5mM dNTP mix: 1.6μL
AzpK2_F (100 μM): 0.6 μL
AzpK2_R (100 μM): 0.6 μL
Genomic DNA (2.5-25μg/μL): 0.4μL
PrimeStar® HS DNA polymerase: 0.2 μL

　制限酵素ＮｄｅＩ及びＸｈｏＩ（タカラバイオ社製）で処理したｐＣｏｌｄ　Ｉベクター（タカラバイオ社製）と上記ＰＣＲ産物をＩｎ－Ｆｕｓｉｏｎ（登録商標）　ＨＤ　Ｃｌｏｎｉｎｇ　Ｋｉｔ（タカラバイオ社製）を用いて常法に従ってライゲーションした。その後、常法に従って大腸菌Ｅ．ｃｏｌｉ　ＪＭ１０９株（タカラバイオ社製）に形質転換した。得られたコロニーからミニプレップ法により常法に従ってプラスミドＤＮＡを抽出し、ｐＣｏｌｄ　Ｉ－ＡｚｐＫ２ベクターを得た。 The pCold I vector (Takara Bio) treated with the restriction enzymes NdeI and XhoI (Takara Bio) and the above PCR product were ligated using the In-Fusion (registered trademark) HD Cloning Kit (Takara Bio) in a standard manner. The E. coli JM109 strain (Takara Bio) was then transformed in a standard manner. Plasmid DNA was extracted from the resulting colonies using the miniprep method in a standard manner to obtain the pCold I-AzpK2 vector.

＜実施例２＞（ａｚｐＪ遺伝子のクローニング）
　ＪＣＭより購入した放線菌Ｓｔｒｅｐｔａｃｉｄｉｐｈｉｌｕｓ　ｇｒｉｓｅｏｐｌａｎｕｓ　ＪＣＭ４３００を用い、実施例１と同様の手法を用いてゲノムＤＮＡを取得し、配列番号１５２と１５３に示すプライマーを用いて、実施例１と同様の手法を用いて、配列番号１４０に示すＡｚｐＪタンパク質（同タンパク質のアミノ酸配列をコードするＤＮＡの塩基配列は、配列番号１３９）を発現するためのｐＣｏｌｄ　Ｉ－ＡｚｐＪベクターを得た。 Example 2 (Cloning of azpJ gene)
Genomic DNA was obtained using the actinomycete Streptacidiphilus griseoplanus JCM4300 purchased from JCM in the same manner as in Example 1, and the primers shown in SEQ ID NOs: 152 and 153 were used in the same manner as in Example 1 to obtain pCold I-AzpJ vector for expressing the AzpJ protein shown in SEQ ID NO: 140 (the base sequence of DNA encoding the amino acid sequence of the protein is SEQ ID NO: 139).

＜実施例３＞（Ｐｐ＿ａｚｐＫ２遺伝子、ａｚｐＫ２ホモログ遺伝子及びＰｐ＿ａｚｐＪ遺伝子の発現ベクター作製）
　下記の表３に示すＰｐ＿ａｚｐＫ２遺伝子、ａｚｐＫ２ホモログ遺伝子及び配列番号１４２に示すＰｐ＿ａｚｐＪタンパク質をコードするＰｐ＿ａｚｐＪ遺伝子について、大腸菌での発現に適したコドン頻度に最適化した後、各配列番号の核酸配列にｐＣｏｌｄ　Ｉベクターと相同な配列として、遺伝子上流にＴＣＧＡＡＧＧＴＡＧＧＣＡＴＡＴＧ（配列番号１５４）、また遺伝子下流にＴＡＡＣＴＣＧＡＧＧＧＡＴＣＣＧＡＡＴ（配列番号１５５）を付与した合成遺伝子を、Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社より購入した。 Example 3 (Preparation of expression vectors for Pp_azpK2 gene, azpK2 homologue gene, and Pp_azpJ gene)
The Pp_azpK2 gene, azpK2 homolog gene, and Pp_azpJ gene encoding the Pp_azpJ protein shown in SEQ ID NO: 142 shown in Table 3 below were optimized to have a codon frequency suitable for expression in E. coli, and synthetic genes in which TCGAAGGTAGGCATATG (SEQ ID NO: 154) was added upstream of the gene and TAACTCGAGGGATCCGAAT (SEQ ID NO: 155) was added downstream of the gene as sequences homologous to the pCold I vector in the nucleic acid sequence of each SEQ ID NO were purchased from Thermo Fisher Scientific.

　ｐＣｏｌｄ　Ｉベクター（タカラバイオ社製）を制限酵素ＮｄｅＩ及びＸｈｏＩ（タカラバイオ社製）で処理し、合成遺伝子と一緒にＩｎ－Ｆｕｓｉｏｎ（登録商標）　ＨＤ　Ｃｌｏｎｉｎｇ　Ｋｉｔ（タカラバイオ社製）を使用して常法に手順に従ってライゲーションした。その後、大腸菌Ｅ．　ｃｏｌｉ　ＪＭ１０９株に常法に従って形質転換を行い、得られたコロニーからミニプレップ法を用いてプラスミドＤＮＡを抽出して下記表３の発現ベクターを作製した。 The pCold I vector (Takara Bio) was treated with the restriction enzymes NdeI and XhoI (Takara Bio), and ligated together with the synthetic gene using the In-Fusion (registered trademark) HD Cloning Kit (Takara Bio) according to standard procedures. After that, E. coli JM109 strain was transformed according to standard procedures, and plasmid DNA was extracted from the resulting colonies using the miniprep method to prepare the expression vectors shown in Table 3 below.

＜実施例４＞（組換えタンパク質水溶液の調製－１）
　実施例１～３により得られた発現ベクターを用い、Ｅ．　ｃｏｌｉ　ＢＬ２１（ＤＥ３）株（Ｍｅｒｃｋ社製）に常法に従って形質転換した。得られたコロニーを終濃度５０μｇ／ｍＬ　のアンピシリンナトリウム（富士フイルム和光純薬工業社製）を含むＬＢ培地（以下、「ＬＢ（Ａｍｐ５０）培地」と称する。）２ｍＬに植菌し、３７℃で一晩振盪培養を行った。 Example 4 (Preparation of recombinant protein aqueous solution-1)
The expression vectors obtained in Examples 1 to 3 were used to transform E. coli BL21 (DE3) strain (Merck) in a conventional manner. The resulting colonies were inoculated into 2 mL of LB medium (hereinafter referred to as "LB (Amp50) medium") containing ampicillin sodium (Fujifilm Wako Pure Chemical Industries, Ltd.) at a final concentration of 50 μg/mL, and cultured overnight at 37° C. with shaking.

　得られた培養液を、終濃度５０μｇ／ｍＬのアンピシリンナトリウムを含むＴｅｒｒｉｆｉｃ　Ｂｒｏｔｈ（以下、「ＴＢ（Ａｍｐ５０）培地」と称する）に１％容量植菌し、３７℃で振盪培養（１５０ｒｐｍ）した。吸光度ＯＤ６００ｎｍが０．５～０．８に達したところで１５℃に冷却し、ＩＰＴＧを終濃度が２０μＭ又は５０μＭとなるように加えて誘導を行い、１５℃で振盪培養（１５０ｒｐｍ）を２４時間行った後、遠心分離（２０,０００×ｇ、１０分、４℃）により培養液から菌体を回収した。Ｌｙｓｉｓ　ｂｕｆｆｅｒ（２０ｍＭ　ＨＥＰＥＳ、１０Ｗ／Ｖ％　グリセロール、２００ｍＭ　塩化ナトリウム（以下、「ＮａＣｌ」と称する。）、ｐＨ８．０）に再懸濁した菌体をＱ５００　Ｓｏｎｉｃａｔｏｒ（ＱＳｏｎｉｃａ社製）により超音波破砕後、遠心分離（２０,０００×ｇ、６０分、４℃）を行い、組換えタンパク質を含む水溶液を取得した。この水溶液にＨｉｓ６０　Ｎｉ　Ｓｕｐｅｒｆｌｏｗ　Ｒｅｓｉｎ（タカラバイオ社製）を加えて撹拌し、組換えタンパク質を結合させ、カラムに移した。 The obtained culture medium was inoculated at 1% volume into Terrific Broth (hereinafter referred to as "TB (Amp50) medium") containing a final concentration of 50 μg/mL sodium ampicillin, and cultured at 37°C with shaking (150 rpm). When the absorbance at OD600nm reached 0.5-0.8, the medium was cooled to 15°C, and induction was performed by adding IPTG to a final concentration of 20 μM or 50 μM. After shaking culture at 15°C for 24 hours (150 rpm), the bacterial cells were collected from the culture medium by centrifugation (20,000 x g, 10 minutes, 4°C). The cells were resuspended in lysis buffer (20 mM HEPES, 10% W/V glycerol, 200 mM sodium chloride (hereinafter referred to as "NaCl"), pH 8.0) and ultrasonically disrupted using a Q500 Sonicator (QSonica), followed by centrifugation (20,000 x g, 60 minutes, 4°C) to obtain an aqueous solution containing the recombinant protein. His60 Ni Superflow Resin (Takara Bio) was added to this aqueous solution and stirred to bind the recombinant protein, which was then transferred to a column.

　Ｅｌｕｔｉｏｎ　ｂｕｆｆｅｒ（２０ｍＭ　ＨＥＰＥＳ、１０％グリセロール、２００ｍＭ　ＮａＣｌ、５００ｍＭ　イミダゾール、ｐＨ８．０）とＬｙｓｉｓ　ｂｕｆｆｅｒを混合し、イミダゾール濃度がそれぞれ２０ｍＭ、５０ｍＭ、１００ｍＭ、２５０ｍＭとなる水溶液を作製した。イミダゾール濃度が低い水溶液から高い水溶液へと段階的にカラムに添加することにより、カラム内の樹脂から組換えタンパク質を溶出させた。 Elution buffer (20 mM HEPES, 10% glycerol, 200 mM NaCl, 500 mM imidazole, pH 8.0) was mixed with lysis buffer to prepare aqueous solutions with imidazole concentrations of 20 mM, 50 mM, 100 mM, and 250 mM, respectively. The recombinant protein was eluted from the resin in the column by gradually adding aqueous solutions with low to high imidazole concentrations to the column.

　各イミダゾール濃度の溶出画分について、ポリアクリルアミドゲル電気泳動（ＳＤＳ－ＰＡＧＥ）により組換えタンパク質の有無を確認し、存在が確認された溶出画分についてＡｍｉｃｏｎ　Ｕｌｔｒａ－１５　Ｃｅｎｔｒｉｆｕｇａｌ　Ｆｉｌｔｅｒｓ（１０ｋＤａ）（メルク社製）を用いて限外濾過（５０００×ｇ、４℃）による濃縮及び脱塩操作を行った。得られたタンパク質水溶液のタンパク質濃度をＮａｎｏＤｒｏｐ（登録商標）微量分光光度計（Ｔｈｅｒｍｏ　ｆｉｓｈｅｒ　ｓｃｉｅｎｔｉｆｉｃ社製）で測定した後に凍結保存した。この方法で調製したタンパク質水溶液の濃度はアミノ酸配列とアミノ酸の予想分子量からＡ２８０ｎｍにおける吸光係数を概算し、その値を用いて算出した。 The eluted fractions at each imidazole concentration were examined for the presence or absence of recombinant protein by polyacrylamide gel electrophoresis (SDS-PAGE), and the eluted fractions in which the presence was confirmed were concentrated and desalted by ultrafiltration (5000 x g, 4°C) using Amicon Ultra-15 Centrifugal Filters (10 kDa) (Merck). The protein concentration of the resulting protein solution was measured using a NanoDrop (registered trademark) microspectrophotometer (Thermo Fisher Scientific) and then frozen and stored. The concentration of the protein solution prepared by this method was calculated using the approximate absorption coefficient at A280 nm calculated from the amino acid sequence and the predicted molecular weight of the amino acid.

＜実施例５＞（ＡｚｐＫ２、Ｐｐ＿ＡｚｐＫ２及びＡｚｐＫ２ホモログのリジン５位水酸化活性の確認）
　０．１ｍＭ　Ｌ－リジン（以下、「Ｌ－Ｌｙｓ」と称する。）、０．１ｍＭ　α－ケトグルタル酸（以下、「αＫＧ」と称することがある）、１．０ｍＭ　アスコルビン酸、２５μＭ　ＦｅＳＯ４、２０ｍＭ　ＨＥＰＥＳ、１０Ｗ／Ｖ％　グリセロール、２００ｍＭ　ＮａＣｌ（ｐＨ８．０）、５．０μＭの実施例４で取得したＡｚｐＫ２、Ｐｐ＿ＡｚｐＫ２又はＡｚｐＫ２ホモログ（ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－１～１－９、２－１～２－３、３－１～３－２）の組換えタンパク質の組成で調製した反応溶液１００μＬを用いて、３０℃で１時間反応させた。反応終了後、１００μＬのメタノールを加えることにより反応を停止させ、遠心分離して得た上清をＬＣ－ＭＳ（分析条件１）で解析した。分析条件１の詳細は以下の通りである。 Example 5 (Confirmation of Lysine 5-hydroxylation Activity of AzpK2, Pp_AzpK2, and AzpK2 Homologs)
The reaction was carried out at 30° C. for 1 hour using 100 μL of a reaction solution prepared with the following composition: 0.1 mM L-lysine (hereinafter referred to as "L-Lys"), 0.1 mM α-ketoglutaric acid (hereinafter sometimes referred to as "αKG"), 1.0 mM ascorbic acid, 25 μM FeSO 4 , 20 mM HEPES, 10 W/V% glycerol, 200 mM NaCl (pH 8.0), and 5.0 μM of the recombinant protein of AzpK2, Pp_AzpK2, or AzpK2 homolog (AzpK2 Homolog 1-1 to 1-9, 2-1 to 2-3, 3-1 to 3-2) obtained in Example 4. After completion of the reaction, the reaction was stopped by adding 100 μL of methanol, and the supernatant obtained by centrifugation was analyzed by LC-MS (analysis condition 1). Details of analysis condition 1 are as follows.

　分析の結果、ＡｚｐＫ２、Ｐｐ＿ＡｚｐＫ２、及びＡｚｐＫ２ホモログ（ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－１～１－９、２－１～２－３、３－１～３－２）の組換えタンパク質水溶液を用いた場合の全てにおいて、５－ヒドロキシ－Ｌ－リジン（メルク社製）と同じ保持時間（４ｍｉｎ）にｍ／ｚ　１６３．２のピークを検出した。 As a result of the analysis, a peak at m/z 163.2 was detected at the same retention time (4 min) as 5-hydroxy-L-lysine (Merck) in all cases where recombinant protein aqueous solutions of AzpK2, Pp_AzpK2, and AzpK2 homologs (AzpK2 Homolog 1-1 to 1-9, 2-1 to 2-3, 3-1 to 3-2) were used.

　また、前記で調製した反応溶液のうち５０μＬをとり、アセトニトリルに溶解した１ｍＭ塩化フルオレニルメチルオキシカルボニル（以下、「Ｆｍｏｃ－Ｃｌ」と称することがある。）５０μＬと混合し３０℃で３０分間反応させた。ここに１Ｍ　ＨＣｌ　５０μＬを加えて反応を停止させ、ここから１５０μＬの酢酸エチルで反応産物を抽出した。遠心エバポレーターで溶媒を除去し、残った固形物を２０μＬのメタノールに溶解してアミノ酸をＦｍｏｃ化したＦｍｏｃ化処理サンプルを得た。このＦｍｏｃ化処理サンプルをＬＣ－ＭＳ（分析条件２）で解析した。分析条件２の詳細は以下の通りである。 Furthermore, 50 μL of the reaction solution prepared above was taken and mixed with 50 μL of 1 mM fluorenylmethyloxycarbonyl chloride (hereinafter sometimes referred to as "Fmoc-Cl") dissolved in acetonitrile, and reacted at 30°C for 30 minutes. 50 μL of 1 M HCl was added to this to stop the reaction, and the reaction product was extracted from this with 150 μL of ethyl acetate. The solvent was removed using a centrifugal evaporator, and the remaining solid was dissolved in 20 μL of methanol to obtain an Fmoc-treated sample in which the amino acid was Fmoc-treated. This Fmoc-treated sample was analyzed by LC-MS (analysis condition 2). Details of analysis condition 2 are as follows.

　分析の結果、ＡｚｐＫ２、Ｐｐ＿ＡｚｐＫ２及びＡｚｐＫ２ホモログ（ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－１～１－９、２－１～２－３、３－１～３－２）の組換えタンパク質水溶液を用いた場合の全てにおいて、５－ヒドロキシ－Ｌ－リジン（メルク社製）のｄｉ－Ｆｍｏｃ化体と同じ保持時間（８ｍｉｎ）にｍ／ｚ　６０７．２のピークを検出した。したがって、ＡｚｐＫ２、Ｐｐ＿ＡｚｐＫ２、及びＡｚｐＫ２ホモログ（ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－１～１－９，２－１～２－３，３－１～３－２）は全てＬ－リジン５位水酸化活性を有することが判明した。 As a result of the analysis, in all cases where recombinant protein aqueous solutions of AzpK2, Pp_AzpK2, and AzpK2 homologs (AzpK2 Homolog 1-1 to 1-9, 2-1 to 2-3, 3-1 to 3-2) were used, a peak of m/z 607.2 was detected at the same retention time (8 min) as the di-Fmoc derivative of 5-hydroxy-L-lysine (Merck). Therefore, it was found that AzpK2, Pp_AzpK2, and AzpK2 homologs (AzpK2 Homolog 1-1 to 1-9, 2-1 to 2-3, 3-1 to 3-2) all have L-lysine 5-position hydroxylation activity.

　図３に、ＡｚｐＫ２を用いた場合の反応生成物のＬＣ－ＭＳ検出結果（ＭＳ　＋６０７．３によるｄｉ－Ｆｍｏｃ化５－ヒドロキシ－Ｌ－リジンの検出結果）を示す。 Figure 3 shows the LC-MS detection results of the reaction product when AzpK2 was used (detection results of di-Fmoc-5-hydroxy-L-lysine with MS +607.3).

　図３中の「ｐｏｓｉｔｉｖｅ　ｃｏｎｔｒｏｌ」は５－ヒドロキシ－Ｌ－リジンのｄｉ－Ｆｍｏｃ化体を分析したポジティブコントロールであり、８．０ｍｉｎのピークが５－ヒドロキシ－Ｌ－リジンのｄｉ－Ｆｍｏｃ化体である。「ｎｏ　ＡｚｐＫ２」は全反応液組成からＡｚｐＫ２を除いたネガティブコントロールである。「Ａｌｌ」はＡｚｐＫ２を含む全反応液組成を入れたものであり、８．０ｍｉｎ付近にポジティブコントロールと同じ５－ヒドロキシ－Ｌ－リジンのｄｉ－Ｆｍｏｃ化体のピークが検出された。 In Figure 3, "positive control" is the positive control analyzed for the di-Fmoc derivative of 5-hydroxy-L-lysine, and the peak at 8.0 min is the di-Fmoc derivative of 5-hydroxy-L-lysine. "No AzpK2" is a negative control in which AzpK2 was omitted from the total reaction solution composition. "All" is the total reaction solution composition including AzpK2, and the same peak of the di-Fmoc derivative of 5-hydroxy-L-lysine as the positive control was detected around 8.0 min.

＜実施例６＞（精製酵素を用いたＡｚｐＫ２、Ｐｐ＿ＡｚｐＫ２及びＡｚｐＫ２ホモログの酵素学的パラメーター測定）
　０．１ｍＭ　α－ケトグルタル酸、１．０ｍＭ　アスコルビン酸、２５μＭ　ＦｅＳＯ４、２０ｍＭ　ＨＥＰＥＳ、１０Ｗ／Ｖ％　グリセロール、２００ｍＭ　ＮａＣｌ（ｐＨ８．０）、１．０ｍＭ　ＮＡＤ＋、５．０μＭ　の実施例４で取得したＡｚｐＫ２、Ｐｐ＿ＡｚｐＫ２又はＡｚｐＫ２ホモログ（ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－１～１－３、２－１～２－３、３－１）の組換えタンパク質水溶液、２．０μＭの実施例４で取得したＡｚｐＪ又はＰｐ＿ＡｚｐＪの組換えタンパク質水溶液、及びＬ－Ｌｙｓの濃度を０．０１ｍＭ、０．０２ｍＭ、０．１ｍＭ、０．２ｍＭ又は０．５ｍＭとした組成で、それぞれ反応溶液１００μＬを調製した。Ｌ－Ｌｙｓは、他の成分を混合した後、最後に添加した。 Example 6 (Measurement of enzymatic parameters of AzpK2, Pp_AzpK2, and AzpK2 homologues using purified enzymes)
100 μL of reaction solution was prepared each having a composition of 0.1 mM α-ketoglutaric acid, 1.0 mM ascorbic acid, 25 μM FeSO 4 , 20 mM HEPES, 10 W/V% glycerol, 200 mM NaCl (pH 8.0), 1.0 mM NAD + , 5.0 μM aqueous solution of recombinant protein of AzpK2, Pp_AzpK2 or AzpK2 homolog (AzpK2 Homolog 1-1 to 1-3, 2-1 to 2-3, 3-1) obtained in Example 4, 2.0 μM aqueous solution of recombinant protein of AzpJ or Pp_AzpJ obtained in Example 4, and L-Lys at a concentration of 0.01 mM, 0.02 mM, 0.1 mM, 0.2 mM or 0.5 mM. L-Lys was added last after mixing the other ingredients.

　反応は、最後に添加したＬ－Ｌｙｓ（基質）により開始した。ＳｐｅｃｔｒａＭａｘ　Ｍ２（Ｍｏｌｅｃｕｌａｒ　Ｄｅｖｉｃｅｓ社製）を用いて、波長３４０ｎｍ（ＮＡＤＨの吸光波長）の吸光度を継時的に測定し、得られる吸光度変化の傾きから、ＮＡＤＨ生成の初速度（反応初速度）を測定した。 The reaction was initiated by the addition of L-Lys (substrate). The absorbance at a wavelength of 340 nm (the absorbance wavelength of NADH) was measured continuously using a SpectraMax M2 (Molecular Devices), and the initial velocity of NADH production (initial reaction velocity) was measured from the slope of the resulting absorbance change.

　得られた反応初速度をＭｉｃｈａｅｌｉｓ－Ｍｅｎｔｅｎ式にフィッティングして、各タンパク質の酵素学的パラメーターであるＫｃａｔ、Ｋｍ及びＫｃａｔ／Ｋｍを算出した。結果を下記表４に示す。 The initial reaction rates obtained were fitted to the Michaelis-Menten equation to calculate the enzymatic parameters Kcat, Km, and Kcat/Km for each protein. The results are shown in Table 4 below.

　表４から明らかなように、ＫｃａｔはＡｚｐＫ２　Ｈｏｍｏｌｏｇ３－１が最も高く、次いでＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－２、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ２－２の順に高かった。なり、また、Ｋｃａｔ／ＫｍはＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－２が最も高く、次いでＰｐ＿ＡｚｐＫ２、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ２－３、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－１の順の順に高かった。 As is clear from Table 4, Kcat was highest for AzpK2 Homolog3-1, followed by AzpK2 Homolog1-2 and AzpK2 Homolog2-2, in that order. Also, Kcat/Km was highest for AzpK2 Homolog1-2, followed by Pp_AzpK2, AzpK2 Homolog2-3 and AzpK2 Homolog1-1, in that order.

＜実施例７＞（Ｌ－リジン５位水酸化酵素による５－ヒドロキシ－Ｌ－リジンに対する水酸化活性の確認）
　ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－１，１－２及び１－３について、５－ヒドロキシ－Ｌ－リジンの５位水酸化活性の有無を測定した。 Example 7 (Confirmation of hydroxylation activity of L-lysine 5-hydroxylase on 5-hydroxy-L-lysine)
AzpK2 Homologs 1-1, 1-2 and 1-3 were assayed for the presence or absence of 5-hydroxylation activity of 5-hydroxy-L-lysine.

　０．１ｍＭ　５－ヒドロキシ－Ｌ－リジン、０．１ｍＭ又は１ｍＭの　α－ケトグルタル酸、１．０ｍＭ　アスコルビン酸、２５μＭ　ＦｅＳＯ４、２０ｍＭ　ＨＥＰＥＳ、１０％　グリセロール、２００ｍＭ　ＮａＣｌ（ｐＨ８．０）、及び５．０μＭの実施例４で取得したＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－１、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－２又はＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－３の組換えタンパク質溶液の組成で調製した反応溶液１００μＬを用いて、３０℃で１時間反応させた。反応液を実施例５と同様の方法でＦｍｏｃ化し、ＨＰＬＣ（分析条件３）で分析を行った。分析条件３の詳細は以下の通りである。 The reaction was carried out at 30° C. for 1 hour using 100 μL of a reaction solution prepared with the composition of 0.1 mM 5-hydroxy-L-lysine, 0.1 mM or 1 mM α-ketoglutaric acid, 1.0 mM ascorbic acid, 25 μM FeSO 4 , 20 mM HEPES, 10% glycerol, 200 mM NaCl (pH 8.0), and 5.0 μM of the recombinant protein solution of AzpK2 Homolog1-1, AzpK2 Homolog1-2, or AzpK2 Homolog1-3 obtained in Example 4. The reaction solution was Fmoc-modified in the same manner as in Example 5, and analyzed by HPLC (analysis condition 3). Details of analysis condition 3 are as follows.

　ＨＰＬＣによる分析結果を図４に示す。
　図４において、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－１、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－２、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－３のそれぞれについて、上段が１．０ｍＭ　αＫＧ条件で反応した結果、下段が０．１ｍＭ　αＫＧ条件で反応した結果である。図４中、「５－ＯＨＬｙｓ－Ｆｍｏｃ」は５－ヒドロキシ－Ｌ－リジンのＦｍｏｃ化体、「５－ｏｘｏＬｙｓ－Ｆｍｏｃ」は５－オキソリジンのＦｍｏｃ化体、「５－ＯＨＬｙｓ－ｄｉＦｍｏｃ」は５－ヒドロキシ－Ｌ－リジンのｄｉ－Ｆｍｏｃ化体のピークを示す。 The results of the HPLC analysis are shown in FIG.
4, for AzpK2 Homolog1-1, AzpK2 Homolog1-2, and AzpK2 Homolog1-3, the upper row shows the results of the reaction under 1.0 mM αKG conditions, and the lower row shows the results of the reaction under 0.1 mM αKG conditions. In FIG. 4, "5-OHLys-Fmoc" indicates the peak of 5-hydroxy-L-lysine Fmoc derivative, "5-oxoLys-Fmoc" indicates the peak of 5-oxolysine Fmoc derivative, and "5-OHLys-diFmoc" indicates the peak of 5-hydroxy-L-lysine di-Fmoc derivative.

　図４から明らかなように、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－１、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－２及びＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－３は全ての条件において５－オキソリジンが検出された。また、いずれもα－ケトグルタル酸添加濃度１ｍＭ条件の方が０．１ｍＭ条件よりも５－オキソリジンの検出ピークは大きかった。 As is clear from Figure 4, 5-oxolidine was detected under all conditions for AzpK2 Homolog1-1, AzpK2 Homolog1-2, and AzpK2 Homolog1-3. In addition, the detection peak of 5-oxolidine was larger in all cases when α-ketoglutaric acid was added at a concentration of 1 mM than at 0.1 mM.

　このことから、Ｌ－リジン５位水酸化酵素を用いてＬ－Ｌｙｓから５－ヒドロキシ－Ｌ－リジンを得ようとする場合、α－ケトグルタル酸添加濃度が低い方が、５－オキソリジンの副生を抑え、５－ヒドロキシ－Ｌ－リジンを高収量で得られることが示唆される。 This suggests that when attempting to obtain 5-hydroxy-L-lysine from L-Lys using L-lysine 5-hydroxylase, adding a lower concentration of α-ketoglutaric acid will suppress the by-production of 5-oxolidine and allow for a higher yield of 5-hydroxy-L-lysine.

＜参考例１＞（５－ヒドロキシ－Ｌ－リジンのヒドロキシ基の立体確認方法）
　約１ｍＭの５－ヒドロキシ－Ｌ－リジンを含む反応液を２０ｍＭ　ＨＥＰＥＳ緩衝液、０．２ｍＭ　ＮＡＤ＋、及び０．４６ｍｇ／ｍＬ　Ｐｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉｄａ由来シクロデアミナ－ゼ（ｐＨ７．０）を加えた反応溶液１．０ｍＬを調製し、３０℃で振盪（２００ｒｐｍ）しながら４～２４時間程度反応させることにより、反応生成物である５－ヒドロキシ－Ｌ－ピペコリン酸を含む反応液を取得する。 Reference Example 1 (Method for confirming the stereochemistry of the hydroxy group in 5-hydroxy-L-lysine)
A reaction solution containing about 1 mM 5-hydroxy-L-lysine is added with 20 mM HEPES buffer, 0.2 mM NAD + , and 0.46 mg/mL Pseudomonas putida-derived cyclodeaminase (pH 7.0) to prepare 1.0 mL of reaction solution, and the solution is reacted at 30°C for about 4 to 24 hours with shaking (200 rpm) to obtain a reaction solution containing the reaction product, 5-hydroxy-L-pipecolic acid.

　得られた反応液５０μＬに対し、５００ｍＭ　ほう酸（ｐＨ９．０）　５０μＬ、１０ｍＭ　Ｎα－（２,４－ジニトロ－５－フルオロフェニル）－Ｌ－アラニンアミド（ＦＤＡＡ）　５０μＬを順次加え、４０℃で１時間保温後、１Ｍ　ＨＣｌ　５０μＬとアセトニトリル２００μＬを加えたのち、０．４５μｍフィルターでろ過することによりＦＤＡＡ誘導体化５－ヒドロキシ－Ｌ－ピペコリン酸を取得する。 50 μL of the resulting reaction solution is added in turn to 50 μL of 500 mM boric acid (pH 9.0), 50 μL of 10 mM Nα-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (FDAA), and after incubating at 40°C for 1 hour, 50 μL of 1 M HCl and 200 μL of acetonitrile are added, and the solution is filtered through a 0.45 μm filter to obtain FDAA-derivatized 5-hydroxy-L-pipecolic acid.

　得られたＦＤＡＡ誘導体化５－ヒドロキシ－Ｌ－ピペコリン酸を含む溶液をＨＰＬＣ（分析条件４又は分析条件５）で分析し、予め準備しておいた（２Ｓ,５Ｓ）－５－ヒドロキシ－Ｌ－ピペコリン酸をＦＤＡＡ誘導体化したもの又は（２Ｓ,５Ｒ）－５－ヒドロキシ－Ｌ－ピペコリン酸をＦＤＡＡ誘導体化したものと比較することによって、反応生成物である５－ヒドロキシ－Ｌ－リジンのヒドロキシ基の絶対配置を決めることができる。分析条件４及び分析条件５の詳細は以下の通りである。 The resulting solution containing FDAA-derivatized 5-hydroxy-L-pipecolic acid is analyzed by HPLC (analysis condition 4 or analysis condition 5) and compared with previously prepared FDAA-derivatized (2S,5S)-5-hydroxy-L-pipecolic acid or FDAA-derivatized (2S,5R)-5-hydroxy-L-pipecolic acid, thereby determining the absolute configuration of the hydroxy group in the reaction product, 5-hydroxy-L-lysine. Details of analysis condition 4 and analysis condition 5 are as follows.

＜実施例８＞（５－ヒドロキシ－Ｌ－リジンのヒドロキシ基の立体確認）
　２０ｍＭ　ＨＥＰＥＳ緩衝液（ｐＨ７．０）、１０ｍＭ　Ｌ－Ｌｙｓ、２０ｍＭ　α－ケトグルタル酸、１．０ｍＭ　アスコルビン酸、０．５ｍＭ　ＦｅＳＯ４、及び、３．３μＭの実施例４で取得したＡｚｐＫ２又はＰｐ＿ＡｚｐＫ２のタンパク質水溶液を含む反応溶液１．０ｍＬを、２８℃、２００ｒｐｍで振盪しながら１４時間反応させて、５－ヒドロキシ－Ｌ－リジン溶液を得た。 Example 8 (Confirmation of the stereochemistry of the hydroxy group of 5-hydroxy-L-lysine)
1.0 mL of a reaction solution containing 20 mM HEPES buffer (pH 7.0), 10 mM L-Lys, 20 mM α-ketoglutaric acid, 1.0 mM ascorbic acid, 0.5 mM FeSO 4 , and 3.3 μM of the aqueous protein solution of AzpK2 or Pp_AzpK2 obtained in Example 4 was reacted at 28° C. for 14 hours with shaking at 200 rpm to obtain a 5-hydroxy-L-lysine solution.

　この５－ヒドロキシ－Ｌ－リジン溶液に対し、参考例１に従ってＰｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉｄａ由来シクロデアミナーゼを用い、ＡｚｐＫ２を用いて得られた５－ヒドロキシ－Ｌ－リジン溶液は２４時間、Ｐｐ＿ＡｚｐＫ２を用いて得られた５－ヒドロキシ－Ｌ－リジン溶液は４時間反応させ、それぞれについて５－ヒドロキシ－Ｌ－ピペコリン酸を含む反応液を取得した。 　This 5-hydroxy-L-lysine solution was reacted with cyclodeaminase derived from Pseudomonas putida according to Reference Example 1, and the 5-hydroxy-L-lysine solution obtained using AzpK2 was reacted for 24 hours, and the 5-hydroxy-L-lysine solution obtained using Pp_AzpK2 was reacted for 4 hours, to obtain reaction solutions containing 5-hydroxy-L-pipecolic acid for each.

　さらに、得られたそれぞれの５－ヒドロキシ－Ｌ－ピペコリン酸を含む反応液について、参考例１に従って、ＦＤＡＡ誘導体化５－ヒドロキシ－Ｌ－ピペコリン酸を取得した。得られたＦＤＡＡ誘導体化５－ヒドロキシ－Ｌ－ピペコリン酸を含む溶液について、ＡｚｐＫ２を用いて得られた反応液は分析条件４で、Ｐｐ＿ＡｚｐＫ２を用いて得られた反応液は分析条件５でそれぞれ分析して、予め準備しておいた（２Ｓ,５Ｓ）－５－ヒドロキシ－Ｌ－ピペコリン酸のＦＤＡＡ誘導化物又は（２Ｓ,５Ｒ）－５－ヒドロキシ－Ｌ－ピペコリン酸のＦＤＡＡ誘導化物と比較した。 Furthermore, FDAA-derivatized 5-hydroxy-L-pipecolic acid was obtained from each of the obtained reaction solutions containing 5-hydroxy-L-pipecolic acid according to Reference Example 1. The obtained solutions containing FDAA-derivatized 5-hydroxy-L-pipecolic acid were analyzed under analytical condition 4 for the reaction solution obtained using AzpK2 and under analytical condition 5 for the reaction solution obtained using Pp_AzpK2, and compared with the FDAA-derivatized product of (2S,5S)-5-hydroxy-L-pipecolic acid or the FDAA-derivatized product of (2S,5R)-5-hydroxy-L-pipecolic acid that had been prepared in advance.

　その結果、ＡｚｐＫ２を用いて得られた５－ヒドロキシ－Ｌ－リジン溶液をシクロデアミナーゼで反応させた際の反応生成物は（２Ｓ,５Ｓ）－５－ヒドロキシ－Ｌ－ピペコリン酸であり、またＰｐ＿ＡｚｐＫ２を用いて得られた５－ヒドロキシ－Ｌ－リジン溶液をシクロデアミナーゼで反応させた際の反応生成物は（２Ｓ,５Ｒ）－５－ヒドロキシ－Ｌ－ピペコリン酸であった。 As a result, when the 5-hydroxy-L-lysine solution obtained using AzpK2 was reacted with cyclodeaminase, the reaction product was (2S,5S)-5-hydroxy-L-pipecolic acid, and when the 5-hydroxy-L-lysine solution obtained using Pp_AzpK2 was reacted with cyclodeaminase, the reaction product was (2S,5R)-5-hydroxy-L-pipecolic acid.

　このことから、ＡｚｐＫ２はＬ－リジンの５位をＳ選択的に水酸化する活性を有しており、またＰｐ＿ＡｚｐＫ２はＬ－リジンの５位をＲ選択的に水酸化する活性を有することが判明した。 This demonstrated that AzpK2 has the activity of S-selectively hydroxylating the 5th position of L-lysine, and that Pp_AzpK2 has the activity of R-selectively hydroxylating the 5th position of L-lysine.

＜実施例９＞（ＡｚｐＪ及びＰｐ＿ＡｚｐＪの基質選択性の確認）
　０．１ｍＭ　Ｌ－Ｌｙｓ、０．１ｍＭ　α－ケトグルタル酸、１．０ｍＭ　アスコルビン酸、１．０ｍＭ　ＮＡＤ＋、２５μＭ　ＦｅＳＯ４、５．０μＭの実施例４で取得した水酸化酵素ＡｚｐＫ２又はＰｐ＿ＡｚｐＫ２の組換えタンパク質、５．０μＭの実施例４で取得した酸化酵素ＡｚｐＪ又はＰｐ＿ＡｚｐＪの組換えタンパク質、２０ｍＭ　ＨＥＰＥＳ、１０％　グリセロール、２００ｍＭ　ＮａＣｌ（ｐＨ８．０）の組成で反応液１００μＬを調製し、ＳｐｅｃｔｒａＭａｘ　Ｍ２（Ｍｏｌｅｃｕｌａｒ　Ｄｅｖｉｃｅｓ社製）を使用して、３０℃で、ＮＡＤＨの吸光を示す波長３４０ｎｍの吸光度を２分間測定した。ここで得られる吸光度変化の傾きから、ＮＡＤＨ生成の初速度を算出した。ＮＡＤＨの吸光度はＮＡＤＨ試薬（ナカライテスク社製）の吸光度データを基に見積もった。 Example 9 (Confirmation of substrate selectivity of AzpJ and Pp_AzpJ)
A reaction solution (100 μL) containing 0.1 mM L-Lys, 0.1 mM α-ketoglutaric acid, 1.0 mM ascorbic acid, 1.0 mM NAD + , 25 μM FeSO 4 , 5.0 μM of recombinant protein of hydroxylase AzpK2 or Pp_AzpK2 obtained in Example 4, 5.0 μM of recombinant protein of oxidase AzpJ or Pp_AzpJ obtained in Example 4, 20 mM HEPES, 10% glycerol, and 200 mM NaCl (pH 8.0) was prepared, and the absorbance at a wavelength of 340 nm, which indicates the absorbance of NADH, was measured for 2 minutes at 30° C. using a SpectraMax M2 (Molecular Devices). The initial rate of NADH production was calculated from the slope of the absorbance change obtained here. The absorbance of NADH was estimated based on the absorbance data of the NADH reagent (manufactured by Nacalai Tesque).

　その結果、Ｓ選択的Ｌ－リジン５位水酸化酵素であるＡｚｐＫ２を用いた場合はＡｚｐＪを添加したときのみＮＡＤＨの蓄積増加による３４０ｎｍの吸光度上昇が確認され、Ｒ選択的Ｌ－リジン５位水酸化酵素であるＰｐ＿ＡｚｐＫ２を用いた場合はＰｐ＿ＡｚｐＪを添加したときのみＮＡＤＨの蓄積増加による３４０ｎｍの吸光度上昇が確認された。 As a result, when AzpK2, an S-selective L-lysine 5-hydroxylase, was used, an increase in absorbance at 340 nm due to increased accumulation of NADH was confirmed only when AzpJ was added, and when Pp_AzpK2, an R-selective L-lysine 5-hydroxylase, was used, an increase in absorbance at 340 nm due to increased accumulation of NADH was confirmed only when Pp_AzpJ was added.

　また、前記反応液をＨＰＬＣ（分析条件６）により分析したところ、Ｋａｗａｉ，Ｓ．らＡｎｇｅｗ．　Ｃｈｅｍｉｎｔ．　Ｉｎｔ．Ｅｄ．　Ｅｎｇｌ．　２０２１，　６０，　ｐ．１０３１９－１０３２５に報告のある５－オキソ－Ｌ－リジンが環化・脱水した化合物のｍ／ｚ　１４３．２と同一の質量ピークが検出された。分析条件６の詳細は下記の通りである。 In addition, when the reaction solution was analyzed by HPLC (analysis condition 6), a mass peak was detected that was identical to the m/z 143.2 of the compound formed by cyclization and dehydration of 5-oxo-L-lysine reported by Kawai, S. et al. in Angew. Chemint. Int. Ed. Engl. 2021, 60, pp. 10319-10325. Details of analysis condition 6 are as follows.

　以上の結果より、ＡｚｐＪは（５Ｓ）－ヒドロキシ－Ｌ－リジン選択的にヒドロキシ基を酸化する酵素であり、Ｐｐ＿ＡｚｐＪは（５Ｒ）－ヒドロキシ－Ｌ－リジン選択的にヒドロキシ基を酸化する酵素であることが分かった。
　したがって、リジン５位水酸化酵素の反応液にＡｚｐＪを用いたときに酸化反応が起きてＮＡＤＨに由来する吸光度が増加した場合には当該リジン５位水酸化酵素はＳ体選択的な水酸化酵素であり、リジン５位水酸化酵素の反応液にＰｐ＿ＡｚｐＪを用いたときに酸化反応が起きてＮＡＤＨに由来する吸光度が増加した場合には当該リジン５位水酸化酵素はＲ体選択的な水酸化酵素であると判別することができる。 The above results demonstrated that AzpJ is an enzyme that selectively oxidizes the hydroxy group in (5S)-hydroxy-L-lysine, and Pp_AzpJ is an enzyme that selectively oxidizes the hydroxy group in (5R)-hydroxy-L-lysine.
Therefore, when AzpJ is used in a reaction solution of lysine 5-hydroxylase, if an oxidation reaction occurs and the absorbance derived from NADH increases, the lysine 5-hydroxylase can be determined to be an S-selective hydroxylase, and when Pp_AzpJ is used in a reaction solution of lysine 5-hydroxylase, if an oxidation reaction occurs and the absorbance derived from NADH increases, the lysine 5-hydroxylase can be determined to be an R-selective hydroxylase.

＜実施例１０＞（ＡｚｐＫ２ホモログの水酸基の立体選択性確認）
　実施例４で得られたＡｚｐＫ２ホモログのタンパク質水溶液を用い、実施例９の手順に従って、ＡｚｐＪとＰｐ＿ＡｚｐＪのどちらの酸化酵素を用いた場合にＮＡＤＨの吸光を示す３４０ｎｍの吸光度が増加するか測定したところ、表５の結果が得られた。 Example 10 (Confirmation of stereoselectivity of hydroxyl group of AzpK2 homologue)
Using the aqueous protein solution of the AzpK2 homolog obtained in Example 4, and following the procedure of Example 9, it was measured whether the increase in absorbance at 340 nm, which indicates the absorbance of NADH, was caused by the use of either AzpJ or Pp_AzpJ oxidase. The results shown in Table 5 were obtained.

　また、前述の実施例９のとおり、ＡｚｐＪで吸光度が増加したＡｚｐＫ２ホモログはＳ選択的なＬ－リジン５位水酸化酵素であり、Ｐｐ＿ＡｚｐＪで吸光度が増加したＡｚｐＫ２ホモログはＲ選択的なＬ－リジン５位水酸化酵素であることから、各水酸化酵素の立体選択性（実施例４で得られたＡｚｐＫ２ホモログ）についても表５に示す。 As described above in Example 9, the AzpK2 homolog whose absorbance increased with AzpJ is an S-selective L-lysine 5-hydroxylase, and the AzpK2 homolog whose absorbance increased with Pp_AzpJ is an R-selective L-lysine 5-hydroxylase. Therefore, the stereoselectivity of each hydroxylase (AzpK2 homolog obtained in Example 4) is also shown in Table 5.

　なお、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ４－１は、ＡｚｐＪとＰｐ＿ＡｚｐＪのどちらの酸化酵素を用いた場合に吸光度が増加するか確認したところ、いずれの場合も吸光度増加量が少なく判別が困難であった。そこで、前述の実施例７と同様の方法でＦｍｏｃ化した５－オキソリジンの検出を試みた。その結果を図５に示す。 When we checked whether the absorbance of AzpK2 Homolog4-1 would increase when AzpJ or Pp_AzpJ was used as the oxidase, the increase in absorbance was small in both cases, making it difficult to distinguish. Therefore, we attempted to detect Fmoc-modified 5-oxolidine using the same method as in Example 7 above. The results are shown in Figure 5.

　図５中、「ＡｚｐＫ２＋ＡｚｐＪ」は、水酸化酵素ＡｚｐＫ２と酸化酵素ＡｚｐＪを用いて５－オキソリジンを生成したポジティブコントロールを表し、「４－１」は水酸化酵素ＡｚｐＫ２　Ｈｏｍｏｌｏｇ４－１を入れているが酸化酵素は入れていないネガティブコントロールである。「４－１＋ＡｚｐＪ」は、水酸化酵素ＡｚｐＫ２　Ｈｏｍｏｌｏｇ４－１と酸化酵素ＡｚｐＪを入れた反応液の分析結果であり、ネガティブコントロールよりやや大きい５－オキソリジンのピークが検出された。「４－１＋Ｐｐ＿ＡｚｐＪ」は、水酸化酵素ＡｚｐＫ２　Ｈｏｍｏｌｏｇ４－１と酸化酵素Ｐｐ＿ＡｚｐＪを入れた反応液の分析結果であり、前記「４－１＋ＡｚｐＪ」よりも明らかに強い５－オキソリジンのピークが検出された。すなわち、酸化酵素Ｐｐ＿ＡｚｐＪを用いた場合の方が酸化酵素ＡｚｐＪを用いた場合よりも多くの５－オキソリジンが検出された。 In Figure 5, "AzpK2 + AzpJ" represents a positive control in which 5-oxolidine was produced using the hydroxylase AzpK2 and oxidase AzpJ, and "4-1" is a negative control in which the hydroxylase AzpK2 Homolog 4-1 was added but no oxidase was added. "4-1 + AzpJ" is the analysis result of a reaction solution containing the hydroxylase AzpK2 Homolog 4-1 and oxidase AzpJ, and a 5-oxolidine peak that was slightly larger than that of the negative control was detected. "4-1 + Pp_AzpJ" is the analysis result of a reaction solution containing the hydroxylase AzpK2 Homolog 4-1 and oxidase Pp_AzpJ, and a 5-oxolidine peak that was clearly stronger than that of "4-1 + AzpJ." In other words, more 5-oxolidine was detected when the oxidase Pp_AzpJ was used than when the oxidase AzpJ was used.

　したがって、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ４－１は、Ｌ－リジンの５位をＳ体とＲ体の両方に水酸化する活性を有しているが、Ｒ体選択的な水酸化活性の方が高いことが分かった。 Therefore, it was found that AzpK2 Homolog4-1 has the activity of hydroxylating the 5-position of L-lysine to both the S- and R-isomers, but that the R-selective hydroxylation activity is higher.

＜実施例１１＞（Ｌ－リジン５位水酸化酵素の反応性比較）
　実施例９の手法では、Ｌ－リジン５位水酸化酵素によるＬ－Ｌｙｓの水酸化反応と比較して、５－ヒドロキシ－Ｌ－リジンの酸化反応の方が反応速度が速かった。したがって、水酸化酵素を変更して実施例９と同様の手法を用いた場合のＮＡＤＨの蓄積速度の差は水酸化酵素によるＬ－リジン水酸化反応速度の差を表す。 Example 11 (Comparison of reactivity of L-lysine 5-hydroxylase)
In the method of Example 9, the reaction rate of the oxidation reaction of 5-hydroxy-L-lysine was faster than that of the hydroxylation reaction of L-Lys by L-lysine 5-position hydroxylase. Therefore, the difference in the rate of NADH accumulation when the same method as in Example 9 is used with a different hydroxylase represents the difference in the rate of L-lysine hydroxylation reaction by the hydroxylase.

　酸化酵素ＡｚｐＪを用いて、実施例９と同様の手法を用いて、１ｍＭ　Ｌ－Ｌｙｓに対する各水酸化酵素の反応性を比較した。最初の２分間のＮＡＤＨ蓄積速度（初速）を測定した結果を表６に示す。 Using the oxidase AzpJ, the reactivity of each hydroxylase to 1 mM L-Lys was compared using the same method as in Example 9. The results of measuring the NADH accumulation rate (initial rate) for the first 2 minutes are shown in Table 6.

　表６から明らかなように、Ｓ体選択的Ｌ－リジン５位水酸化酵素の中では、ＮＡＤＨ蓄積速度（初速）はＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－２が最も高く、次いでＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－９、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－１、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－３の順に高い結果であった。したがって、Ｓ体選択的Ｌ－リジン５位水酸化酵素の水酸化活性についても同じ順に高いと考えられる。また、Ｒ体選択的Ｌ－リジン５位水酸化酵素の中では、ＮＡＤＨ蓄積速度（初速）はＡｚｐＫ２　Ｈｏｍｏｌｏｇ３－１が最も高く、次いでＰｐ＿ＡｚｐＫ２、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ２－２、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ２－３の順に高い結果であった。したがって、Ｒ体選択的Ｌ－リジン５位水酸化酵素の水酸化活性についても同じ順に高いと考えられる。 As is clear from Table 6, among the S-selective L-lysine 5-hydroxylases, AzpK2 Homolog1-2 had the highest NADH accumulation rate (initial velocity), followed by AzpK2 Homolog1-9, AzpK2 Homolog1-1, and AzpK2 Homolog1-3 in that order. Therefore, it is considered that the hydroxylation activity of the S-selective L-lysine 5-hydroxylases also follows the same order of increasing. Furthermore, among the R-selective L-lysine 5-hydroxylases, AzpK2 Homolog3-1 had the highest NADH accumulation rate (initial velocity), followed by Pp_AzpK2, AzpK2 Homolog2-2, and AzpK2 Homolog2-3 in that order. Therefore, it is considered that the hydroxylation activity of the R-selective L-lysine 5-hydroxylases also follows the same order of increasing.

＜実施例１２＞（遺伝子組換え大腸菌を用いたＡｚｐＫ２、Ｐｐ＿ＡｚｐＫ２及びＡｚｐＫ２ホモログの活性評価－１）
＜実施例１２－１：ｐＫＷ３２発現ベクターの作製＞
　下記表７に示すＡｚｐＫ２、Ｐｐ＿ＡｚｐＫ２、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－１、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－２及びＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－３について、各配列番号に示す遺伝子配列の上流にＮｄｅＩサイト、下流にＸｈｏＩサイトを付与した配列の直鎖ＤＮＡ断片をユ－ロフィンジェノミクス社より購入した。 Example 12 (Evaluation of the activities of AzpK2, Pp_AzpK2, and AzpK2 homologues using recombinant Escherichia coli-1)
<Example 12-1: Construction of pKW32 expression vector>
For AzpK2, Pp_AzpK2, AzpK2 Homolog1-1, AzpK2 Homolog1-2 and AzpK2 Homolog1-3 shown in Table 7 below, linear DNA fragments having an NdeI site upstream and an XhoI site downstream of the gene sequence shown in each SEQ ID NO were purchased from Europhin Genomics.

　ここで得られたＰｐ＿ＡｚｐＫ２、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－１、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－２及びＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－３の合成遺伝子に対し、下記表８に示す各遺伝子に制限酵素ＥｃｏＲＩ及びＸｂａＩサイトを付与するために設計した各プライマー配列を用い、ＰｒｉｍｅＳｔａｒ　ｍａｘ　ＤＮＡ　ｐｏｌｙｍｅｒａｓｅ（タカラバイオ社製）を使用して常法に従ってＰＣＲを行い、制限酵素ＥｃｏＲＩとＸｂａＩサイトを有する遺伝子断片を増幅した。 For the synthetic genes Pp_AzpK2, AzpK2 Homolog1-1, AzpK2 Homolog1-2, and AzpK2 Homolog1-3 obtained here, PCR was carried out according to standard methods using PrimeStar max DNA polymerase (Takara Bio) with each primer sequence designed to provide restriction enzyme EcoRI and XbaI sites to each gene shown in Table 8 below, to amplify gene fragments having restriction enzyme EcoRI and XbaI sites.

　また、ｐＫＷ３２ベクター（特許第５６１３６６０号を参考に取得）を制限酵素ＭｕｎＩ及びＸｂａＩ（共にタカラバイオ社製）で切断後、ＴＳＡＰ（Ｔｈｅｒｍｏｓｅｎｓｉｔｉｖｅ　Ａｌｋａｌｉｎｅ　Ｐｈｏｓｐｈａｔａｓｅ、プロメガ社製）を用いた脱リン酸化を行い調製したＤＮＡ断片と、上記で得られたＰＣＲ産物を制限酵素ＥｃｏＲＩとＸｂａＩ（タカラバイオ社製）で切断したＤＮＡ断片とを、ＤＮＡ　Ｌｉｇａｔｉｏｎ　Ｋｉｔ　＜Ｍｉｇｈｔｙ　Ｍｉｘ＞（タカラバイオ社製）を用いて常法に従ってライゲーションした。その後、常法に従って大腸菌ＪＭ１０９株（タカラバイオ社製）に形質転換したものを５０μｇ／ｍＬのカナマイシン硫酸塩（富士フイルム和光純薬社製）を含むＬＢ寒天培地（以下ＬＢ（Ｋｍ５０）寒天培地）上で、３０℃で一晩培養した。得られたコロニーからＱＩＡｐｒｅｐ　Ｓｐｉｎ　ｍｉｎｉｐｒｅｐ　ｋｉｔ（キアゲン社製）を使用して、常法に従って表８の作製ベクター名に示す発現ベクターを取得した。 In addition, a pKW32 vector (obtained with reference to Patent No. 5613660) was cleaved with the restriction enzymes MunI and XbaI (both manufactured by Takara Bio Inc.), and then dephosphorylated using TSAP (Thermosensitive Alkaline Phosphatase, manufactured by Promega Inc.) to prepare a DNA fragment. This was then ligated to a DNA fragment obtained by cleaving the PCR product obtained above with the restriction enzymes EcoRI and XbaI (manufactured by Takara Bio Inc.) using the DNA Ligation Kit <Mighty Mix> (manufactured by Takara Bio Inc.) in accordance with standard methods. Then, the resulting strain was transformed into E. coli JM109 (Takara Bio) in the usual manner and cultured overnight at 30°C on LB agar medium (hereinafter referred to as LB (Km50) agar medium) containing 50 μg/mL kanamycin sulfate (Fujifilm Wako Pure Chemical Industries, Ltd.). From the resulting colonies, the expression vectors shown in the names of the vectors prepared in Table 8 were obtained in the usual manner using a QIAprep Spin miniprep kit (Qiagen).

＜実施例１２－２：ｐＣｏｌｄ－ＰＳ２Ｋ発現ベクターの作製＞
　実施例１２－１でユ－ロフィンジェノミクス社より購入したＡｚｐＫ２及びＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－２のそれぞれの直鎖ＤＮＡ断片を、制限酵素ＮｄｅＩとＸｈｏＩ（共にタカラバイオ社製）で切断して、それぞれの挿入遺伝子断片を得た。 <Example 12-2: Preparation of pCold-PS2K expression vector>
The linear DNA fragments of AzpK2 and AzpK2 Homolog1-2 purchased from Europhin Genomics in Example 12-1 were each cleaved with restriction enzymes NdeI and XhoI (both manufactured by Takara Bio Inc.) to obtain the respective inserted gene fragments.

　次に、ｐＣｏｌｄ－ＰｒｏＳ２ベクター（タカラバイオ社製）に対して配列番号１６４と１６５のプライマー、ｐＫＷ３２ベクターに対して配列番号１６６と１６７のプライマーをそれぞれ用いて、常法に従ってＰＣＲを行い、それぞれのＰＣＲ増幅断片を取得した。 Next, PCR was performed according to standard methods using primers of sequence numbers 164 and 165 for the pCold-ProS2 vector (Takara Bio), and primers of sequence numbers 166 and 167 for the pKW32 vector, to obtain the respective PCR amplified fragments.

　得られたこれらのＰＣＲ増幅断片をＩｎ－Ｆｕｓｉｏｎ（登録商標）　ＨＤ　Ｃｌｏｎｉｎｇ　Ｋｉｔ（タカラバイオ社製）を用いて常法に従ってライゲーションした。その後、大腸菌Ｅ．　ｃｏｌｉ　ＪＭ１０９株に常法に従って形質転換して、ＬＢ（Ｋｍ５０）寒天培地上で、３０℃で一晩培養した。得られたコロニーからミニプレップ法によりプラスミドＤＮＡを抽出し、ｐＣｏｌｄ－ＰｒｏＳ２ベクターの薬剤耐性遺伝子をカナマイシンに変更したｐＣｏｌｄ－ＰＳ２Ｋベクターを得た。 These PCR-amplified fragments were ligated using the In-Fusion (registered trademark) HD Cloning Kit (Takara Bio Inc.) in the usual manner. The E. coli JM109 strain was then transformed in the usual manner and cultured overnight at 30°C on LB (Km50) agar medium. Plasmid DNA was extracted from the resulting colonies by the miniprep method, and the pCold-PS2K vector was obtained, in which the drug resistance gene of the pCold-ProS2 vector was changed to kanamycin.

　得られたｐＣｏｌｄ－ＰＳ２Ｋベクターを制限酵素ＮｄｅＩとＸｈｏＩ（共にタカラバイオ社製）で切断後、実施例１２－１と同様にして脱リン酸化を行い、さらにＡｚｐＫ２又はＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－２の各挿入遺伝子断片を用いて、実施例１２－１と同様にライゲーションして、それぞれの発現ベクターであるｐＣｏｌｄ－ＰＳ２Ｋ－ＡｚｐＫ２及びｐＣｏｌｄ－ＰＳ２Ｋ－１－２を得た。 The resulting pCold-PS2K vector was cleaved with the restriction enzymes NdeI and XhoI (both manufactured by Takara Bio Inc.), and then dephosphorylated in the same manner as in Example 12-1. The inserted gene fragments of AzpK2 or AzpK2 Homolog1-2 were then used for ligation in the same manner as in Example 12-1 to obtain the respective expression vectors pCold-PS2K-AzpK2 and pCold-PS2K-1-2.

＜実施例１２－３：フラスコ培養＞
　実施例１２－１及び実施例１２－２で得た発現ベクターを用いて、常法に従ってｐＫＷ３２のプラスミドを大腸菌ＪＭ１０９株に、ｐＣｏｌｄ－ＰＳ２ＫのプラスミドをＥ．　ｃｏｌｉ　ＢＬ２１　ｓｔａｒ（ＤＥ３）（Ｔｈｅｒｍｏ　ｆｉｓｈｅｒ　ｓｃｉｅｎｔｉｆｉｃ社製）にそれぞれ導入した。 <Example 12-3: Flask culture>
Using the expression vectors obtained in Examples 12-1 and 12-2, the pKW32 plasmid was introduced into E. coli JM109 strain, and the pCold-PS2K plasmid was introduced into E. coli BL21 star (DE3) (Thermo Fisher Scientific) according to a conventional method.

　得られたコロニーをＬＢ（Ｋｍ５０）培地２ｍＬに植菌して振盪培養し、培養液に終濃度１５Ｗ／Ｖ％となるように７５Ｗ／Ｖ％のグリセロールを添加・混合し、凍結菌ストックを作製した。凍結菌ストックは－８０℃で保存した。各凍結菌ストックをそれぞれＬＢ（Ｋｍ５０）培地２ｍＬに植菌し、３０℃、１８０ｒｐｍで一晩振盪培養したものを前培養液とした。 The colonies obtained were inoculated into 2 mL of LB (Km50) medium and cultured with shaking, and 75 W/V% glycerol was added and mixed to the culture solution to a final concentration of 15 W/V% to prepare frozen bacterial stocks. The frozen bacterial stocks were stored at -80°C. Each frozen bacterial stock was inoculated into 2 mL of LB (Km50) medium and cultured overnight with shaking at 30°C and 180 rpm to prepare preculture solutions.

　Ｅ．　ｃｏｌｉ　ＪＭ１０９／ｐＫＷ３２はＬＢ（Ｋｍ５０）培地４０ｍＬに前培養液４０μＬを植菌後、イソプロピル－β－Ｄ－チオガラクトピラノシド（以下、「ＩＰＴＧ」と称する。）を終濃度０．２ｍＭとなるように添加し、３０℃、２３０ｒｐｍで一晩振盪培養した。Ｅ．　ｃｏｌｉ　ＢＬ２１　ｓｔａｒ（ＤＥ３）／ｐＣｏｌｄ－ＰＳ２ＫはＬＢ（Ｋｍ５０）培地４０ｍＬに前培養液１ｍＬを植菌し、３０℃、２３０ｒｐｍで振盪培養した。ＯＤ６３０ｎｍが０．５に達した後、１５℃で３０分静置し、ＩＰＴＧを終濃度０．２ｍＭとなるように添加して、１５℃、１８０ｒｐｍで２４時間振盪培養した。 For E. coli JM109/pKW32, 40 μL of preculture was inoculated into 40 mL of LB (Km50) medium, isopropyl-β-D-thiogalactopyranoside (hereinafter referred to as "IPTG") was added to a final concentration of 0.2 mM, and the mixture was cultured overnight at 30°C and 230 rpm with shaking. For E. coli BL21 star (DE3)/pCold-PS2K, 1 mL of preculture was inoculated into 40 mL of LB (Km50) medium, and cultured at 30°C and 230 rpm with shaking. After OD630nm reached 0.5, the mixture was left to stand at 15°C for 30 minutes, IPTG was added to a final concentration of 0.2 mM, and the mixture was cultured at 15°C and 180 rpm with shaking for 24 hours.

＜実施例１２－４：遺伝子組換え大腸菌を用いた反応性評価＞
　実施例１２－３で得られた培養液を遠心分離（６５００ｒｐｍ、１０分、４℃）して集菌し、集菌した菌体の湿菌体重量を測定して１００ｇ／Ｌとなるように５０ｍＭ　ＭＥＳ－ＮａＯＨ　ｂｕｆｆｅｒ（ｐＨ７．０）（以下、「ＭＥＳ　ｂｕｆｆｅｒ（ｐＨ７．０）」と表現する場合がある。）で懸濁後、超音波ホモジナイザーＳＯＮＩＦＩＥＲ（登録商標）　２５０Ｄ（ＢＲＡＮＳＯＮ社製）を使用して超音波破砕した。得られた菌体破砕液を用いて、反応液組成表－１の反応液組成１～３のそれぞれについて、容量１４ｍＬのＦａｌｃｏｎ（登録商標）ラウンドチューブで反応液を調製した。 <Example 12-4: Reactivity evaluation using recombinant Escherichia coli>
The culture solution obtained in Example 12-3 was centrifuged (6500 rpm, 10 minutes, 4°C) to collect the bacteria, and the wet weight of the collected bacteria was measured and suspended in 50 mM MES-NaOH buffer (pH 7.0) (hereinafter sometimes referred to as "MES buffer (pH 7.0)") so as to be 100 g/L, followed by ultrasonic disruption using an ultrasonic homogenizer SONIFIER (registered trademark) 250D (manufactured by BRANSON). Using the obtained disrupted bacteria solution, reaction solutions were prepared in 14 mL Falcon (registered trademark) round tubes for each of reaction solution compositions 1 to 3 in Reaction Solution Composition Table-1.

　反応液組成表－１中、菌体破砕液の濃度は、遠心分離して集菌した菌体をどの程度含むかを表す。また、これ以降も「・・ｇ／Ｌの菌体破砕液」や「・・ｇ／Ｌの菌体懸濁液」と表現する場合には同様の意味を表す。 In the reaction solution composition table 1, the concentration of the bacterial cell lysate indicates the amount of bacterial cells collected by centrifugation that are contained in the solution. In the following, the same meaning will be expressed when it is expressed as "... g/L bacterial cell lysate" or "... g/L bacterial cell suspension."

　調製した反応液を三段恒温振盪培養機ＴＡＬ－ＲＳ３１０（トーマス科学器械社製）を使用して、１５℃、２００ｒｐｍで１９時間振盪して反応を行った。 The prepared reaction solution was shaken at 15°C and 200 rpm for 19 hours using a three-stage constant temperature shaking incubator TAL-RS310 (manufactured by Thomas Scientific Instruments).

　なお、振盪開始（反応開始）後０．５時間、１時間、２時間及び４時間経過後、それぞれのラウンドチューブから反応液の一部を採取し、精製水で５倍に希釈後、フィルターユニット（Ａｍｉｃｏｎ（登録商標）　Ｕｌｔｒａ－０．５　１０ｋＤａ（メルク社製））を用いて、室温で、１０,０００×ｇ、１０分間限外ろ過し、得られたサンプルをＨＰＬＣ（分析条件７）で分析した。分析条件７の詳細は以下の通りである。 Furthermore, 0.5 hours, 1 hour, 2 hours, and 4 hours after the start of shaking (start of reaction), a portion of the reaction solution was taken from each round tube, diluted 5-fold with purified water, and ultrafiltered at room temperature for 10 minutes at 10,000 x g using a filter unit (Amicon (registered trademark) Ultra-0.5 10 kDa (Merck)). The obtained sample was analyzed by HPLC (analysis condition 7). Details of analysis condition 7 are as follows.

　反応液組成表－１の反応液組成１で反応させた反応液について、各水酸化酵素を発現した大腸菌の菌体破砕液の比活性を測定した。ＡｚｐＫ２、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－１、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－２及びＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－３については反応時間０．５時間、Ｐｐ＿ＡｚｐＫ２については反応時間４時間のサンプルにおける５－ヒドロキシ－Ｌ－リジンの蓄積濃度を測定し、菌体１ｇあたりの５－ヒドロキシ－Ｌ－リジン生産活性すなわち比活性とした。比活性は、１分間の水酸化反応で１μｍｏｌの５－ヒドロキシ－Ｌ－リジンが蓄積する酵素活性を１ユニット（以下「Ｕ」と称する。）と定義し、Ｕ／ｇ－ｃｅｌｌで表す。比活性の測定結果を表９に示す。 The specific activity of the cell lysate of E. coli expressing each hydroxylase was measured for the reaction solution reacted with reaction solution composition 1 in Reaction Solution Composition Table 1. The accumulated concentration of 5-hydroxy-L-lysine in the samples with a reaction time of 0.5 hours for AzpK2, AzpK2 Homolog1-1, AzpK2 Homolog1-2, and AzpK2 Homolog1-3, and with a reaction time of 4 hours for Pp_AzpK2, was measured and used as the 5-hydroxy-L-lysine production activity per gram of cells, i.e., specific activity. The enzyme activity that accumulates 1 μmol of 5-hydroxy-L-lysine in a hydroxylation reaction for 1 minute is defined as 1 unit (hereinafter referred to as "U"), and is expressed in U/g-cell. The measurement results of the specific activity are shown in Table 9.

　表９から明らかなように、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－１、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－２及びＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－３は、ＡｚｐＫ２及びＰｐ＿ＡｚｐＫ２よりも高い比活性を示した。その中でも、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－１及びＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－２は特に高い比活性を示した。 As is clear from Table 9, AzpK2 Homolog1-1, AzpK2 Homolog1-2, and AzpK2 Homolog1-3 showed higher specific activity than AzpK2 and Pp_AzpK2. Among them, AzpK2 Homolog1-1 and AzpK2 Homolog1-2 showed particularly high specific activity.

　なお、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－２については、ｐＫＷ３２ベクター（Ｅ．　ｃｏｌｉ　ＪＭ１０９株）とｐＣｏｌｄ－ＰＳ２Ｋベクター（Ｅ．　ｃｏｌｉ　ＢＬ２１　ｓｔａｒ（ＤＥ３）（株））の２種類の培養菌体を用いて比活性を比較したところ、ｐＫＷ３２ベクター（Ｅ．　ｃｏｌｉ　ＪＭ１０９株）の方が高い比活性を示した。 When the specific activity of AzpK2 Homolog1-2 was compared using two types of cultured bacterial cells, pKW32 vector (E. coli JM109 strain) and pCold-PS2K vector (E. coli BL21 star (DE3) strain), the pKW32 vector (E. coli JM109 strain) showed a higher specific activity.

　また、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－１、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－２及びＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－３については、反応液組成１～３で反応させた各反応液について、反応時間１時間のサンプルにおける５－ヒドロキシ－Ｌ－リジン（５ＨＬ）の濃度（蓄積量）を比較した結果を表１０に示す。 Also, for AzpK2 Homolog 1-1, AzpK2 Homolog 1-2, and AzpK2 Homolog 1-3, the concentration (accumulation amount) of 5-hydroxy-L-lysine (5HL) in samples reacted for 1 hour in each reaction solution reacted with reaction solution compositions 1 to 3 is compared. The results are shown in Table 10.

　なお、表１０において、相対値は、各酵素のＬ－Ｌｙｓ　２０ｍＭ仕込みの５－ヒドロキシ－Ｌ－リジン蓄積量を基準として、それぞれの５－ヒドロキシ－Ｌ－リジン蓄積量を除したものであり、また蓄積量と蓄積濃度はどちらも同じ意味を示す。 In Table 10, the relative values are calculated by dividing the amount of 5-hydroxy-L-lysine accumulated by the amount of 5-hydroxy-L-lysine accumulated in the 20 mM L-Lys preparation for each enzyme, and the terms accumulated amount and accumulated concentration have the same meaning.

　表１０から明らかなように、いずれの酵素においてもＬ－Ｌｙｓ仕込み濃度２０ｍＭの場合が最も５－ヒドロキシ－Ｌ－リジン蓄積量が多く、Ｌ－Ｌｙｓ仕込み濃度が５０ｍＭ、１００ｍＭと高くなるほど５－ヒドロキシ－Ｌ－リジン蓄積量は減少した。したがって、いずれの酵素においても基質であるＬ－Ｌｙｓ仕込み濃度が高くなると反応阻害が生じて反応生成物が少なくなる基質阻害の現象が生じていることが示唆される。 As is clear from Table 10, for both enzymes, the amount of 5-hydroxy-L-lysine accumulated was greatest when the L-Lys concentration was 20 mM, and the amount of 5-hydroxy-L-lysine accumulated decreased as the L-Lys concentration increased to 50 mM and 100 mM. This suggests that for both enzymes, the phenomenon of substrate inhibition occurred, in which reaction inhibition occurs and reaction products decrease when the concentration of L-Lys, the substrate, is increased.

　ここで、基質阻害による反応速度低下の影響度は、基質濃度を高くした場合の相対値が高いほど基質阻害の影響は少なく、反対に基質濃度を高くした場合の相対値が低いほど基質阻害の影響を大きく受けることを表す。 Here, the degree of influence of substrate inhibition on the decrease in reaction rate is such that the higher the relative value when the substrate concentration is increased, the less the influence of substrate inhibition is, and conversely, the lower the relative value when the substrate concentration is increased, the greater the influence of substrate inhibition is.

　表１０の結果より、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－３が最も基質阻害による影響が低い酵素であり、次いでＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－２、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－１の順で低かった。 The results in Table 10 show that AzpK2 Homolog 1-3 is the enzyme least affected by substrate inhibition, followed by AzpK2 Homolog 1-2 and AzpK2 Homolog 1-1.

＜実施例１３＞（遺伝子組換え大腸菌を用いたＡｚｐＫ２ホモログの活性評価－２）
　ｐＣｏｌｄ　Ｉベクター（タカラバイオ社製）及び実施例３で取得したｐＣｏｌｄ　Ｉ－１－１～１－９の９種の発現ベクターについて、それぞれ常法に従ってＥ．　ｃｏｌｉ　ＢＬ２１　ｓｔａｒ（ＤＥ３）株の形質転換体を取得した。 Example 13 (Evaluation of activity of AzpK2 homologue using recombinant E. coli-2)
For each of the nine expression vectors pCold I vector (manufactured by Takara Bio Inc.) and pCold I-1-1 to 1-9 obtained in Example 3, transformants of the E. coli BL21 star (DE3) strain were obtained according to a conventional method.

　ｐＣｏｌｄ　Ｉベクター及びｐＣｏｌｄ　Ｉ－１－１～１－９の各形質転換体は、１００　μｇ／ｍＬのアンピシリンナトリウムを含むＴｅｒｒｉｆｉｃ　Ｂｒｏｔｈ培地（以下「ＴＢ（Ａｍｐ１００）培地」と称する。）２ｍＬに植菌し、３０℃で一晩振盪培養した（前培養）。 The pCold I vector and each of the pCold I-1-1 to 1-9 transformants were inoculated into 2 mL of Terrific Broth medium containing 100 μg/mL sodium ampicillin (hereinafter referred to as "TB (Amp100) medium") and cultured overnight at 30°C with shaking (preculture).

　別のＴＢ（Ａｍｐ１００）培地４０ｍＬに、得られた各前培養液０．４ｍＬを植菌し、３０℃で振盪培養した。ＯＤ６３０ｎｍが０．５に達するまで培養し、１５℃で３０分静置後、ＩＰＴＧを終濃度０．０２ｍＭとなるように添加し、さらに１５℃、２００ｒｐｍで２４時間振盪培養して各培養液を得た。 0.4 mL of each preculture solution was inoculated into 40 mL of another TB (Amp100) medium and cultured with shaking at 30°C. Culture was continued until the OD630nm reached 0.5, and after standing at 15°C for 30 minutes, IPTG was added to a final concentration of 0.02 mM, and cultured with shaking at 15°C and 200 rpm for 24 hours to obtain each culture solution.

　得られた各培養液について、実施例１２－４と同様に菌体破砕液を取得した。得られた菌体破砕液を用いて、２０ｍＭ　Ｌ－Ｌｙｓ、３０ｍＭ　α－ケトグルタル酸、２ｍＭ　アスコルビン酸ナトリウム、０．５ｍＭ　ＦｅＳＯ４、２．５ｍＭ　クエン酸、５０ｍＭ　ＨＥＰＥＳ（ｐＨ７．０）、５０ｇ／Ｌ菌体破砕液の組成で反応溶液０．４ｍＬを調製した。得られた各反応溶液を、小型恒温振盪培養機ＢＲ－２２ＦＰ（タイテック社製）を使用して、１５℃、２００ｒｐｍで一晩振盪して反応を行った。 A cell lysate was obtained from each of the culture solutions obtained in the same manner as in Example 12-4. Using the resulting cell lysate, 0.4 mL of a reaction solution was prepared with the following composition: 20 mM L-Lys, 30 mM α-ketoglutaric acid, 2 mM sodium ascorbate, 0.5 mM FeSO 4 , 2.5 mM citric acid, 50 mM HEPES (pH 7.0), and 50 g/L cell lysate. Each of the resulting reaction solutions was reacted overnight at 15° C. and 200 rpm with a small thermostatic shaking incubator BR-22FP (manufactured by Taitec Corporation).

　なお、振盪開始（反応開始）後１時間、２時間、４時間及び２４時間経過後に、それぞれサンプリングし、精製水で２５倍に希釈後、フィルターユニット（Ａｍｉｃｏｎ（登録商標）　Ｕｌｔｒａ－０．５　１０ｋＤａ（メルク社製））を用いて、室温で、１０,０００×ｇ、１０分間限外ろ過し、得られたたサンプルをＨＰＬＣ（分析条件８）で５－ヒドロキシ－Ｌ－リジン蓄積量を分析した。分析条件８の詳細は以下の通りである。 Samples were taken 1 hour, 2 hours, 4 hours, and 24 hours after the start of shaking (start of reaction), diluted 25-fold with purified water, and ultrafiltered at 10,000 x g for 10 minutes at room temperature using a filter unit (Amicon (registered trademark) Ultra-0.5 10 kDa (Merck)). The resulting samples were analyzed for the amount of 5-hydroxy-L-lysine accumulated by HPLC (analysis condition 8). Details of analysis condition 8 are as follows.

　ネガティブコントロールであるｐＣｏｌｄ　Ｉベクター、及び各ＡｚｐＫ２ホモログを発現した遺伝子組換え大腸菌の菌体破砕液の５－ヒドロキシ－Ｌ－リジン蓄積量の測定結果を図６に示す。図６中、縦軸は、５－ヒドロキシリジン濃度（ｍＭ）を表す。 The results of measuring the amount of 5-hydroxy-L-lysine accumulated in the cell lysates of recombinant E. coli expressing the negative control pCold I vector and each AzpK2 homolog are shown in Figure 6. In Figure 6, the vertical axis represents the 5-hydroxylysine concentration (mM).

　図６から明らかなように、ネガティブコントロールであるｐＣｏｌｄ　Ｉベクターを除き、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－１～１－９の全てにおいて５－ヒドロキシリジンの蓄積が見られた。５－ヒドロキシ－Ｌ－リジン蓄積量は、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－１～１－９の中ではＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－３が最も高く、次いでＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－６とＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－２、さらに次にＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－１、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－５及びＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－４の順に高かった。 As is clear from Figure 6, accumulation of 5-hydroxylysine was observed in all of AzpK2 Homolog1-1 to 1-9, except for the negative control pCold I vector. The amount of 5-hydroxy-L-lysine accumulated was highest in AzpK2 Homolog1-3 among AzpK2 Homolog1-1 to 1-9, followed by AzpK2 Homolog1-6 and AzpK2 Homolog1-2, and then AzpK2 Homolog1-1, AzpK2 Homolog1-5, and AzpK2 Homolog1-4.

＜実施例１４＞（遺伝子組換え大腸菌を用いたＳ選択的リジン水酸化酵素の反応性評価－３）
　実施例１３で取得したｐＣｏｌｄ　Ｉベクター、ｐＣｏｌｄ　Ｉ－１－２、ｐＣｏｌｄ　Ｉ－１－３又はｐＣｏｌｄ　Ｉ－１－６を形質転換した組換え大腸菌の各菌体破砕液を用いて、酵素の基質阻害の影響及び５－ヒドロキシ－Ｌ－リジンの水酸化活性を評価した。 Example 14 (Reactivity evaluation of S-selective lysine hydroxylase using recombinant Escherichia coli-3)
The effect of substrate inhibition on the enzyme and the hydroxylation activity of 5-hydroxy-L-lysine were evaluated using each cell lysate of recombinant E. coli transformed with the pCold I vector, pCold I-1-2, pCold I-1-3, or pCold I-1-6 obtained in Example 13.

＜実施例１４－１：Ｓ選択的リジン水酸化酵素の基質阻害による影響評価＞
　反応液組成表－２の反応組成４～７で反応液を調製し、それぞれ実施例１３と同様に反応を実施して、５－ヒドロキシ－Ｌ－リジン蓄積濃度を測定した。 Example 14-1: Evaluation of the effect of substrate inhibition on S-selective lysine hydroxylase
Reaction solutions were prepared using reaction compositions 4 to 7 in Table 2, and reactions were carried out in the same manner as in Example 13 to measure the accumulated concentration of 5-hydroxy-L-lysine.

　反応液組成４～７で反応させた各反応液について、実施例１３と同様にして、測定及び算出した、反応時間１時間のサンプルにおける５－ヒドロキシ－Ｌ－リジン蓄積量及び相対値を表１１に示す。 The amount and relative value of 5-hydroxy-L-lysine accumulated in the sample after 1 hour of reaction, measured and calculated in the same manner as in Example 13 for each reaction solution reacted with reaction solution compositions 4 to 7, are shown in Table 11.

　なお、表１１において、相対値は、各酵素のＬ－Ｌｙｓ　２０ｍＭ仕込みの５－ヒドロキシ－Ｌ－リジン蓄積量を基準として、それぞれの５－ヒドロキシ－Ｌ－リジン蓄積量を除したものである。 In Table 11, the relative values are calculated by dividing the amount of 5-hydroxy-L-lysine accumulated by the amount of 5-hydroxy-L-lysine accumulated in the 20 mM L-Lys preparation for each enzyme.

　表１１から明らかなように、基質阻害の影響を表す相対値はＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－２が最も高く、次いでＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－３、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－６の順に高かった。したがって、基質阻害の影響を最も受けづらい酵素はＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－２であり、次にＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－３、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－６の順に影響を受けづらいことが分かった。 As is clear from Table 11, the relative value indicating the effect of substrate inhibition was highest for AzpK2 Homolog 1-2, followed by AzpK2 Homolog 1-3 and AzpK2 Homolog 1-6. Therefore, it was found that the enzyme least susceptible to the effects of substrate inhibition was AzpK2 Homolog 1-2, followed by AzpK2 Homolog 1-3 and AzpK2 Homolog 1-6.

　一方、５－ヒドロキシ－Ｌ－リジン蓄積量は、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－３が最も高く、次いでＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－２、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－６の順に高かった。また、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－３は、５－ヒドロキシ－Ｌ－リジンの生産性を表す５－ヒドロキシ－Ｌ－リジン蓄積濃度は、基質であるＬ－リジンの濃度が２０～１００ｍＭのいずれの条件においてもＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－２及びＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－６より高かった。このことから、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－３は基質阻害の影響をＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－２よりも受けやすいものの、５－ヒドロキシ－Ｌ－リジンの生産性はＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－２やＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－６と比べて優れていることが分かった。 On the other hand, the amount of 5-hydroxy-L-lysine accumulated was highest in AzpK2 Homolog1-3, followed by AzpK2 Homolog1-2 and AzpK2 Homolog1-6. Furthermore, the 5-hydroxy-L-lysine accumulation concentration, which indicates the productivity of 5-hydroxy-L-lysine, in AzpK2 Homolog1-3 was higher than that of AzpK2 Homolog1-2 and AzpK2 Homolog1-6 under all conditions where the concentration of the substrate L-lysine was 20 to 100 mM. This indicates that although AzpK2 Homolog1-3 is more susceptible to substrate inhibition than AzpK2 Homolog1-2, its productivity of 5-hydroxy-L-lysine is superior to that of AzpK2 Homolog1-2 and AzpK2 Homolog1-6.

＜実施例１４－２：Ｓ選択的リジン水酸化酵素の５－ヒドロキシ－Ｌ－リジンの水酸化活性評価＞
　Ｌ－リジン５位水酸化酵素は、Ｌ－リジンの水酸化活性を有するが、一方で実施例１０に示したように、生成物である５－ヒドロキシリジンの５位をさらに水酸化する活性も有している。また、ここで５－オキソリジンが生成すると、目的生成物である５－ヒドロキシリジンの収量が低下することから、５－ヒドロキシリジンを合成する製法を確立しようとする場合には、水酸化酵素による５－ヒドロキシリジンの水酸化活性は低い方が好ましい。 Example 14-2: Evaluation of 5-hydroxy-L-lysine hydroxylation activity of S-selective lysine hydroxylase
L-lysine 5-hydroxylase has the activity of hydroxylating L-lysine, but also has the activity of further hydroxylating the 5-position of the product, 5-hydroxylysine, as shown in Example 10. Furthermore, if 5-oxolidine is produced here, the yield of the target product, 5-hydroxylysine, decreases. Therefore, when attempting to establish a method for synthesizing 5-hydroxylysine, it is preferable that the hydroxylase has a low hydroxylating activity of 5-hydroxylysine.

　そこで、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－２、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－３及びＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－６について、Ｌ－リジンに対する水酸化活性と５－ヒドロキシリジンに対する水酸化活性を比較した。 Then, the hydroxylation activity of L-lysine and the hydroxylation activity of 5-hydroxylysine were compared for AzpK2 Homolog1-2, AzpK2 Homolog1-3, and AzpK2 Homolog1-6.

　２０ｍＭ　ラセミ体５－ヒドロキシリジン、３０ｍＭ　α－ケトグルタル酸、２ｍＭ　アスコルビン酸、０．５ｍＭ　ＦｅＳＯ４、２．５ｍＭ　クエン酸及び５０ｍＭ　ＨＥＰＥＳ（ｐＨ　７．０）、５０ｇ／Ｌの菌体破砕液の組成で反応溶液０．４ｍＬを調製し、反応時間を３時間とし、その他の条件は実施例１３と同様にして反応させ、５－ヒドロキシ－Ｌ－リジン濃度を測定した。測定した５－ヒドロキシ－Ｌ－リジン濃度を反応時間で除して、単位時間あたりの５－ヒドロキシ－Ｌ－リジン蓄積速度を算出した。 A 0.4 mL reaction solution was prepared with a composition of 20 mM racemic 5-hydroxylysine, 30 mM α-ketoglutaric acid, 2 mM ascorbic acid, 0.5 mM FeSO 4 , 2.5 mM citric acid, 50 mM HEPES (pH 7.0), and 50 g/L of cell lysate, and the reaction was carried out for 3 hours under the same conditions as in Example 13, and the 5-hydroxy-L-lysine concentration was measured. The measured 5-hydroxy-L-lysine concentration was divided by the reaction time to calculate the 5-hydroxy-L-lysine accumulation rate per unit time.

　各酵素の反応３時間後の５－ヒドロキシ－Ｌ－リジン残量を、ネガティブコントロ－ルであるｐＣｏｌｄ　Ｉベクター導入株の５－ヒドロキシ－Ｌ－リジン残量の差で減算して、単位時間当たりの５－ヒドロキシ－Ｌ－リジン消費速度を算出した。 The remaining amount of 5-hydroxy-L-lysine after 3 hours of reaction with each enzyme was subtracted by the difference in the remaining amount of 5-hydroxy-L-lysine in the negative control pCold I vector-introduced strain to calculate the 5-hydroxy-L-lysine consumption rate per unit time.

　また、５－ヒドロキシ－Ｌ－リジン消費速度を、実施例１４－１で測定した５－ヒドロキシ－Ｌ－リジン蓄積速度で除したものを５－ヒドロキシ－Ｌ－リジン／Ｌ－Ｌｙｓ水酸化活性比として算出し、表１２に表した。 The 5-hydroxy-L-lysine consumption rate was divided by the 5-hydroxy-L-lysine accumulation rate measured in Example 14-1 to calculate the 5-hydroxy-L-lysine/L-Lys hydroxylation activity ratio, which is shown in Table 12.

　５－ヒドロキシ－Ｌ－リジン／Ｌ－Ｌｙｓ水酸化活性比が高いほど、５－ヒドロキシ－Ｌ－リジンを基質として反応した際に５－オキソリジンを生成しやすいため、５－ヒドロキシ－Ｌ－リジン／Ｌ－Ｌｙｓ水酸化活性比は低い方が５－ヒドロキシ－Ｌ－リジン収量を高くすることができる。表１２から明らかなように、５－ヒドロキシ－Ｌ－リジン／Ｌ－Ｌｙｓ水酸化活性比はＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－３が最も低く、次いでＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－２、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－６の順に低かった。このことから、５－ヒドロキシ－Ｌ－リジン収量は、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－３が最も高く、次いでＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－２、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－６の順に高いと考えられる。 The higher the 5-hydroxy-L-lysine/L-Lys hydroxylation activity ratio, the easier it is to produce 5-oxolidine when reacting with 5-hydroxy-L-lysine as a substrate, so a lower 5-hydroxy-L-lysine/L-Lys hydroxylation activity ratio can increase the 5-hydroxy-L-lysine yield. As is clear from Table 12, the 5-hydroxy-L-lysine/L-Lys hydroxylation activity ratio was lowest for AzpK2 Homolog1-3, followed by AzpK2 Homolog1-2 and AzpK2 Homolog1-6 in that order. From this, it is considered that the 5-hydroxy-L-lysine yield is highest for AzpK2 Homolog1-3, followed by AzpK2 Homolog1-2 and AzpK2 Homolog1-6 in that order.

＜実施例１５＞（反応条件の検討）
＜実施例１５－１＞
　実施例１２－１で作製したｐＫＷ３２－１－３ベクターを常法に従って大腸菌ＪＭ１０９株に導入してＥ．　ｃｏｌｉ　ＪＭ１０９－ｐＫＷ３２－１－３株を作製した。得られたＥ．　ｃｏｌｉ　ＪＭ１０９－ｐＫＷ３２－１－３株を常法に従ってＩＰＴＧを終濃度０．２ｍＭとなるように酵素発現誘導を行って培養し、Ｅ．　ｃｏｌｉ　ＪＭ１０９－ｐＫＷ３２－１－３株の培養菌体を取得した。 Example 15 (Study of reaction conditions)
<Example 15-1>
The pKW32-1-3 vector prepared in Example 12-1 was introduced into the E. coli JM109 strain according to a conventional method to prepare the E. coli JM109-pKW32-1-3 strain. The obtained E. coli JM109-pKW32-1-3 strain was cultured by inducing enzyme expression with IPTG to a final concentration of 0.2 mM according to a conventional method, and cultured cells of the E. coli JM109-pKW32-1-3 strain were obtained.

＜実施例１５－２：菌体破砕条件の検討＞
　実施例１５－１で取得したＥ．　ｃｏｌｉ　ＪＭ１０９－ｐＫＷ３２－１－３株の培養菌体を、濃度１００ｇ／Ｌになるように、下記条件Ｉ～IIIの緩衝溶液にそれぞれ懸濁し、ＳＯＮＩＦＩＥＲ（登録商標）　２５０Ｄ（ＢＲＡＮＳＯＮ社製）を使用して超音波破砕した。 Example 15-2: Examination of cell disruption conditions
The cultured cells of E. coli JM109-pKW32-1-3 strain obtained in Example 15-1 were suspended in each of the buffer solutions under the following conditions I to III to a concentration of 100 g/L, and were ultrasonically disrupted using a SONIFIER (registered trademark) 250D (manufactured by BRANSON).

　得られた菌体破砕液と破砕未処理の菌体懸濁液を用いて、実施例１４－１の反応組成５として、１５℃で０．５時間、実施例１３と同様に反応させた。得られた反応液の５－ヒドロキシ－Ｌ－リジン蓄積濃度をＨＰＬＣ（分析条件８）にて解析した。解析結果を図７に示す。図７中、縦軸は、５－ヒドロキシリジン濃度（ｍＭ）を表す。 The resulting cell disruption solution and the undisrupted cell suspension were used to react in the same manner as in Example 13 for 0.5 hours at 15°C, using reaction composition 5 of Example 14-1. The concentration of 5-hydroxy-L-lysine accumulated in the resulting reaction solution was analyzed by HPLC (analysis condition 8). The analysis results are shown in Figure 7. In Figure 7, the vertical axis represents the 5-hydroxylysine concentration (mM).

　図７から明らかなように、条件IIのＨＥＰＥＳ－ｐＨ緩衝液にＮａＣｌを添加したものが最も高い５－ヒドロキシ－Ｌ－リジン蓄積濃度を示し、次に条件IIIのＨＥＰＥＳ－ｐＨ緩衝液にＮａＣｌとグリセロールを添加したもの、条件ＩのＨＥＰＥＳ－ｐＨ緩衝液のみの順に高かった。このことから、条件II又は条件IIIのＮａＣｌを添加した条件において、菌体破砕時のＬ－リジン５位水酸化酵素の活性低下が軽減できることが示唆された。 As is clear from Figure 7, the highest 5-hydroxy-L-lysine accumulation concentration was observed under condition II, where NaCl was added to the HEPES-pH buffer, followed by condition III, where NaCl and glycerol were added to the HEPES-pH buffer, and condition I, where only the HEPES-pH buffer was used. This suggests that the decrease in activity of L-lysine 5-hydroxylase during cell disruption can be reduced under condition II or III, where NaCl was added.

＜実施例１５－３：反応液のｐＨ検討＞
　実施例１５－２の条件IIで得た菌体破砕液を用いて、反応液組成表－３の反応組成７～１３で反応液を調製し、それぞれ１５℃で０．５時間、実施例１３と同様に反応させた。 Example 15-3: pH study of reaction solution
Using the cell lysate obtained under Condition II of Example 15-2, reaction solutions were prepared according to reaction compositions 7 to 13 in Table 3 of Reaction Solution Composition. Each reaction solution was reacted at 15° C. for 0.5 hours in the same manner as in Example 13.

　得られた反応液の５－ヒドロキシ－Ｌ－リジン蓄積濃度をＨＰＬＣ（分析条件８）にて解析した。解析結果を図８に示す。図８中、縦軸は、５－ヒドロキシリジン濃度（ｍＭ）を表す。 The concentration of 5-hydroxy-L-lysine accumulated in the resulting reaction solution was analyzed by HPLC (analysis condition 8). The analysis results are shown in Figure 8. In Figure 8, the vertical axis represents the 5-hydroxylysine concentration (mM).

　図８から明らかなように、ｐＨ６．０～８．０の反応組成においては同程度の５－ヒドロキシ－Ｌ－リジン蓄積濃度が得られており、反応組成がｐＨ６．０～８．０の範囲ではｐＨによる影響をほぼ受けないことが分かった。 As is clear from Figure 8, the same levels of 5-hydroxy-L-lysine accumulation were obtained in reaction compositions with pH values between 6.0 and 8.0, indicating that the reaction composition is hardly affected by pH in the range of pH 6.0 to 8.0.

＜実施例１５－４：基質濃度の検討－１＞
　実施例１５－１で取得したＥ．　ｃｏｌｉ　ＪＭ１０９－ｐＫＷ３２－１－３株の培養菌体を、濃度１００ｇ／Ｌ又は２００ｇ／Ｌになるように、実施例１５－２の条件IIの緩衝溶液にそれぞれ懸濁し、ＳＯＮＩＦＩＥＲ（登録商標）　２５０Ｄ（ＢＲＡＮＳＯＮ社製）を使用して超音波破砕した。得られた各菌体破砕液を用いて、反応液組成表－４で反応液を調製し、それぞれ１５℃で２２．５時間、実施例１３と同様に反応させた。 <Example 15-4: Examination of substrate concentration-1>
The cultured cells of E. coli JM109-pKW32-1-3 strain obtained in Example 15-1 were suspended in the buffer solution of Condition II in Example 15-2 to a concentration of 100 g/L or 200 g/L, and then ultrasonically disrupted using a SONIFIER (registered trademark) 250D (manufactured by BRANSON). Using each of the resulting disrupted cell solutions, reaction solutions were prepared according to Table 4 on the reaction solution composition, and each was reacted at 15° C. for 22.5 hours in the same manner as in Example 13.

　反応中に適宜反応液を採取し、反応液中の５－ヒドロキシ－Ｌ－リジン蓄積濃度をＨＰＬＣ（分析条件８）にて解析した。解析結果を図９に示す。図９において、縦軸は５－ヒドロキシリジン濃度（ｍＭ）又はＬ－リジン濃度（ｍＭ）を表す。
　反応開始後２２．５時間の５－ヒドロキシ－Ｌ－リジン蓄積濃度を測定した結果、反応組成１３で反応した場合は５０ｍＭ、反応組成１４で反応した場合は約９５ｍＭ、反応組成１５で反応した場合は約１８０ｍＭであった。 The reaction solution was appropriately sampled during the reaction, and the concentration of accumulated 5-hydroxy-L-lysine in the reaction solution was analyzed by HPLC (analysis condition 8). The analysis results are shown in Figure 9. In Figure 9, the vertical axis represents the 5-hydroxylysine concentration (mM) or the L-lysine concentration (mM).
The accumulated concentration of 5-hydroxy-L-lysine was measured 22.5 hours after the start of the reaction. As a result, it was 50 mM when the reaction was performed with reaction composition 13, about 95 mM when the reaction was performed with reaction composition 14, and about 180 mM when the reaction was performed with reaction composition 15.

＜実施例１５－５：基質濃度の検討－２＞
　実施例１５－２の条件IIで得た菌体破砕液を用いて、反応液組成表－５の反応組成１６～１７で反応液０．３５Ｌを調製し、１Ｌジャーファーメンター（バイオット社製）を用い、それぞれ１５℃、ｐＨ７．０（１Ｍ　ＨＣｌを適宜添加して調整）、通気量０．３５Ｌ／ｍｉｎ、溶存酸素濃度２０％、攪拌速度４００～１０００ｒｐｍ（溶存酸素濃度が２０％以上になるように攪拌速度調整）で２４時間反応させた。 <Example 15-5: Examination of substrate concentration-2>
Using the cell disruption solution obtained under condition II in Example 15-2, 0.35 L of reaction solution was prepared according to reaction compositions 16 to 17 in Table 5 of reaction solution composition. The reaction solution was reacted for 24 hours in a 1 L jar fermenter (manufactured by Biot) at 15° C., pH 7.0 (adjusted by adding 1 M HCl appropriately), aeration rate of 0.35 L/min, dissolved oxygen concentration of 20%, and stirring speed of 400 to 1000 rpm (stirring speed adjusted so that the dissolved oxygen concentration was 20% or more).

　反応中に適宜反応液を採取し、反応液中の５－ヒドロキシ－Ｌ－リジン蓄積濃度をＨＰＬＣ（分析条件７）にて解析した。解析結果を図１０に示す。図１０において、縦軸は、５－ヒドロキシリジン濃度（ｍＭ）又はＬ－リジン濃度（ｍＭ）を表す。
　反応組成１６の条件ではＬ－Ｌｙｓはほぼ消費され、５－ヒドロキシ－Ｌ－リジン蓄積濃度は１８．２ｍＭ（２．９５ｇ／Ｌ）であり、約１．０ｇの５－ヒドロキシ－Ｌ－リジンを含む高純度な５－ヒドロキシ－Ｌ－リジン溶液が得られた。反応組成１７の条件では、Ｌ－Ｌｙｓは４．３ｍＭ（０．６２ｇ／Ｌ）残存したが、５－ヒドロキシ－Ｌ－リジン蓄積濃度は４０．６ｍＭ（６．５８ｇ／Ｌ）であり、約２．３ｇの５－ヒドロキシ－Ｌ－リジンを含む５－ヒドロキシ－Ｌ－リジン溶液が得られた。 The reaction solution was appropriately sampled during the reaction, and the accumulated concentration of 5-hydroxy-L-lysine in the reaction solution was analyzed by HPLC (analysis condition 7). The analysis results are shown in Figure 10. In Figure 10, the vertical axis represents the 5-hydroxylysine concentration (mM) or the L-lysine concentration (mM).
Under the conditions of reaction composition 16, L-Lys was almost completely consumed, the accumulated 5-hydroxy-L-lysine concentration was 18.2 mM (2.95 g/L), and a high-purity 5-hydroxy-L-lysine solution containing about 1.0 g of 5-hydroxy-L-lysine was obtained. Under the conditions of reaction composition 17, 4.3 mM (0.62 g/L) of L-Lys remained, but the accumulated 5-hydroxy-L-lysine concentration was 40.6 mM (6.58 g/L), and a 5-hydroxy-L-lysine solution containing about 2.3 g of 5-hydroxy-L-lysine was obtained.

＜実施例１６＞（Ｃｌａｄｅ２～４に属するＬ－リジン５位水酸化酵素ホモログの活性評価）
＜実施例１６－１：Ｌ－リジン５位水酸化酵素ホモログを発現するｐＣ１Ｋ発現ベクターの作製＞
　ｐＣｏｌｄ　Ｉベクター（タカラバイオ社製）に対しては配列番号１６４と１６５のプライマー、ｐＫＷ３２ベクターに対しては配列番号１６６と１６７のプライマーをそれぞれ用いて、実施例１２－２と同様にして、ｐＣｏｌｄ　Ｉベクターの薬剤耐性遺伝子をカナマイシンに変更したｐＣ１Ｋベクターを得た。 Example 16 (Evaluation of activity of L-lysine 5-hydroxylase homologs belonging to Classes 2 to 4)
<Example 16-1: Construction of pC1K expression vector expressing L-lysine 5-hydroxylase homolog>
A pCold I vector (Takara Bio) was treated with primers of SEQ ID NOs: 164 and 165, and a pKW32 vector was treated with primers of SEQ ID NOs: 166 and 167, respectively, to obtain a pC1K vector in which the drug resistance gene of the pCold I vector was changed to kanamycin, in the same manner as in Example 12-2.

　得られたｐＣ１Ｋベクターを制限酵素ＮｄｅＩとＸｈｏＩ（共にタカラバイオ社製）で切断後、実施例１２－１と同様に脱リン酸化を行い、さらにユーロフィンジェノミクス社より購入した配列番号３３、３５、３７、３９、４１、４３、４５、４７、４９、５１、５３、５５、５７、５９、６１、６３、６５、６７、６９、７１、７３、７５、７７、７９、８１、８３、８５、８７、８９、９１、９３、９５、９７、９９、１０１、１０３、１０５、１０７、１０９、１１１、１１３、１１５、１１７、１１９、１２１、１２３、１２５、１２７、１２９、１３１及び１３３のそれぞれの直鎖ＤＮＡ断片を、制限酵素ＮｄｅＩとＸｈｏＩ（共にタカラバイオ社製）で切断した。得られた挿入遺伝子断片を、実施例１２－１と同様にライゲーションして、それぞれ下記表１３に示すＬ－リジン５位水酸化酵素を発現するｐＣ１Ｋ発現ベクターを得た。 The resulting pC1K vector was cleaved with restriction enzymes NdeI and XhoI (both manufactured by Takara Bio Inc.), dephosphorylated in the same manner as in Example 12-1, and then ligated with the following DNA fragments (SEQ ID NOs: 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150 Each of the linear DNA fragments 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, and 133 was cleaved with the restriction enzymes NdeI and XhoI (both manufactured by Takara Bio Inc.). The resulting inserted gene fragments were ligated in the same manner as in Example 12-1 to obtain pC1K expression vectors expressing the L-lysine 5-hydroxylase enzymes shown in Table 13 below.

＜実施例１６－２：Ｃｌａｄｅ２～４に属するＬ－リジン５位水酸化酵素の活性評価＞
　実施例１６－１で得たＬ－リジン５位水酸化酵素を発現するｐＣ１Ｋ発現ベクターについて、それぞれＩＰＴＧの終濃度が０．０５ｍＭになるように添加したこと以外は実施例１３と同様の条件でフラスコ培養を行った。得られた培養菌体を濃度１００ｇ／Ｌとなるように、２００ｍＭ　ＮａＣｌを含む５０ｍＭ　ＨＥＰＥＳ－ＮａＯＨ　ｂｕｆｆｅｒ（ｐＨ８．０）に懸濁し、ＳＯＮＩＦＩＥＲ（登録商標）　２５０Ｄ（ＢＲＡＮＳＯＮ社製）を使用して超音波破砕を行い、菌体破砕液を取得した。得られた菌体破砕液を実施例１４－１の反応組成４に調製して、１５℃で２４時間、実施例１３と同様に反応させた。 Example 16-2: Evaluation of activity of L-lysine 5-hydroxylase belonging to Classes 2 to 4
The pC1K expression vector expressing L-lysine 5-hydroxylase obtained in Example 16-1 was cultured in a flask under the same conditions as in Example 13, except that IPTG was added to a final concentration of 0.05 mM. The cultured cells were suspended in a 50 mM HEPES-NaOH buffer (pH 8.0) containing 200 mM NaCl to a concentration of 100 g/L, and ultrasonically disrupted using a SONIFIER (registered trademark) 250D (manufactured by BRANSON) to obtain a cell disruption solution. The resulting cell disruption solution was prepared to the reaction composition 4 of Example 14-1, and reacted at 15° C. for 24 hours in the same manner as in Example 13.

　反応開始から２４時間経過後に反応液を採取し、反応液中の５－ヒドロキシ－Ｌ－リジン蓄積濃度をＨＰＬＣ（分析条件８）にて解析した。また、ポジティブコントロールとしてｐＫＷ３２－１－３、ネガティブコントロールとしてｐＣ１Ｋベクターを用い、Ｌ－リジン５位水酸化酵素を発現するｐＣ１Ｋ発現ベクターと同様に培養、菌体破砕及び反応を行った。それぞれの５－ヒドロキシ－Ｌ－リジン蓄積濃度を表１４に示す。 The reaction solution was sampled 24 hours after the start of the reaction, and the concentration of 5-hydroxy-L-lysine accumulated in the reaction solution was analyzed by HPLC (analysis condition 8). In addition, pKW32-1-3 was used as a positive control, and the pC1K vector was used as a negative control, and the cultivation, cell disruption, and reaction were carried out in the same manner as for the pC1K expression vector expressing L-lysine 5-hydroxylase. The respective 5-hydroxy-L-lysine accumulation concentrations are shown in Table 14.

　表１４中の記号の意味は、それぞれ以下の通りである。
　－：５－ヒドロキシ－Ｌ－リジンが検出されなかったもの
　＊：５－ヒドロキシ－Ｌ－リジン蓄積濃度が３ｍＭ以下のもの
　＊＊：５－ヒドロキシ－Ｌ－リジン蓄積濃度が３ｍＭより高く１０ｍＭ以下のもの
　＊＊＊：５－ヒドロキシ－Ｌ－リジン蓄積濃度が１０ｍＭより高いもの The symbols in Table 14 have the following meanings:
-: 5-hydroxy-L-lysine was not detected *: 5-hydroxy-L-lysine accumulation concentration was 3 mM or less **: 5-hydroxy-L-lysine accumulation concentration was more than 3 mM and less than 10 mM ***: 5-hydroxy-L-lysine accumulation concentration was more than 10 mM

　表１４から明らかなように、Ｃｌａｄｅ２～４に属するＬ－リジン５位水酸化酵素の活性評価を行った結果、活性が高かったのはＡｚｐＫ２　Ｈｏｍｏｌｏｇ－Ｃ３－４及びＡｚｐＫ２　Ｈｏｍｏｌｏｇ－Ｃ３－１５であり、次いで活性が高かったのはＡｚｐＫ２　Ｈｏｍｏｌｏｇ－Ｃ２－２、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ－Ｃ２－３、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ－Ｃ２－８、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ－Ｃ２－９、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ－Ｃ２－１１、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ－Ｃ２－２５、ＡｚｐＫ２　Ｈｏｍｏｌｏｇ－Ｃ３－１０及びＡｚｐＫ２　Ｈｏｍｏｌｏｇ－Ｃ３－１２であった。 As is clear from Table 14, the activity of L-lysine 5-hydroxylases belonging to Clades 2 to 4 was evaluated, and the enzymes with the highest activity were AzpK2 Homolog-C3-4 and AzpK2 Homolog-C3-15, followed by AzpK2 Homolog-C2-2, AzpK2 Homolog-C2-3, AzpK2 Homolog-C2-8, AzpK2 Homolog-C2-9, AzpK2 Homolog-C2-11, AzpK2 Homolog-C2-25, AzpK2 Homolog-C3-10, and AzpK2 Homolog-C3-12.

＜実施例１７＞（Ｌ－リジン５位水酸化酵素のアミノ酸配列の同一性比較）
　実施例１０及び実施例１６において、Ｓ選択的なＬ－リジン５位水酸化活性が検出されたＬ－リジン５位水酸化酵素について、ＣｌｕｓｔａｌＷを用いてアミノ酸配列の同一性を比較した結果を図１１に示す。 Example 17 (Identity comparison of amino acid sequences of L-lysine 5-hydroxylase)
FIG. 11 shows the results of a comparison of amino acid sequence identity using ClustalW for the L-lysine 5-hydroxylases in which S-selective L-lysine 5-hydroxylation activity was detected in Examples 10 and 16.

　図１１において、背景色が灰色（参考図面では、赤字かつ背景色がピンク色）になっている箇所は同一性の数値が５５％以上のものを示す。 In Figure 11, the areas with a gray background (red text with a pink background in the reference drawing) indicate areas with an identity value of 55% or more.

　図１１中、Ｃｌａｄｅ１に属するＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－１～１－９はいずれもＡｚｐＫ２との同一性が５５％である。 In Figure 11, AzpK2 Homologs 1-1 to 1-9 belonging to Clade 1 all have 55% identity with AzpK2.

　図１１の結果より、Ｃｌａｄｅ１に属するＡｚｐＫ２及びＡｚｐＫ２　Ｈｏｍｏｌｏｇ１－１～１－９は、ＡｚｐＫ２を基準とした場合の同一性はいずれも５５％以上であった。 The results in Figure 11 show that AzpK2 and AzpK2 Homolog1-1 to 1-9, which belong to Class 1, all have identities of 55% or more when AzpK2 is used as the standard.

　また、実施例１０及び実施例１６において、Ｒ選択的なＬ－リジン５位水酸化活性が検出されたＬ－リジン５位水酸化酵素について、ＣｌｕｓｔａｌＷを用いてアミノ酸配列の同一性を比較した結果を図１２に示す。 In addition, for the L-lysine 5-hydroxylases in which R-selective L-lysine 5-hydroxylation activity was detected in Examples 10 and 16, the results of comparing the identity of the amino acid sequences using ClustalW are shown in Figure 12.

　図１２において、背景色が薄い灰色（参考図面では水色）になっている箇所は同一性の数値が５８％以上のものを示し、また背景色が濃い灰色（参考図面では黄色）になっている箇所は同一性の数値が５０％以上のものを示している。 In Figure 12, areas with a light gray background (light blue in the reference drawing) indicate areas with an identity value of 58% or more, and areas with a dark gray background (yellow in the reference drawing) indicate areas with an identity value of 50% or more.

　図１２の結果より、Ｃｌａｄｅ２及びＣｌａｄｅ３に属するＬ－リジン５位水酸化活性を有するアミノ酸配列はＡｚｐＫ２　Ｈｏｍｏｌｏｇ　Ｃ２－２２を基準として５０％以上の同一性を有することが分かった。また、Ｃｌａｄｅ４については、Ｌ－リジン５位水酸化活性を有するアミノ酸配列は互いに５８％以上の同一性を有することが分かった。 The results in Figure 12 show that the amino acid sequences with L-lysine 5-hydroxylation activity belonging to Clade 2 and Clade 3 have 50% or more identity with AzpK2 Homolog C2-22 as a reference. In addition, for Clade 4, the amino acid sequences with L-lysine 5-hydroxylation activity have 58% or more identity with each other.

＜実施例１８＞（ＡｚｐＫ２の結晶構造解析）
＜実施例１８－１：ＡｚｐＫ２タンパク質の精製＞
　実施例４と同様の方法で取得したＡｚｐＫ２の組換えタンパク質水溶液をAKTA pure（Ｃｙｔｉｖａ社製）を用いた陰イオン交換クロマトグラフィーにより精製した。溶出は、ＮａＣｌ濃度勾配（Ａ液（２０ｍＭ　ＨＥＰＥＳ、１０Ｗ／Ｖ％　グリセロール、ｐＨ８．０）及びＢ液（２０ｍＭ　ＨＥＰＥＳ、１０Ｗ／Ｖ％　グリセロール、１Ｍ　ＮａＣｌ、ｐＨ８．０））を利用し、カラムにはＲＥＳＯＵＲＣＥ（登録商標）　Ｑ　１ｍＬ（Ｃｙｔｉｖａ社製）を用いた。溶出したタンパク質を含む溶液の溶媒を限外濾過（Ａｍｉｃｏｎ　Ｕｌｔｒａ―１５　１０ｋＤａ、メルク社製）によりＡ液で置換し、タンパク質濃度を１０ｍｇ／ｍＬに調製してＡｚｐＫ２タンパク質の精製溶液を取得した。 Example 18 (Crystal structure analysis of AzpK2)
Example 18-1: Purification of AzpK2 protein
The recombinant protein aqueous solution of AzpK2 obtained in the same manner as in Example 4 was purified by anion exchange chromatography using AKTA pure (manufactured by Cytiva). Elution was performed using a NaCl concentration gradient (solution A (20 mM HEPES, 10 W/V% glycerol, pH 8.0) and solution B (20 mM HEPES, 10 W/V% glycerol, 1 M NaCl, pH 8.0)) and a RESOURCE (registered trademark) Q 1 mL (manufactured by Cytiva) column. The solvent of the solution containing the eluted protein was replaced with solution A by ultrafiltration (Amicon Ultra-15 10 kDa, manufactured by Merck), and the protein concentration was adjusted to 10 mg/mL to obtain a purified solution of AzpK2 protein.

＜実施例１８－２：ＡｚｐＫ２タンパク質結晶の調製＞
　結晶化用のスクリーニングキットＣｒｙｓｔａｌ　Ｓｃｒｅｅｎ（Ｈａｍｐｔｏｎ　Ｒｅｓｅａｒｃｈ社製）、Ｉｎｄｅｘ（Ｈａｍｐｔｏｎ　Ｒｅｓｅａｒｃｈ社製）、Ｗｉｚａｒｄ　Ｉ（Ｍｏｌｅｃｕｌａｒ　Ｄｉｍｅｎｓｉｏｎｓ社製）及びＷｉｚａｒｄ　ＩＩ（Ｍｏｌｅｃｕｌａｒ　Ｄｉｍｅｎｓｉｏｎｓ社製）を用いて初期スクリーニングを行った。 <Example 18-2: Preparation of AzpK2 protein crystals>
Initial screening was performed using crystallization screening kits Crystal Screen (Hampton Research), Index (Hampton Research), Wizard I (Molecular Dimensions), and Wizard II (Molecular Dimensions).

　スクリーニングにおいては、結晶化用バッファーとタンパク質溶液を１μＬずつ混合し、シッティングドロップ法により２０℃で静置して結晶化を行った。その結果、いくつかの条件で良好な結晶が得られることが分かった。そこで、良好な結晶が得られた条件については、さらに結晶化用バッファーとタンパク質溶液を２μＬずつ混合し、ハンギングドロップ法を用いた結晶化により条件の最適化を行った。この際、ｐＨと沈殿剤の量を検討した。その結果、結晶化用バッファーの組成が２４Ｗ／Ｖ％　ＰＥＧ３３５０、１００ｍＭ　Ｂｉｓ－Ｔｒｉｓ　ｐＨ　６．５、２００ｍＭ　ＮａＣｌを利用した条件でもっとも良好なＡｚｐＫ２単独結晶が得られた。 In the screening, 1 μL each of the crystallization buffer and protein solution were mixed, and crystallization was performed by the sitting drop method at 20°C. As a result, it was found that good crystals could be obtained under several conditions. Therefore, for the conditions under which good crystals were obtained, 2 μL each of the crystallization buffer and protein solution were mixed, and the conditions were optimized by crystallization using the hanging drop method. At this time, the pH and amount of precipitant were examined. As a result, the best AzpK2 single crystals were obtained under the conditions of a crystallization buffer composition of 24 W/V% PEG3350, 100 mM Bis-Tris pH 6.5, 200 mM NaCl.

　次に、前記と同様にして、５－ヒドロキシ－Ｌ－リジン及びα－ケトグルタル酸との共結晶の取得を試みた。ＡｚｐＫ２の保存バッファーに５－ヒドロキシ－Ｌ－リジン及びα－ケトグルタル酸を濃度１ｍＭになるように加え、氷上で３０分間静置した後、前記と同様にハンギングドロップ法により結晶化を行った。その結果、保存バッファーの組成が２２Ｗ／Ｖ％　ＰＥＧ３３５０、１００ｍＭ　Ｂｉｓ－Ｔｒｉｓ　ｐＨ６．５、２００ｍＭ　ＮａＣｌを利用した条件で共結晶が得られた。 Next, we attempted to obtain co-crystals with 5-hydroxy-L-lysine and α-ketoglutaric acid in the same manner as above. 5-hydroxy-L-lysine and α-ketoglutaric acid were added to the storage buffer of AzpK2 to a concentration of 1 mM, and after leaving it to stand on ice for 30 minutes, crystallization was performed using the hanging drop method in the same manner as above. As a result, co-crystals were obtained using a storage buffer with a composition of 22 W/V% PEG3350, 100 mM Bis-Tris pH 6.5, and 200 mM NaCl.

＜実施例１８－３：ＡｚｐＫ２タンパク質結晶のＸ線構造解析＞
　実施例１８－２で得られたＡｚｐＫ２単独結晶をリソループで掬い取り、液体窒素により凍結した。その後、高エネルギー加速器研究機構にあるビームラインＰＦ　ＢＬ１Ａを用いて、１００Ｋの窒素ガス気流下で、凍結した前記結晶にＸ線を照射し、回折像を得た。その結果、最高分解能１．６０オングストロームの回折像が得られた、 <Example 18-3: X-ray structure analysis of AzpK2 protein crystal>
The AzpK2 single crystal obtained in Example 18-2 was scooped up with a litho loop and frozen with liquid nitrogen. Thereafter, the frozen crystal was irradiated with X-rays under a nitrogen gas flow at 100 K using the beamline PF BL1A at the High Energy Accelerator Research Organization to obtain a diffraction image. As a result, a diffraction image with a maximum resolution of 1.60 angstroms was obtained.

　得られたデータをＸＤＳソフトウェア（Ｍａｘ　Ｐｌａｎｃｋ　Ｉｎｓｔｉｔｕｔｅ　ｆｏｒ　Ｍｅｄｉｃａｌ　Ｒｅｓｅａｒｃｈ製）を用いて処理し、さらにＡＩＭＬＥＳＳソフトウェアを用いてスケーリングを行った。さらに、Ｐｈｅｎｉｘのｐｈａｓｅｒを利用した分子置換による位相決定を行い、ＡｕｔｏＢｕｉｌｄを利用して初期モデルを構築した。 The data obtained was processed using XDS software (Max Planck Institute for Medical Research) and further scaled using AIMLESS software. Phase determination was then performed by molecular replacement using Phoenix phaser, and an initial model was constructed using AutoBuild.

　構築した初期モデルをもとにＣＯＯＴによる手動の構造の調整とｐｈｅｎｉｘ．ｒｅｆｉｎｅを利用したｒｅｆｉｎｅｍｅｎｔを繰り返すことにより構造のモデルを作成した。電子密度が希薄であったため、一部のモデル（図１３左、(1)Ｇｌｙ６０～Ｍｅｔ７８，(2)Ａｒｇ１３２～Ｈｉｓ１３６，(3)Ａｓｐ２３０以降）を構築することができず、酵素の活性中心と思われる領域が露出していた。 A structural model was created based on the initial model constructed by repeatedly performing manual structural adjustments using COOT and refinement using phenix. refine. Due to sparse electron density, some models (Figure 13, left, (1) Gly60-Met78, (2) Arg132-His136, (3) Asp230 onward) could not be constructed, and the region thought to be the active center of the enzyme was exposed.

　次に、５－ヒドロキシ－Ｌ－リジン及びα－ケトグルタル酸との共結晶を用いて同様の実験を試みた結果、最高分解能２．２４Åの回折像が得られた。得られたデータを同様に処理し、モデル構築を行った。５－ヒドロキシ－Ｌ－リジン及びα－ケトグルタル酸に該当する電子密度を明瞭に観察することはできなかったが、基質結合なしの構造では観察できなかった領域もモデルを構築することができた（図１３右）。しかしながら、Ｃ末端の一部のＧｌｕ２３６以降は構造が観測されず、基質結合様式を完全に明らかにすることはできなかった。 Next, a similar experiment was attempted using co-crystals with 5-hydroxy-L-lysine and α-ketoglutaric acid, resulting in a diffraction image with a maximum resolution of 2.24 Å. The obtained data was similarly processed and a model was constructed. Although the electron density corresponding to 5-hydroxy-L-lysine and α-ketoglutaric acid could not be clearly observed, a model could be constructed for the region that could not be observed in the structure without substrate binding (Figure 13, right). However, no structure was observed beyond Glu236, a part of the C-terminus, and the substrate binding mode could not be fully elucidated.

　図１３の左側の図は、ＡｚｐＫ２単結晶から得られた構造モデルであり、右側の図は、ＡｚｐＫ２の５－ヒドロキシ－Ｌ－リジン及びα－ケトグルタル酸との結晶から得られた構造モデルである。リボン状に表しているのがＡｚｐＫ２タンパク質であり、灰色の（参考図面でオレンジ色の）球は鉄原子を表す。 The diagram on the left in Figure 13 is a structural model obtained from a single crystal of AzpK2, and the diagram on the right is a structural model obtained from a crystal of AzpK2 with 5-hydroxy-L-lysine and α-ketoglutaric acid. The ribbon-shaped representation is the AzpK2 protein, and the gray spheres (orange in the reference diagram) represent iron atoms.

＜実施例１９＞（ＡｚｐＫ２とＢｅｓＤの同一性比較）
　Ｃｌａｄｅ１に属するＡｚｐＫ２とＣｌａｄｅ５に属するＢｅｓＤについて、ＣｌｕｓｔａｌＷ法を用いてアミノ酸の同一性比較を行った。同一性の比較結果を図１４に示す。 Example 19 (Identity comparison between AzpK2 and BesD)
Amino acid identity comparison was performed using the ClustalW method for AzpK2 belonging to Clade 1 and BesD belonging to Clade 5. The results of the identity comparison are shown in FIG.

　ＢｅｓＤは、ＡｚｐＫ２と比較的近い配列を有しており、かつ同じリジンを基質とするαＫＧ依存型ジオキシゲナーゼであるが、リジンをハロゲン化する点でＡｚｐＫ２とは機能が異なる。ＢｅｓＤは非特許文献８において活性に重要なアミノ酸残基の位置が報告されている。 BesD has a sequence relatively similar to AzpK2 and is an αKG-dependent dioxygenase that uses the same lysine as a substrate, but its function differs from that of AzpK2 in that it halogenates lysine. The positions of amino acid residues important for the activity of BesD have been reported in Non-Patent Document 8.

　図１４から明らかなように、ＡｚｐＫ２においてもＢｅｓＤと同様に、α－ケトグルタル酸、鉄原子、基質であるリジンのαアミノ基及びカルボン酸と相互作用するアミノ酸残基は保存されていた。 As is clear from Figure 14, the amino acid residues that interact with α-ketoglutarate, iron atoms, the α-amino group of the substrate lysine, and carboxylic acids are conserved in AzpK2, just like in BesD.

　図１４中、〇はＬ－リジンのカルボン酸と相互作用する残基、●は鉄原子と相互作用する残基、◎はＬ－リジンのαアミノ基周囲に配位する水分子と相互作用する残基、▽はαＫＧと相互作用する残基である。 In Figure 14, circles represent residues that interact with the carboxylic acid of L-lysine, circles represent residues that interact with the iron atom, double circles represent residues that interact with the water molecules that coordinate around the α-amino group of L-lysine, and ▽ represent residues that interact with αKG.

＜実施例２０＞（ＡｚｐＫ２の立体構造予測による酵素活性に対する重要モチーフの特定）
　ＡｚｐＫ２について、タンパク質構造予測ソフトであるＡｌｐｈａＦｏｌｄ２により立体構造予測を行ったところ、図１５に示すように、Ｘ線構造解析モデル構築ができなかった部分についても構造予測が可能であった。さらに、立体構造が報告されているＢｅｓＤの立体構造とＡｚｐＫ２の予測構造を比較した。 Example 20 (Identification of important motifs for enzyme activity by prediction of the three-dimensional structure of AzpK2)
When the three-dimensional structure of AzpK2 was predicted using AlphaFold2, a protein structure prediction software, it was possible to predict the structure even for parts for which an X-ray structure analysis model could not be constructed, as shown in Figure 15. Furthermore, the three-dimensional structure of BesD, the three-dimensional structure of which has been reported, was compared with the predicted structure of AzpK2.

　その結果、Ｘ線構造解析でモデル構築できなかった部分は、いずれも基質であるＬ－リジン、αＫＧあるいは鉄原子との距離が近く、反応に重要なことが予想できるアミノ酸残基に該当していた。 As a result, the parts that could not be modeled using X-ray structural analysis all corresponded to amino acid residues that were close to the substrates L-lysine, αKG, or iron atoms and were predicted to be important for the reaction.

　図１５において、左上の図はＡｚｐＫ２単結晶のＸ線構造解析によるモデル構築結果（活性中心付近）、右上の図はＡｚｐＫ２、αＫＧ、５－ヒドロキシリジン共結晶のＸ線構造解析によるモデル構築結果（活性中心付近）、左下の図はＢｅｓＤのＡｌｐｈａＦｏｌｄ２での予測構造、また、右下の図はＡｚｐＫ２のＡｌｐｈａＦｏｌｄ２での予測構造（ＢｅｓＤ由来のリジンとαＫＧを重ねて表示）である。 In Figure 15, the top left figure shows the model construction results from X-ray structural analysis of AzpK2 single crystals (near the active center), the top right figure shows the model construction results from X-ray structural analysis of AzpK2, αKG, and 5-hydroxylysine co-crystals (near the active center), the bottom left figure shows the predicted structure of BesD in AlphaFold2, and the bottom right figure shows the predicted structure of AzpK2 in AlphaFold2 (BesD-derived lysine and αKG are superimposed).

　図１５中のそれぞれの図において、中心付近の灰色の球（参考図面のオレンジ色の球）は電子密度の高い領域を表しており、αＫＧ依存型ジオキシゲナーゼでは一般的に鉄原子（Ｆｅ２＋）が該当することが知られている。中心付近の薄い灰色の球（参考図面の緑色の球）はＢｅｓＤにおける鉄原子イオンを表す。 In each diagram in Figure 15, the gray sphere near the center (orange sphere in the reference diagram) represents a region of high electron density, which is generally known to correspond to an iron atom (Fe2 + ) in αKG-dependent dioxygenases, and the light gray sphere near the center (green sphere in the reference diagram) represents an iron atom ion in BesD.

　具体的には、ＡｚｐＫ２の領域(2)は、図１５において、ＢｅｓＤにおけるＬ－リジンのカルボニル基及びアミノ基、及びαＫＧに近接した領域に該当しており、特にＨｉｓ１３６－Ａｓｐ１４１の領域はリジンのアミノ基、カルボン酸、又は鉄原子に近いことが示唆され、基質の選択性や活性に寄与する重要な領域であることが示された。 Specifically, in Figure 15, region (2) of AzpK2 corresponds to the carbonyl group and amino group of L-lysine in BesD, and the region close to αKG. In particular, the His136-Asp141 region is suggested to be close to the amino group of lysine, carboxylic acid, or iron atom, and is shown to be an important region that contributes to substrate selectivity and activity.

　次にＡｚｐＫ２の領域(3)、その中でも特にＴｈｒ２３７～Ｐｈｅ２４２の領域は図１５において、ＢｅｓＤにおける酵素反応によって修飾を受けるＬ－リジンの３～５位のアルキル鎖に近接した領域に該当しており、リジンの修飾箇所及び水酸基の光学選択性の決定に寄与する重要な領域であることが示された。 Next, the AzpK2 region (3), particularly the region from Thr237 to Phe242, corresponds to the region close to the alkyl chain at positions 3 to 5 of L-lysine, which is modified by the enzymatic reaction in BesD in Figure 15, and was shown to be an important region that contributes to determining the modification site of lysine and the optical selectivity of the hydroxyl group.

　また、酵素内のアミノ酸残基において酵素以外の要因（例：基質、αＫＧ、鉄原子など）と相互作用している場合に、これらの相互作用は隣接するアミノ酸残基の性質（例：分子の大きさや電気的性質など）に影響を受けることが知られている。立体構造予測により、領域(2)ではＡｓｐ１４２、領域(3)ではＨｉｓ２３５とＡｓｎ２３６が相互作用に関与する可能性が示唆された。 In addition, it is known that when amino acid residues within an enzyme interact with factors other than the enzyme (e.g., substrate, αKG, iron atoms, etc.), these interactions are influenced by the properties of adjacent amino acid residues (e.g., molecular size and electrical properties, etc.). Three-dimensional structure prediction suggests that Asp142 in region (2) and His235 and Asn236 in region (3) may be involved in the interaction.

　以上のことから、ＡｚｐＫ２のＬ－リジン５位水酸化活性において、酵素活性に対する重要なアミノ酸残基はＨｉｓ１３６～Ａｓｐ１４２であり、またＬ－リジン５位水酸化活性の光学選択性の決定に重要なアミノ酸残基はＨｉｓ２３５～Ｐｈｅ２４２であることが明らかになった。 From the above, it was revealed that in the L-lysine 5-hydroxylation activity of AzpK2, the important amino acid residues for the enzyme activity are His136 to Asp142, and the important amino acid residues for determining the optical selectivity of the L-lysine 5-hydroxylation activity are His235 to Phe242.

＜実施例２１＞（ＡｚｐＫ２のアミノ酸置換体の活性測定）
　実施例１において調製したｐＣｏｌｄ　Ｉ－ＡｚｐＫ２を鋳型にして、表１５に示すアミノ酸置換変異導入用のプライマーセットを用い、またＰｒｉｍｅＳｔａｒ　ＨＳ　ＤＮＡ　ｐｏｌｙｍｅｒａｓｅ（タカラバイオ社製）を用いて常法に従って対応する各アミノ酸置換変異体をコードするｐＣｏｌｄ　Ｉ－ＡｚｐＫ２ベクターを得た。 Example 21 (Measurement of activity of AzpK2 amino acid substituted derivatives)
Using pCold I-AzpK2 prepared in Example 1 as a template, a primer set for introducing amino acid substitution mutations shown in Table 15 was used, and PrimeStar HS DNA polymerase (manufactured by Takara Bio Inc.) was used according to a standard method to obtain pCold I-AzpK2 vectors encoding the corresponding amino acid substitution mutants.

　得られたＡｚｐＫ２のアミノ酸置換体について、実施例４と同様の方法で各ＡｚｐＫ２アミノ酸置換体のタンパク質水溶液を調製した。得られたタンパク質溶液を用いて実施例５と同様の方法で酵素アッセイを行い、Ｆｍｏｃ化サンプルをＬＣ－ＭＳ（分析条件２）で解析した。解析結果を図１６に示す。図１６中、ＷＴは野生型のＡｚｐＫ２を表す。 A protein aqueous solution of each of the obtained amino acid substituted AzpK2 was prepared in the same manner as in Example 4. Using the obtained protein solution, an enzyme assay was performed in the same manner as in Example 5, and the Fmoc-conjugated sample was analyzed by LC-MS (analysis condition 2). The analysis results are shown in Figure 16. In Figure 16, WT represents wild-type AzpK2.

　図１６に示すように、ＡｚｐＫ２のアミノ酸置換体（Ｒ７６Ａ、Ｅ１２２Ａ、Ｈ１３６Ａ、Ｄ１４１Ｇ及びＦ２４２Ｗ）においては、５－ヒドロキシリジンは検出されなかったことから活性が消失していることが分かり、これらのアミノ酸残基が活性に重要であることが示唆された。 As shown in Figure 16, 5-hydroxylysine was not detected in the amino acid substitutions of AzpK2 (R76A, E122A, H136A, D141G, and F242W), indicating that activity was lost, suggesting that these amino acid residues are important for activity.

＜実施例２２＞（ＡｚｐＫ２及びホモログにおける酵素活性に対する重要モチーフの特定）
　実施例２０で明らかになったＡｚｐＫ２の酵素活性に関連する重要なアミノ酸残基について、Ｌ－リジン５位水酸化酵素であるＣｌａｄｅ１～４及びＣｌａｄｅ１０に属するＰｐ＿ＡｚｐＫ２、並びにＣｌａｄｅ４に近接するがＬ－リジン５位水酸化活性を有さないＣｌａｄｅ５のアミノ酸配列を比較した。 Example 22 (Identification of important motifs for enzyme activity in AzpK2 and homologues)
Regarding important amino acid residues related to the enzyme activity of AzpK2 clarified in Example 20, the amino acid sequences of Pp_AzpK2 belonging to Clades 1 to 4 and Clade 10, which are L-lysine 5-hydroxylase, and Clade 5, which is adjacent to Clade 4 but does not have L-lysine 5-hydroxylation activity, were compared.

　まず、Ｃｌａｄｅ１～５に属するアミノ酸配列について、ＣｌｕｓｔａｌＷを用いて各Ｃｌａｄｅ内において同一性を比較した。この同一性の比較結果のうち、実施例２０で特定したＡｚｐＫ２の重要な領域であるＨｉｓ１３６～Ａｓｐ１４２及びＨｉｓ２３５～Ｐｈｅ２４２のアミノ酸配列について、Ｃｌａｄｅ１～５に属するアミノ酸配列それぞれにおけるアミノ酸の出現頻度をロゴプロットで表したものを図１７及び図１８に示した。 First, the amino acid sequences belonging to Clades 1 to 5 were compared for identity within each Clade using ClustalW. From the results of this identity comparison, the occurrence frequency of amino acids in each of the amino acid sequences belonging to Clades 1 to 5 for the amino acid sequences of His136 to Asp142 and His235 to Phe242, which are important regions of AzpK2 identified in Example 20, is shown in Figures 17 and 18 as logo plots.

　またＰｐ＿ＡｚｐＫ２については、Ｃｌａｄｅ内での同一性比較を行わずにＰｐ＿ＡｚｐＫ２のアミノ酸配列をＣｌａｄｅ１～５に属するアミノ酸配列と比較した。 Furthermore, for Pp_AzpK2, the amino acid sequence of Pp_AzpK2 was compared with the amino acid sequences belonging to Clades 1 to 5 without comparing identity within the Clade.

　１３６～１４２番目のアミノ酸を比較した結果、図１７に示すようにＣｌａｄｅ１～４に属するものは、１３６～１４２番目のアミノ酸として、全て同じアミノ酸が保存されており、１３６～１４２番目のアミノ酸の配列は「ＨＧＷＨＷＤＤ」であった。 Comparing the amino acids at positions 136 to 142, it was found that the same amino acids were conserved in all of the Clades 1 to 4 at positions 136 to 142, as shown in Figure 17, and the sequence of amino acids 136 to 142 was "HGWHWDD."

　また、Ｐｐ＿ＡｚｐＫ２の１３６～１４２番目のアミノ酸は「ＨＧＷＨＬＤＤ」であり、一方でＣｌａｄｅ５の１３６～１４２番目のアミノ酸は「ＨＧＷＨＷＧＤ」であった。 The amino acids 136 to 142 of Pp_AzpK2 were "HGWHLDD", while the amino acids 136 to 142 of Clade5 were "HGWHWGD".

　ここで、非特許文献８において、１３９番目と１４１番目のアミノ酸の組み合わせが水酸化酵素では「ＨＸＤ」、ハロゲン化酵素では「ＨＸＧ」を特徴とすることが報告されている。さらに、同じ非特許文献８において、「ＨＸＤ」又は「ＨＸＧ」からＮ末端側に３番目のアミノ酸に位置する「Ｈ」は酵素活性に重要であることが報告されている。また、前記実施例２１においてもＡｚｐＫ２のＨｉｓ１３６にアミノ酸変異を入れると酵素活性が失われることが明らかになっている。 Here, Non-Patent Document 8 reports that the combination of amino acids at positions 139 and 141 is characterized as "HXD" in hydroxylases and "HXG" in halogenases. Furthermore, Non-Patent Document 8 reports that the "H" located third amino acid on the N-terminal side from "HXD" or "HXG" is important for enzyme activity. Also, Example 21 above reveals that introducing an amino acid mutation into His136 of AzpK2 causes the enzyme activity to be lost.

　このことから、Ｌ－リジン５位水酸化酵素の水酸化活性の発現には、Ｈｉｓ１３６～Ａｓｐ１４２までのアミノ酸残基のまとまりである「ＨＧＷＨＷＤＤ」又は「ＨＧＷＨＬＤＤ」モチーフが重要であることが示唆される。 This suggests that the "HGWHWDD" or "HGWHLDD" motif, which is a group of amino acid residues from His136 to Asp142, is important for the expression of the hydroxylation activity of L-lysine 5-hydroxylase.

　次に、２３５～２４２番目のアミノ酸を比較した結果を図１８に示す。また、図１８の結果から、同一Ｃｌａｄｅ内で保存されていないアミノ酸を「Ｘ」と表記して、Ｃｌａｄｅ１～５及びＰｐ＿ＡｚｐＫ２の２３５～２４２番目のアミノ酸を比較した結果を表１６に示す。 Next, the results of comparing amino acids 235-242 are shown in Figure 18. Furthermore, from the results of Figure 18, amino acids that are not conserved within the same Clade are indicated with "X", and the results of comparing amino acids 235-242 of Clades 1-5 and Pp_AzpK2 are shown in Table 16.

　前記実施例２０の結果から、Ｌ－リジン５位水酸化活性の光学選択性の決定に重要な役割を果たす２３５～２４２番目のアミノ酸について、この領域自体がアミノ酸残基のまとまりであるモチーフと言える。 The results of Example 20 above indicate that the region from amino acids 235 to 242, which plays an important role in determining the optical selectivity of L-lysine 5-hydroxylation activity, is itself a motif that is a group of amino acid residues.

　そこでＣｌａｄｅ１～４について、同一性比較の結果からモチーフのアミノ酸を特定した。 Then, the amino acid motifs for Clades 1 to 4 were identified based on the results of identity comparison.

　まず、アミノ酸２３５番目と２３６番目はＣｌａｄｅ１～４で完全に保存されている。次にアミノ酸２３７番目について、１種を除いてスレオニン（Ｔ）が保存されており、Ｃｌａｄｅ３に属する1種でグリシン（Ｇ）であったがＣｌａｄｅ３の中で相対的に活性は低く、保存アミノ酸としてはスレオニン（Ｔ）として特定できる。 First, amino acids 235 and 236 are completely conserved in Clades 1 to 4. Next, for amino acid 237, threonine (T) is conserved in all but one case, and one of the cases belongs to Clade 3, where it is glycine (G), but its activity is relatively low among Clade 3, and it can be identified as threonine (T) as a conserved amino acid.

　アミノ酸２３８番目は、Ｃｌａｄｅ１～３ではメチオニン（Ｍ）、Ｃｌａｄｅ４ではアスパラギン（Ｎ）が保存されていた。アミノ酸２３９番目は、Ｃｌａｄｅ１～４においてアスパラギン酸（Ｄ）、グルタミン酸（Ｅ）、アスパラギン（Ｎ）の３種であり、一定の保存性が見られ、Ｃｌａｄｅ１ではグルタミン酸（Ｅ）、Ｃｌａｄｅ２～３ではグルタミン酸（Ｅ）もしくはアスパラギン酸（Ｄ）、Ｃｌａｄｅ４ではアスパラギン酸（Ｄ）もしくはアスパラギン（Ｎ）であった。 Amino acid position 238 was conserved as methionine (M) in Clades 1 to 3 and asparagine (N) in Clade 4. Amino acid position 239 was conserved as aspartic acid (D), glutamic acid (E), and asparagine (N) in Clades 1 to 4, showing a certain degree of conservation, with glutamic acid (E) in Clade 1, glutamic acid (E) or aspartic acid (D) in Clades 2 to 3, and aspartic acid (D) or asparagine (N) in Clade 4.

　アミノ酸２４０番目については、Ｃｌａｄｅ１～４でアミノ酸が保存されていない。アミノ酸２４１番目と２４２番目は、Ｃｌａｄｅ１ではロイシン（Ｌ）＋フェニルアラニン（Ｆ）、Ｃｌａｄｅ２～３ではロイシン（Ｌ）＋トリプトファン（Ｗ）、Ｃｌａｄｅ４ではロイシン（Ｌ）＋フェニルアラニン（Ｆ）が保存されていた。 At amino acid 240, no amino acid is conserved in Clades 1 to 4. At amino acids 241 and 242, leucine (L) + phenylalanine (F) is conserved in Clade 1, leucine (L) + tryptophan (W) in Clades 2 and 3, and leucine (L) + phenylalanine (F) in Clade 4.

　以上より、酵素活性Ｓ選択的なＬ－リジン５位水酸化活性を有するＣｌａｄｅ　１では「ＨＥＴＭＥＸＬＦ」が保存されたモチーフとして特定され、Ｒ選択的なＬ－リジン５位水酸化活性を有するＣｌａｄｅ２とＣｌａｄｅ３では「ＨＥＴＭＤＸＬＷ」又は「ＨＥＴＭＥＸＬＷ」モチーフ、同じくＲ選択的なＬ－リジン５位水酸化活性を有するＣｌａｄｅ４では「ＨＥＴＮＤＸＬＦ」又は「ＨＥＴＮＮＸＬＦ」モチーフが特定された。また、Ｐｐ＿ＡｚｐＫ２はＣｌａｄｅ１～４のモチーフとは異なる特徴を有するアミノ酸配列であり、さらに近接するがＬ－リジン５位水酸化活性を有さないＣｌａｄｅ５は、Ｃｌａｄｅ１～４及びＰｐ＿ＡｚｐＫ２のいずれとも異なるアミノ酸配列であった。 From the above, the "HETMEXLF" motif was identified as a conserved motif in Clade 1, which has S-selective L-lysine 5-hydroxylation activity, while the "HETMDXLW" or "HETMEXLW" motif was identified in Clade 2 and Clade 3, which have R-selective L-lysine 5-hydroxylation activity, and the "HETNDXLF" or "HETNNXLF" motif was identified in Clade 4, which also has R-selective L-lysine 5-hydroxylation activity. Furthermore, Pp_AzpK2 has an amino acid sequence with different characteristics from the motifs of Clades 1 to 4, and Clade 5, which is close but does not have L-lysine 5-hydroxylation activity, has an amino acid sequence different from both Clades 1 to 4 and Pp_AzpK2.

　ここで、実施例２１において、ＡｚｐＫ２のＦ２４２Ｗ変異体はＬ－リジン５位水酸化活性が消失しており、Ｃｌａｄｅ１とＣｌａｄｅ２で異なる２４２番目のアミノ酸のみを入れ替えてもＬ－リジンの水酸基の光学選択性を変更できなかった。 Here, in Example 21, the F242W mutant of AzpK2 lost the activity of hydroxylating L-lysine at position 5, and even when only the 242nd amino acid, which differs between Clade 1 and Clade 2, was swapped, the optical selectivity of the hydroxyl group of L-lysine could not be changed.

　したがって、２３６～２４２番目アミノ酸を構成する１つのアミノ酸だけではなく、２３６～２４２番目のアミノ酸のモチーフがＬ－リジン５位水酸化酵素の機能発現に重要であることが示唆される。 This suggests that not just one amino acid that constitutes amino acids 236-242, but the motif of amino acids 236-242 is important for the functional expression of L-lysine 5-hydroxylase.

＜実施例２３＞（Ｌ－リジン５位水酸化酵素活性を有する遺伝子の探索）
　非特許文献７の情報を基に、Ｌ－リジン５位水酸化酵素活性を有する遺伝子の探索を行った。具体的には、非特許文献７に記載されているアラゾペプチン生合成遺伝子クラスターを有するＳｔｒｅｐｔａｃｉｄｉｐｈｉｌｕｓ　ｇｒｉｓｅｏｐｌａｎｕｓ株及びＫｉｔａｓａｔｏｓｐｏｒａ　ａｚａｔｉｃａ株の遺伝子情報を基にａｚｐ遺伝子クラスターを有する微生物を探索した。 Example 23 (Search for genes having L-lysine 5-hydroxylase activity)
A search was conducted for a gene having L-lysine 5-hydroxylase activity based on the information in Non-Patent Document 7. Specifically, a search was conducted for a microorganism having an azp gene cluster based on the genetic information of a Streptacidiphilus griseoplanus strain and a Kitasatospora azatica strain having an arazopeptin biosynthetic gene cluster described in Non-Patent Document 7.

　その結果、図１に示すようにＡｃｔｉｎｏｓｙｎｎｅｍａ　ｐｒｅｔｉｏｓｕｍ株およびＡｃｔｉｎｏｓｙｎｎｅｍａ　ｍｉｒｕｍ　ＤＳＭ　４３８２７株のａｚｐ遺伝子クラスターのホモログクラスターにおいて、ａｚｐＫ遺伝子を持たないが、代わりに機能未知の２－オキソグルタル酸依存型水酸化酵素遺伝子が存在することを見出した（図１中、★で示す）。 As a result, as shown in Figure 1, it was found that the homologue clusters of the azp gene cluster in Actinosynnema pretiosum and Actinosynnema mirum DSM 43827 strains do not have the azpK gene, but instead contain a 2-oxoglutarate-dependent hydroxylase gene with unknown function (indicated by an asterisk in Figure 1).

　この２－オキソグルタル酸依存型水酸化酵素をＡｚｐＫ２、また当該ＡｚｐＫ２タンパク質をコードするＤＮＡをａｚｐＫ２遺伝子と命名した。 This 2-oxoglutarate-dependent hydroxylase was named AzpK2, and the DNA encoding the AzpK2 protein was named the azpK2 gene.

　さらに、ａｚｐ遺伝子クラスター内に存在する６－ｄｉａｚｏ－５－ｏｘｏ－Ｌ－ｎｏｒｌｅｕｃｉｎｅの生合成遺伝子群（ａｚｐＪ，ａｚｐＫ，ａｚｐＬ）について同様に探索したところ、Ｐｓｅｕｄｏｍｏｎａｓ　ｐｓｙｃｈｒｏｔｏｌｅｒａｎｓ　ＮＳ３８３株において、ａｚｐＪ遺伝子とａｚｐＬ遺伝子の間に機能未知のａｚｐＫ２遺伝子ホモログがあることを見出した（図１中、☆で示す）。 Furthermore, a similar search was conducted for the 6-diazo-5-oxo-L-norleucine biosynthetic genes (azpJ, azpK, azpL) present in the azp gene cluster, and it was found that in the Pseudomonas psychotolerans NS383 strain, there is an azpK2 gene homologue with unknown function between the azpJ and azpL genes (indicated by a star in Figure 1).

　これをＰｐ＿ａｚｐＫ２遺伝子、Ｐｐ＿ａｚｐＫ２遺伝子がコードするタンパク質をＰｐ＿ＡｚｐＫ２と命名した。 This was named the Pp_azpK2 gene, and the protein encoded by the Pp_azpK2 gene was named Pp_AzpK2.

　図１は、ａｚｐ遺伝子クラスターを比較した図である。図１中の各記号の意味は前述の図１中の記号の説明のとおりである。 Figure 1 shows a comparison of azp gene clusters. The meanings of the symbols in Figure 1 are as explained above.

　前述の実施例において示したように、ＡｚｐＫ２タンパク質は、Ｌ－リジンの５位に対してＳ選択的に水酸化する活性を有するものであった。また、Ｐｐ＿ＡｚｐＫ２タンパク質は、Ｌ－リジンの５位に対してＲ選択的に水酸化する活性を有するものであった。さらに、ＡｚｐＫ２タンパク質をコードするＤＮＡ（ａｚｐＫ２遺伝子）及びＰｐ＿ＡｚｐＫ２タンパク質をコードするＤＮＡ（Ｐｐ＿ａｚｐＫ２遺伝子）について、それぞれの形質転換体を用いてＬ－リジンから５－ヒドロキシ－Ｌ－リジンを合成したところ、高い蓄積濃度で５－ヒドロキシ－Ｌ－リジンを合成することができた。 As shown in the above examples, the AzpK2 protein had the activity of selectively hydroxylating the 5-position of L-lysine in an S-selective manner. The Pp_AzpK2 protein also had the activity of selectively hydroxylating the 5-position of L-lysine in an R-selective manner. Furthermore, when 5-hydroxy-L-lysine was synthesized from L-lysine using transformants of DNA encoding the AzpK2 protein (azpK2 gene) and DNA encoding the Pp_AzpK2 protein (Pp_azpK2 gene), 5-hydroxy-L-lysine was synthesized at a high accumulated concentration.

＜実施例２４＞（ＡｚｐＫ２、Ｐｐ＿ＡｚｐＫ２ホモログの探索）
　ａｚｐＫ２遺伝子及びＰｐ＿ａｚｐＫ２遺伝子について、それぞれＤＮＡ　Ｄａｔａｂａｎｋ　ｏｆ　ＪＡＰＡＮ　（ＤＤＢＪ）等のデータベースを用いてホモロジー検索を行って、ａｚｐＫ２遺伝子及びＰｐ＿ａｚｐＫ２遺伝子のそれぞれのホモログ遺伝子１０００種の遺伝子配列情報を取得した。 Example 24 (Search for AzpK2 and Pp_AzpK2 homologues)
A homology search was carried out for each of the azpK2 gene and the Pp_azpK2 gene using databases such as DNA Databank of JAPAN (DDBJ) to obtain gene sequence information for 1000 homologous genes of each of the azpK2 gene and the Pp_azpK2 gene.

　次いで、ａｚｐＫ２遺伝子のホモログ遺伝子とＰｐ＿ａｚｐＫ２遺伝子のホモログ遺伝子の重複を排除して、ａｚｐＫ２遺伝子およびＰｐ＿ａｚｐＫ２遺伝子を含めたホモログ遺伝子１１１５種の遺伝子配列情報を取得した。 Next, overlapping homologous genes of the azpK2 gene and the Pp_azpK2 gene were eliminated, and gene sequence information for 1,115 homologous genes, including the azpK2 gene and the Pp_azpK2 gene, was obtained.

　これらのホモログ遺伝子がコードするアミノ酸配列をＮｅｉｇｈｂｏｒ－ｊｏｉｎｉｎｇ法を用いて作成した分子系統樹を元に分類したところ、１０個のＣｌａｄｅに分類された。分類された各ＣｌａｄｅをそれぞれＣｌａｄｅ１～１０と命名した。１０個のＣｌａｄｅに分類されたホモログ遺伝子１１１５種（ａｚｐＫ２およびＰｐ＿ａｚｐＫ２遺伝子を含む）がコードするタンパク質のうち、代表的なものを図２に示す。 The amino acid sequences encoded by these homolog genes were classified based on a molecular phylogenetic tree created using the Neighbor-joining method, and were classified into 10 Clades. Each classified Clade was named Clades 1 to 10. Representative proteins encoded by the 1,115 homolog genes (including the azpK2 and Pp_azpK2 genes) classified into the 10 Clades are shown in Figure 2.

　前述の実施例において示したように、Ｃｌａｄｅ１～４に属するタンパク質はＬ－リジン５位水酸化酵素活性を有するものであった。このうち、Ｃｌａｄｅ１に属するタンパク質はＬ－リジンの５位に対してＳ選択的に水酸化する活性を有するものであり、Ｃｌａｄｅ２～４に属するタンパク質はＬ－リジンの５位に対してＲ選択的に水酸化する活性を有するものであった。 As shown in the previous examples, proteins belonging to Clades 1 to 4 have L-lysine 5-hydroxylase activity. Of these, proteins belonging to Clade 1 have the activity of S-selectively hydroxylating the 5th position of L-lysine, and proteins belonging to Clades 2 to 4 have the activity of R-selectively hydroxylating the 5th position of L-lysine.

　本発明のＬ－リジン５位水酸化酵素及び酵素剤組成物は医薬品の中間体等として有用な５－ヒドロキシ－Ｌ－リジンの製造に使用できる。また、本発明の５－ヒドロキシ－Ｌ－リジンの製造法によれば、Ｌ－リジンから高収率かつ低コストで安価に５－ヒドロキシ－Ｌ－リジンを製造することができる。さらに、Ｌ－リジン５位水酸化酵素を使い分けることにより、所望の（２Ｓ，５Ｓ）－５－ヒドロキシ－Ｌ－リジン又は（２Ｓ，５Ｒ）－５－ヒドロキシ－Ｌ－リジンを製造することができる。 The L-lysine 5-hydroxylase and enzyme composition of the present invention can be used to produce 5-hydroxy-L-lysine, which is useful as an intermediate for pharmaceuticals, etc. Furthermore, according to the method for producing 5-hydroxy-L-lysine of the present invention, 5-hydroxy-L-lysine can be produced from L-lysine at a high yield and low cost. Furthermore, by using different L-lysine 5-hydroxylase enzymes, the desired (2S,5S)-5-hydroxy-L-lysine or (2S,5R)-5-hydroxy-L-lysine can be produced.

Claims (21)

　以下の（Ａ）、（Ｂ）、（Ｃ）、（Ｄ）、（Ｅ）又は（Ｆ）に示すポリペプチドを含むＬ－リジン５位水酸化酵素：
（Ａ）配列番号２、４、６、８、１０、１２、１４、１６、１８、２０、２２、２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８、１１０、１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列を有するポリペプチド；
（Ｂ）配列番号２、４、６、８、１０、１２、１４、１６、１８、２０、２２、２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８、１１０、１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換及び／又は付加されたアミノ酸配列を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチド；
（Ｃ）配列番号２、６、８、１０、１２、１４、１６、１８、２０又は２２に示すアミノ酸配列全長と５５％以上の配列同一性を有するアミノ酸配列を有し、かつ、Ｌ－リジン５位水酸化酵素活性を有するポリペプチド；
（Ｄ）配列番号２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８又は１１０に示すアミノ酸配列全長と５０％以上の配列同一性を有するアミノ酸配列を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチド；
（Ｅ）配列番号１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列全長と５８％以上の配列同一性を有するアミノ酸配列を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチド；
（Ｆ）連続したアミノ酸配列のモチーフである「ＨＧＷＨＷＤＤ」；と、「ＨＥＴＭＥＸＬＦ」、「ＨＥＴＭＤＸＬＷ」、「ＨＥＴＭＥＸＬＷ」、「ＨＥＴＮＤＸＬＦ」及び「ＨＥＴＮＮＸＬＦ」よりなる群から選ばれるいずれかの連続したアミノ酸配列のモチーフ；を有し、かつ、Ｌ－リジン５位水酸化活性を保持するポリペプチド（ここで、Ｘは、Ａ、Ｒ、Ｎ、Ｄ、Ｃ、Ｑ、Ｅ、Ｇ、Ｈ、Ｉ、Ｌ、Ｋ、Ｍ、Ｆ、Ｐ、Ｓ、Ｔ、Ｗ、Ｙ又はＶのいずれかのアミノ酸を示す）。 An L-lysine 5-hydroxylase comprising a polypeptide shown in (A), (B), (C), (D), (E) or (F) below:
(A) a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134;
(B) a polypeptide having an amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134 in which one or more amino acids have been deleted, substituted and/or added, and having L-lysine 5-hydroxylation activity;
(C) a polypeptide having an amino acid sequence having 55% or more sequence identity to the full-length amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20, or 22, and having L-lysine 5-hydroxylase activity;
(D) a polypeptide having an amino acid sequence having 50% or more sequence identity to the full-length amino acid sequence shown in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110, and having L-lysine 5-hydroxylation activity;
(E) a polypeptide having an amino acid sequence having 58% or more sequence identity to the full-length amino acid sequence shown in SEQ ID NO: 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134, and having L-lysine 5-hydroxylation activity;
(F) A polypeptide having a consecutive amino acid sequence motif of "HGWHWDD" and any consecutive amino acid sequence motif selected from the group consisting of "HETMEXLF", "HETMDXLW", "HETMEXLW", "HETNDXLF" and "HETNNXLF", and having L-lysine 5-hydroxylation activity (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V). 　請求項１に記載のＬ－リジン５位水酸化酵素をコードするＤＮＡ。 　DNA encoding the L-lysine 5-hydroxylase according to claim 1. 　前記ＤＮＡが、以下の（Ｇ）、（Ｈ）、（Ｉ）、（Ｊ）、（Ｋ）又は（Ｌ）である、請求項２に記載のＤＮＡ：
（Ｇ）配列番号１、３、５、７、９、１１、１３、１５、１７、１９、２１、２３、２５、２７、２９、３１、３３、３５、３７、３９、４１、４３、４５、４７、４９、５１、５５、５７、６７、６９、７１、７３、７５、７９、８１、８３、８５、８７、８９、９１、９３、９５、９７、９９、１０１、１０３、１０５、１０７、１０９、１１１、１１３、１１５、１１９、１２１、１２３、１２５、１２７、１２９、１３１又は１３３に示す塩基配列を含むＤＮＡ；
（Ｈ）配列番号１、３、５、７、９、１１、１３、１５、１７、１９、２１、２３、２５、２７、２９、３１、３３、３５、３７、３９、４１、４３、４５、４７、４９、５１、５５、５７、６７、６９、７１、７３、７５、７９、８１、８３、８５、８７、８９、９１、９３、９５、９７、９９、１０１、１０３、１０５、１０７、１０９、１１１、１１３、１１５、１１９、１２１、１２３、１２５、１２７、１２９、１３１又は１３３に示す塩基配列において、１又は複数個の塩基が置換、欠失、及び／又は付加された塩基配列を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ；
（Ｉ）配列番号１、５、７、９、１１、１３、１５、１７、１９又は２１に示す塩基配列がコードするポリペプチドと５５％以上の配列同一性を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ；
（Ｊ）配列番号２３、２５、２７、２９、３１、３３、３５、３７、３９、４１、４３、４５、４７、４９、５１、５５、６７、６９、７１、７３、７５、７９、８１、８３、８５、８７、８９、９１、９３、９５、９７、９９、１０１、１０３、１０５、１０７又は１０９に示す塩基配列がコードするポリペプチドと５０％以上の配列同一性を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ；
（Ｋ）配列番号１１１、１１３、１１５、１１９、１２１、１２３、１２５、１２７、１２９、１３１又は１３３に示す塩基配列がコードするポリペプチドと５８％以上の配列同一性を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ；
（Ｌ）連続したアミノ酸配列のモチーフである「ＨＧＷＨＷＤＤ」；と、「ＨＥＴＭＥＸＬＦ」、「ＨＥＴＭＤＸＬＷ」、「ＨＥＴＭＥＸＬＷ」、「ＨＥＴＮＤＸＬＦ」及び「ＨＥＴＮＮＸＬＦ」よりなる群から選ばれるいずれかの連続したアミノ酸配列のモチーフ；を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ（ここで、Ｘは、Ａ、Ｒ、Ｎ、Ｄ、Ｃ、Ｑ、Ｅ、Ｇ、Ｈ、Ｉ、Ｌ、Ｋ、Ｍ、Ｆ、Ｐ、Ｓ、Ｔ、Ｗ、Ｙ又はＶのいずれかのアミノ酸を示す）。 The DNA according to claim 2, which is the following (G), (H), (I), (J), (K) or (L):
(G) DNA containing the base sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 57, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133;
(H) a DNA encoding a polypeptide having an L-lysine 5-hydroxylating activity, comprising a nucleotide sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 57, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133 in which one or more nucleotides have been substituted, deleted, and/or added;
(I) a DNA encoding a polypeptide having a sequence identity of 55% or more to a polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, or 21 and having an activity of hydroxylating L-lysine at the 5-position;
(J) a DNA encoding a polypeptide having 50% or more sequence identity to a polypeptide encoded by the base sequence shown in SEQ ID NO: 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107 or 109, and having an activity of hydroxylating L-lysine at position 5;
(K) a DNA encoding a polypeptide having a sequence identity of 58% or more to a polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133 and having L-lysine 5-hydroxylation activity;
(L) A DNA encoding a polypeptide having a consecutive amino acid sequence motif of "HGWHWDD" and any consecutive amino acid sequence motif selected from the group consisting of "HETMEXLF", "HETMDXLW", "HETMEXLW", "HETNDXLF" and "HETNNXLF" and having L-lysine 5-hydroxylation activity (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V). 　Ｌ－リジンに、以下の（Ａ）、（Ｂ）、（Ｃ）、（Ｄ）、（Ｅ）又は（Ｆ）に示すポリペプチド又はそれを含む細胞、前記細胞の調製物若しくは前記細胞を培養して得られた培養液を接触させて、５－ヒドロキシ－Ｌ－リジンを生成させることを特徴とする、５－ヒドロキシ－Ｌ－リジンの製造方法：
（Ａ）配列番号２、４、６、８、１０、１２、１４、１６、１８、２０、２２、２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８、１１０、１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列を有するポリペプチド；
（Ｂ）配列番号２、４、６、８、１０、１２、１４、１６、１８、２０、２２、２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８、１１０、１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換及び／又は付加されたアミノ酸配列を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチド；
（Ｃ）配列番号２、６、８、１０、１２、１４、１６、１８、２０又は２２に示すアミノ酸配列全長と５５％以上の配列同一性を有するアミノ酸配列を有し、かつ、Ｌ－リジン５位水酸化酵素活性を有するポリペプチド；
（Ｄ）配列番号２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８又は１１０に示すアミノ酸配列全長と５０％以上の配列同一性を有するアミノ酸配列を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチド；
（Ｅ）配列番号１１２，１１４，１１６，１２０，１２２，１２４，１２６，１２８，１３０，１３２又は１３４に示すアミノ酸配列と５８％以上の同一性を有するアミノ酸配列を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチド；
（Ｆ）連続したアミノ酸配列のモチーフである「ＨＧＷＨＷＤＤ」；と、「ＨＥＴＭＥＸＬＦ」、「ＨＥＴＭＤＸＬＷ」「ＨＥＴＭＥＸＬＷ」、「ＨＥＴＮＤＸＬＦ」及び「ＨＥＴＮＮＸＬＦ」よりなる群から選ばれるいずれかの連続したアミノ酸配列のモチーフ；を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチド（ここで、Ｘは、Ａ、Ｒ、Ｎ、Ｄ、Ｃ、Ｑ、Ｅ、Ｇ、Ｈ、Ｉ、Ｌ、Ｋ、Ｍ、Ｆ、Ｐ、Ｓ、Ｔ、Ｗ、Ｙ又はＶのいずれかのアミノ酸を示す）。 A method for producing 5-hydroxy-L-lysine, comprising contacting L-lysine with a polypeptide shown in the following (A), (B), (C), (D), (E) or (F), or a cell containing the polypeptide, a preparation of the cell, or a culture medium obtained by culturing the cell, to produce 5-hydroxy-L-lysine:
(A) a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134;
(B) a polypeptide having an amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134 in which one or more amino acids have been deleted, substituted and/or added, and having L-lysine 5-hydroxylation activity;
(C) a polypeptide having an amino acid sequence having 55% or more sequence identity to the full-length amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20, or 22, and having L-lysine 5-hydroxylase activity;
(D) a polypeptide having an amino acid sequence having 50% or more sequence identity to the full-length amino acid sequence shown in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110, and having L-lysine 5-hydroxylation activity;
(E) a polypeptide having an amino acid sequence having 58% or more identity to the amino acid sequence shown in SEQ ID NO: 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134, and having L-lysine 5-hydroxylation activity;
(F) A polypeptide having a consecutive amino acid sequence motif of "HGWHWDD" and any consecutive amino acid sequence motif selected from the group consisting of "HETMEXLF", "HETMDXLW", "HETMEXLW", "HETNDXLF" and "HETNNXLF", and having L-lysine 5-hydroxylation activity (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V). 　前記細胞が、前記Ｌ－リジン５位水酸化酵素をコードするＤＮＡで形質転換された細胞である、請求項４に記載の５－ヒドロキシ－Ｌ－リジンの製造方法。 The method for producing 5-hydroxy-L-lysine according to claim 4, wherein the cell is a cell transformed with DNA encoding the L-lysine 5-hydroxylase. 　前記ＤＮＡが、以下の（Ｇ）、（Ｈ）、（Ｉ）、（Ｊ）、（Ｋ）又は（Ｌ）である、請求項５に記載の５－ヒドロキシ－Ｌ－リジンの製造方法：
（Ｇ）配列番号１、３、５、７、９、１１、１３、１５、１７、１９、２１、２３、２５、２７、２９、３１、３３、３５、３７、３９、４１、４３、４５、４７、４９、５１、５５、５７、６７、６９、７１、７３、７５、７９、８１、８３、８５、８７、８９、９１、９３、９５、９７、９９、１０１、１０３、１０５、１０７、１０９、１１１、１１３、１１５、１１９、１２１、１２３、１２５、１２７、１２９、１３１又は１３３に示す塩基配列を含むＤＮＡ；
（Ｈ）配列番号１、３、５、７、９、１１、１３、１５、１７、１９、２１、２３、２５、２７、２９、３１、３３、３５、３７、３９、４１、４３、４５、４７、４９、５１、５５、５７、６７、６９、７１、７３、７５、７９、８１、８３、８５、８７、８９、９１、９３、９５、９７、９９、１０１、１０３、１０５、１０７、１０９、１１１、１１３、１１５、１１９、１２１、１２３、１２５、１２７、１２９、１３１又は１３３に示す塩基配列において、１又は複数個の塩基が置換、欠失、及び／又は付加された塩基配列を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ；
（Ｉ）配列番号１、５、７、９、１１、１３、１５、１７、１９又は２１に示す塩基配列がコードするポリペプチドと５５％以上の配列同一性を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ；
（Ｊ）配列番号２３、２５、２７、２９、３１、３３、３５、３７、３９、４１、４３、４５、４７、４９、５１、５５、６７、６９、７１、７３、７５、７９、８１、８３、８５、８７、８９、９１、９３、９５、９７、９９、１０１、１０３、１０５、１０７又は１０９に示す塩基配列がコードするポリペプチドと５０％以上の配列同一性を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ；
（Ｋ）配列番号１１１、１１３、１１５、１１９、１２１、１２３、１２５、１２７、１２９、１３１又は１３３に示す塩基配列がコードするポリペプチドと５８％以上の配列同一性を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ；
（Ｌ）連続したアミノ酸配列のモチーフである「ＨＧＷＨＷＤＤ」；と、「ＨＥＴＭＥＸＬＦ」、「ＨＥＴＭＤＸＬＷ」、「ＨＥＴＭＥＸＬＷ」、「ＨＥＴＮＤＸＬＦ」及び「ＨＥＴＮＮＸＬＦ」よりなる群から選ばれるいずれかの連続したアミノ酸配列のモチーフ；とを有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ（ここで、Ｘは、Ａ、Ｒ、Ｎ、Ｄ、Ｃ、Ｑ、Ｅ、Ｇ、Ｈ、Ｉ、Ｌ、Ｋ、Ｍ、Ｆ、Ｐ、Ｓ、Ｔ、Ｗ、Ｙ又はＶのいずれかのアミノ酸を示す）。 The method for producing 5-hydroxy-L-lysine according to claim 5, wherein the DNA is the following (G), (H), (I), (J), (K) or (L):
(G) DNA containing the base sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 57, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133;
(H) a DNA encoding a polypeptide having an L-lysine 5-hydroxylating activity, comprising a nucleotide sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 57, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133 in which one or more nucleotides have been substituted, deleted, and/or added;
(I) a DNA encoding a polypeptide having a sequence identity of 55% or more to a polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, or 21 and having an activity of hydroxylating L-lysine at the 5-position;
(J) a DNA encoding a polypeptide having 50% or more sequence identity to a polypeptide encoded by the base sequence shown in SEQ ID NO: 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107 or 109, and having an activity of hydroxylating L-lysine at position 5;
(K) a DNA encoding a polypeptide having a sequence identity of 58% or more to a polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133 and having L-lysine 5-hydroxylation activity;
(L) A DNA encoding a polypeptide having a consecutive amino acid sequence motif "HGWHWDD" and any consecutive amino acid sequence motif selected from the group consisting of "HETMEXLF", "HETMDXLW", "HETMEXLW", "HETNDXLF" and "HETNNXLF", and having L-lysine 5-hydroxylation activity (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V). 　以下の（Ａ）、（Ｂ）、（Ｃ）、（Ｄ）、（Ｅ）又は（Ｆ）に示すポリペプチド又はそれを含む細胞、前記細胞の調製物若しくは前記細胞を培養して得られた培養液を含み、Ｌ－リジン５位水酸化活性を有する酵素剤組成物：
（Ａ）配列番号２、４、６、８、１０、１２、１４、１６、１８、２０、２２、２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８、１１０、１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列を有するポリペプチド；
（Ｂ）配列番号２、４、６、８、１０、１２、１４、１６、１８、２０、２２、２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８、１１０、１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換及び／又は付加されたアミノ酸配列を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチド；
（Ｃ）配列番号２、６、８、１０、１２、１４、１６、１８、２０又は２２に示すアミノ酸配列全長と５５％以上の配列同一性を有するアミノ酸配列を有し、かつ、Ｌ－リジン５位水酸化酵素活性を有するポリペプチド；
（Ｄ）配列番号２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８又は１１０に示すアミノ酸配列全長と５０％以上の配列同一性を有するアミノ酸配列を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチド；
（Ｅ）配列番号１１２，１１４，１１６，１２０，１２２，１２４，１２６，１２８，１３０，１３２又は１３４に示すアミノ酸配列と５８％以上の同一性を有するアミノ酸配列を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチド；
（Ｆ）連続したアミノ酸配列のモチーフである「ＨＧＷＨＷＤＤ」；と、「ＨＥＴＭＥＸＬＦ」、「ＨＥＴＭＤＸＬＷ」「ＨＥＴＭＥＸＬＷ」、「ＨＥＴＮＤＸＬＦ」及び「ＨＥＴＮＮＸＬＦ」よりなる群から選ばれるいずれかの連続したアミノ酸配列のモチーフ；を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチド（ここで、Ｘは、Ａ、Ｒ、Ｎ、Ｄ、Ｃ、Ｑ、Ｅ、Ｇ、Ｈ、Ｉ、Ｌ、Ｋ、Ｍ、Ｆ、Ｐ、Ｓ、Ｔ、Ｗ、Ｙ又はＶのいずれかのアミノ酸を示す）。 An enzyme composition having L-lysine 5-hydroxylation activity, comprising a polypeptide represented by the following (A), (B), (C), (D), (E) or (F), or a cell containing the same, a preparation of the cell, or a culture medium obtained by culturing the cell:
(A) a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134;
(B) a polypeptide having an amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134 in which one or more amino acids have been deleted, substituted and/or added, and having L-lysine 5-hydroxylation activity;
(C) a polypeptide having an amino acid sequence having 55% or more sequence identity to the full-length amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20, or 22, and having L-lysine 5-hydroxylase activity;
(D) a polypeptide having an amino acid sequence having 50% or more sequence identity to the full-length amino acid sequence shown in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110, and having L-lysine 5-hydroxylation activity;
(E) a polypeptide having an amino acid sequence having 58% or more identity to the amino acid sequence shown in SEQ ID NO: 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134, and having L-lysine 5-hydroxylation activity;
(F) A polypeptide having a consecutive amino acid sequence motif of "HGWHWDD" and any consecutive amino acid sequence motif selected from the group consisting of "HETMEXLF", "HETMDXLW", "HETMEXLW", "HETNDXLF" and "HETNNXLF", and having L-lysine 5-hydroxylation activity (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V). 　以下の（Ａ－１）、（Ｂ－１）、（Ｃ－１）又は（Ｆ－１）に示すポリペプチドを含むＳ選択的Ｌ－リジン５位水酸化酵素：
（Ａ－１）配列番号２、６、８、１０、１２、１４、１６、１８、２０又は２２に示すアミノ酸配列を有するポリペプチド；
（Ｂ－１）配列番号２、６、８、１０、１２、１４、１６、１８、２０又は２２に示すアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換及び／又は付加されたアミノ酸配列を有し、かつ、Ｓ選択的Ｌ－リジン５位水酸化活性を有するポリペプチド；
（Ｃ－１）配列番号２、６、８、１０、１２、１４、１６、１８、２０又は２２に示すアミノ酸配列全長と５５％以上の配列同一性を有するアミノ酸配列を有し、かつ、Ｓ選択的Ｌ－リジン５位水酸化酵素活性を有するポリペプチド；
（Ｆ－１）連続したアミノ酸配列のモチーフである「ＨＧＷＨＷＤＤ」及び「ＨＥＴＭＥＸＬＦ」を有し、かつ、Ｓ選択的Ｌ－リジン５位水酸化活性を有するポリペプチド（ここで、Ｘは、Ａ、Ｒ、Ｎ、Ｄ、Ｃ、Ｑ、Ｅ、Ｇ、Ｈ、Ｉ、Ｌ、Ｋ、Ｍ、Ｆ、Ｐ、Ｓ、Ｔ、Ｗ、Ｙ又はＶのいずれかのアミノ酸を示す）。 An S-selective L-lysine 5-hydroxylase comprising a polypeptide shown in (A-1), (B-1), (C-1) or (F-1) below:
(A-1) a polypeptide having an amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22;
(B-1) a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted and/or added in the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22, and having S-selective L-lysine 5-hydroxylation activity;
(C-1) a polypeptide having an amino acid sequence having 55% or more sequence identity to the full-length amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20, or 22, and having S-selective L-lysine 5-hydroxylase activity;
(F-1) A polypeptide having consecutive amino acid sequence motifs of "HGWHWDD" and "HETMEXLF" and having S-selective L-lysine 5-hydroxylation activity (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V). 　請求項８に記載のＳ選択的Ｌ－リジン５位水酸化酵素をコードするＤＮＡ。 　DNA encoding the S-selective L-lysine 5-hydroxylase described in claim 8. 　前記ＤＮＡが、以下の（Ｇ－１）、（Ｈ－１）、（Ｉ－１）又は（Ｌ－１）である、請求項９に記載のＤＮＡ：
（Ｇ－１）配列番号１、５、７、９、１１、１３、１５、１７、１９又は２１に示す塩基配列を含むＤＮＡ；
（Ｈ－１）配列番号１、５、７、９、１１、１３、１５、１７、１９又は２１に示す塩基配列において、１又は複数個の塩基が置換、欠失、及び／又は付加された塩基配列を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ；
（Ｉ－１）配列番号１、５、７、９、１１、１３、１５、１７、１９又は２１に示す塩基配列がコードするポリペプチドと５５％以上の配列同一性を有し、かつ、Ｓ選択的Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ；
（Ｌ－１）連続したアミノ酸配列のモチーフである「ＨＧＷＨＷＤＤ」及び「ＨＥＴＭＥＸＬＦ」を有し、かつ、Ｓ選択的Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ（ここで、Ｘは、Ａ、Ｒ、Ｎ、Ｄ、Ｃ、Ｑ、Ｅ、Ｇ、Ｈ、Ｉ、Ｌ、Ｋ、Ｍ、Ｆ、Ｐ、Ｓ、Ｔ、Ｗ、Ｙ又はＶのいずれかのアミノ酸を示す）。 The DNA according to claim 9, which is the following (G-1), (H-1), (I-1) or (L-1):
(G-1) DNA comprising the nucleotide sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, or 21;
(H-1) DNA encoding a polypeptide having a nucleotide sequence in which one or more nucleotides have been substituted, deleted, and/or added in the nucleotide sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, or 21 and having an activity of hydroxylating L-lysine at the 5-position;
(I-1) DNA encoding a polypeptide having 55% or more sequence identity to a polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, or 21 and having S-selective L-lysine 5-hydroxylation activity;
(L-1) A DNA encoding a polypeptide having consecutive amino acid sequence motifs "HGWHWDD" and "HETMEXLF" and having S-selective L-lysine 5-hydroxylation activity (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V). 　Ｌ－リジンに、以下の（Ａ－１）、（Ｂ－１）、（Ｃ－１）又は（Ｆ－１）に示すポリペプチド又はそれを含む細胞、前記細胞の調製物若しくは前記細胞を培養して得られた培養液を接触させて、（２Ｓ，５Ｓ）－５－ヒドロキシ－Ｌ－リジンを生成させることを特徴とする、（２Ｓ，５Ｓ）－５－ヒドロキシ－Ｌ－リジンの製造方法：
（Ａ－１）配列番号２、６、８、１０、１２、１４、１６、１８、２０又は２２に示すアミノ酸配列を有するポリペプチド；
（Ｂ－１）配列番号２、６、８、１０、１２、１４、１６、１８、２０又は２２に示すアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換及び／又は付加されたアミノ酸配列を有し、かつ、Ｓ選択的Ｌ－リジン５位水酸化活性を有するポリペプチド；
（Ｃ－１）配列番号２、６、８、１０、１２、１４、１６、１８、２０又は２２に示すアミノ酸配列全長と５５％以上の配列同一性を有するアミノ酸配列を有し、かつ、Ｓ選択的Ｌ－リジン５位水酸化酵素活性を有するポリペプチド；
（Ｆ－１）連続したアミノ酸配列のモチーフである「ＨＧＷＨＷＤＤ」及び「ＨＥＴＭＥＸＬＦ」を有し、かつ、Ｓ選択的Ｌ－リジン５位水酸化活性を保持するポリペプチド（ここで、Ｘは、Ａ、Ｒ、Ｎ、Ｄ、Ｃ、Ｑ、Ｅ、Ｇ、Ｈ、Ｉ、Ｌ、Ｋ、Ｍ、Ｆ、Ｐ、Ｓ、Ｔ、Ｗ、Ｙ又はＶのいずれかのアミノ酸を示す）。 A method for producing (2S,5S)-5-hydroxy-L-lysine, comprising contacting L-lysine with a polypeptide shown in the following (A-1), (B-1), (C-1) or (F-1), or a cell containing the polypeptide, a preparation of the cell, or a culture solution obtained by culturing the cell, to produce (2S,5S)-5-hydroxy-L-lysine:
(A-1) a polypeptide having an amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22;
(B-1) a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted and/or added in the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22, and having S-selective L-lysine 5-hydroxylation activity;
(C-1) a polypeptide having an amino acid sequence having 55% or more sequence identity to the full-length amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20, or 22, and having S-selective L-lysine 5-hydroxylase activity;
(F-1) A polypeptide having consecutive amino acid sequence motifs "HGWHWDD" and "HETMEXLF" and having S-selective L-lysine 5-hydroxylation activity (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V). 　前記細胞が、前記Ｌ－リジン５位水酸化酵素をコードするＤＮＡで形質転換された細胞である、請求項１１に記載の（２Ｓ，５Ｓ）－５－ヒドロキシ－Ｌ－リジンの製造方法。 The method for producing (2S,5S)-5-hydroxy-L-lysine according to claim 11, wherein the cell is a cell transformed with DNA encoding the L-lysine 5-hydroxylase. 　前記ＤＮＡが、以下の（Ｇ－１）、（Ｈ－１）、（Ｉ－１）又は（Ｌ－１）である、請求項１２に記載の（２Ｓ，５Ｓ）－５－ヒドロキシ－Ｌ－リジンの製造方法：
（Ｇ－１）配列番号１、５、７、９、１１、１３、１５、１７、１９又は２１に示す塩基配列を含むＤＮＡ；
（Ｈ－１）配列番号１、５、７、９、１１、１３、１５、１７、１９又は２１に示す塩基配列において、１又は複数個の塩基が置換、欠失、及び／又は付加された塩基配列を有し、かつ、Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ；
（Ｉ－１）配列番号１、５、７、９、１１、１３、１５、１７、１９又は２１に示す塩基配列がコードするポリペプチドと５５％以上の配列同一性を有し、かつ、Ｓ選択的Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ；
（Ｌ－１）連続したアミノ酸配列のモチーフである「ＨＧＷＨＷＤＤ」及び「ＨＥＴＭＥＸＬＦ」を有し、かつ、Ｓ選択的Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ（ここで、Ｘは、Ａ、Ｒ、Ｎ、Ｄ、Ｃ、Ｑ、Ｅ、Ｇ、Ｈ、Ｉ、Ｌ、Ｋ、Ｍ、Ｆ、Ｐ、Ｓ、Ｔ、Ｗ、Ｙ又はＶのいずれかのアミノ酸を示す）。 The method for producing (2S,5S)-5-hydroxy-L-lysine according to claim 12, wherein the DNA is the following (G-1), (H-1), (I-1) or (L-1):
(G-1) DNA comprising the nucleotide sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19 or 21;
(H-1) DNA encoding a polypeptide having a nucleotide sequence in which one or more nucleotides have been substituted, deleted, and/or added in the nucleotide sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, or 21 and having an activity of hydroxylating L-lysine at the 5-position;
(I-1) DNA encoding a polypeptide having 55% or more sequence identity to a polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, or 21 and having S-selective L-lysine 5-hydroxylation activity;
(L-1) A DNA encoding a polypeptide having consecutive amino acid sequence motifs "HGWHWDD" and "HETMEXLF" and having S-selective L-lysine 5-hydroxylation activity (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V). 　以下の（Ａ－１）、（Ｂ－１）、（Ｃ－１）又は（Ｆ－１）に示すポリペプチド又はそれを含む細胞、前記細胞の調製物若しくは前記細胞を培養して得られた培養液を含み、かつ、Ｓ選択的Ｌ－リジン５位水酸化活性を有する酵素剤組成物：
（Ａ－１）配列番号２、６、８、１０、１２、１４、１６、１８、２０又は２２に示すアミノ酸配列を有するポリペプチド；
（Ｂ－１）配列番号２、６、８、１０、１２、１４、１６、１８、２０又は２２に示すアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換及び／又は付加されたアミノ酸配列を有し、かつ、Ｓ選択的Ｌ－リジン５位水酸化活性を有するポリペプチド；
（Ｃ－１）配列番号２、６、８、１０、１２、１４、１６、１８、２０又は２２に示すアミノ酸配列全長と５５％以上の配列同一性を有するアミノ酸配列を有し、かつ、Ｓ選択的Ｌ－リジン５位水酸化酵素活性を有するポリペプチド；
（Ｆ－１）連続したアミノ酸配列のモチーフである「ＨＧＷＨＷＤＤ」及び「ＨＥＴＭＥＸＬＦ」を有し、かつ、Ｓ選択的Ｌ－リジン５位水酸化活性を保持するポリペプチド（ここで、Ｘは、Ａ、Ｒ、Ｎ、Ｄ、Ｃ、Ｑ、Ｅ、Ｇ、Ｈ、Ｉ、Ｌ、Ｋ、Ｍ、Ｆ、Ｐ、Ｓ、Ｔ、Ｗ、Ｙ又はＶのいずれかのアミノ酸を示す）。 An enzyme composition comprising a polypeptide shown in (A-1), (B-1), (C-1) or (F-1) below, a cell containing the polypeptide, a preparation of the cell, or a culture medium obtained by culturing the cell, and having S-selective L-lysine 5-hydroxylation activity:
(A-1) a polypeptide having an amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22;
(B-1) a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted and/or added in the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22, and having S-selective L-lysine 5-hydroxylation activity;
(C-1) a polypeptide having an amino acid sequence having 55% or more sequence identity to the full-length amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20, or 22, and having S-selective L-lysine 5-hydroxylase activity;
(F-1) A polypeptide having consecutive amino acid sequence motifs "HGWHWDD" and "HETMEXLF" and having S-selective L-lysine 5-hydroxylation activity (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V). 　以下の（Ａ－２）、（Ｂ－２）、（Ｄ－２）、（Ｅ－２）又は（Ｆ－２）に示すポリペプチドを含むＲ選択的Ｌ－リジン５位水酸化酵素：
（Ａ－２）配列番号４、２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８、１１０、１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列を有するポリペプチド；
（Ｂ－２）配列番号４、２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８、１１０、１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換及び／又は付加されたアミノ酸配列を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチド；
（Ｄ－２）配列番号２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８又は１１０に示すアミノ酸配列全長と５０％以上の配列同一性を有するアミノ酸配列を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチド；
（Ｅ－２）配列番号１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列全長と５８％以上の配列同一性を有するアミノ酸配列を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチド；（Ｆ－２）連続したアミノ酸配列のモチーフである「ＨＧＷＨＷＤＤ」；と、「ＨＥＴＭＤＸＬＷ」、「ＨＥＴＭＥＸＬＷ」、「ＨＥＴＮＤＸＬＦ」及び「ＨＥＴＮＮＸＬＦ」よりなる群から選ばれるいずれかの連続したアミノ酸配列のモチーフ；を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を保持するポリペプチド（ここで、Ｘは、Ａ、Ｒ、Ｎ、Ｄ、Ｃ、Ｑ、Ｅ、Ｇ、Ｈ、Ｉ、Ｌ、Ｋ、Ｍ、Ｆ、Ｐ、Ｓ、Ｔ、Ｗ、Ｙ又はＶのいずれかのアミノ酸を示す）。 An R-selective L-lysine 5-hydroxylase comprising a polypeptide shown in (A-2), (B-2), (D-2), (E-2) or (F-2) below:
(A-2) a polypeptide having an amino acid sequence shown in SEQ ID NO: 4, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134;
(B-2) a polypeptide having an amino acid sequence represented by SEQ ID NO: 4, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134 in which one or more amino acids have been deleted, substituted, and/or added, and having R-selective L-lysine 5-hydroxylation activity;
(D-2) a polypeptide having an amino acid sequence having 50% or more sequence identity to the full length amino acid sequence shown in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110, and having R-selective L-lysine 5-hydroxylation activity;
(E-2) A polypeptide having an amino acid sequence having 58% or more sequence identity with the full length amino acid sequence shown in SEQ ID NO: 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134, and having R-selective L-lysine 5-position hydroxylation activity; (F-2) A polypeptide having a motif of a consecutive amino acid sequence "HGWHWDD"; and a motif of any consecutive amino acid sequence selected from the group consisting of "HETMDXLW", "HETMEXLW", "HETNDXLF" and "HETNNXLF"; and having R-selective L-lysine 5-position hydroxylation activity (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V). 　請求項１５に記載のＲ選択的Ｌ－リジン５位水酸化酵素をコードするＤＮＡ。 DNA encoding the R-selective L-lysine 5-hydroxylase described in claim 15. 　前記ＤＮＡが、以下の（Ｇ－２）、（Ｈ－２）、（Ｊ－２）、（Ｋ－２）又は（Ｌ－２）である、請求項１６に記載のＤＮＡ：
（Ｇ－２）配列番号３、２３、２５、２７、２９、３１、３３、３５、３７、３９、４１、４３、４５、４７、４９、５１、５５、６７、６９、７１、７３、７５、７９、８１、８３、８５、８７、８９、９１、９３、９５、９７、９９、１０１、１０３、１０５、１０７、１０９、１１１、１１３、１１５、１１９、１２１、１２３、１２５、１２７、１２９、１３１又は１３３に示す塩基配列を含むＤＮＡ；
（Ｈ－２）配列番号３、２３、２５、２７、２９、３１、３３、３５、３７、３９、４１、４３、４５、４７、４９、５１、５５、６７、６９、７１、７３、７５、７９、８１、８３、８５、８７、８９、９１、９３、９５、９７、９９、１０１、１０３、１０５、１０７、１０９、１１１、１１３、１１５、１１９、１２１、１２３、１２５、１２７、１２９、１３１又は１３３に示す塩基配列において、１又は複数個の塩基が置換、欠失、及び／又は付加された塩基配列を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ；
（Ｊ－２）配列番号２３、２５、２７、２９、３１、３３、３５、３７、３９、４１、４３、４５、４７、４９、５１、５５、６７、６９、７１、７３、７５、７９、８１、８３、８５、８７、８９、９１、９３、９５、９７、９９、１０１、１０３、１０５、１０７又は１０９に示す塩基配列がコードするポリペプチドと５０％以上の配列同一性を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ；
（Ｋ－２）配列番号１１１、１１３、１１５、１１９、１２１、１２３、１２５、１２７、１２９、１３１又は１３３に示す塩基配列がコードするポリペプチドと５８％以上の配列同一性を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ；
（Ｌ－２）連続したアミノ酸配列のモチーフである「ＨＧＷＨＷＤＤ」；と、「ＨＥＴＭＤＸＬＷ」、「ＨＥＴＭＥＸＬＷ」、「ＨＥＴＮＤＸＬＦ」及び「ＨＥＴＮＮＸＬＦ」よりなる群から選ばれるいずれかの連続したアミノ酸配列のモチーフ；を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ（ここで、Ｘは、Ａ、Ｒ、Ｎ、Ｄ、Ｃ、Ｑ、Ｅ、Ｇ、Ｈ、Ｉ、Ｌ、Ｋ、Ｍ、Ｆ、Ｐ、Ｓ、Ｔ、Ｗ、Ｙ又はＶのいずれかのアミノ酸を示す）。 The DNA according to claim 16, which is the following (G-2), (H-2), (J-2), (K-2) or (L-2):
(G-2) DNA comprising the base sequence shown in SEQ ID NO: 3, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133;
(H-2) DNA encoding a polypeptide having an R-selective L-lysine 5-hydroxylation activity, comprising a nucleotide sequence represented by SEQ ID NO: 3, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133, in which one or more nucleotides have been substituted, deleted and/or added;
(J-2) a DNA encoding a polypeptide having 50% or more sequence identity to a polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107 or 109, and having R-selective L-lysine 5-hydroxylation activity;
(K-2) a DNA having a sequence identity of 58% or more to a polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133, and encoding a polypeptide having R-selective L-lysine 5-hydroxylation activity;
(L-2) A DNA encoding a polypeptide having a consecutive amino acid sequence motif of "HGWHWDD" and any consecutive amino acid sequence motif selected from the group consisting of "HETMDXLW", "HETMEXLW", "HETNDXLF" and "HETNNXLF" and having R-selective L-lysine 5-hydroxylation activity (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V). 　Ｌ－リジンに、以下の（Ａ－２）、（Ｂ－２）、（Ｄ－２）、（Ｅ－２）又は（Ｆ－２）に示すポリペプチド又はそれを含む細胞、前記細胞の調製物若しくは前記細胞を培養して得られた培養液を接触させて、（２Ｓ，５Ｒ）－５－ヒドロキシ－Ｌ－リジンを生成させることを特徴とする、（２Ｓ，５Ｒ）－５－ヒドロキシ－Ｌ－リジンの製造方法：
（Ａ－２）配列番号４、２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８、１１０、１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列を有するポリペプチド；
（Ｂ－２）配列番号４、２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８、１１０、１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換及び／又は付加されたアミノ酸配列を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチド；
（Ｄ－２）配列番号２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８又は１１０に示すアミノ酸配列全長と５０％以上の配列同一性を有するアミノ酸配列を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチド；
（Ｅ－２）配列番号１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列全長と５８％以上の配列同一性を有するアミノ酸配列を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチド；（Ｆ－２）連続したアミノ酸配列のモチーフである「ＨＧＷＨＷＤＤ」；と、「ＨＥＴＭＤＸＬＷ」、「ＨＥＴＭＥＸＬＷ」、「ＨＥＴＮＤＸＬＦ」及び「ＨＥＴＮＮＸＬＦ」よりなる群から選ばれるいずれかの連続したアミノ酸配列のモチーフ；を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を保持するポリペプチド（ここで、Ｘは、Ａ、Ｒ、Ｎ、Ｄ、Ｃ、Ｑ、Ｅ、Ｇ、Ｈ、Ｉ、Ｌ、Ｋ、Ｍ、Ｆ、Ｐ、Ｓ、Ｔ、Ｗ、Ｙ又はＶのいずれかのアミノ酸を示す）。 A method for producing (2S,5R)-5-hydroxy-L-lysine, comprising contacting L-lysine with a polypeptide shown in the following (A-2), (B-2), (D-2), (E-2) or (F-2), or a cell containing the same, a preparation of the cell, or a culture solution obtained by culturing the cell, to produce (2S,5R)-5-hydroxy-L-lysine:
(A-2) a polypeptide having an amino acid sequence shown in SEQ ID NO: 4, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134;
(B-2) a polypeptide having an amino acid sequence represented by SEQ ID NO: 4, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134 in which one or more amino acids have been deleted, substituted, and/or added, and having R-selective L-lysine 5-hydroxylation activity;
(D-2) a polypeptide having an amino acid sequence having 50% or more sequence identity to the full length amino acid sequence shown in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110, and having R-selective L-lysine 5-hydroxylation activity;
(E-2) A polypeptide having an amino acid sequence having 58% or more sequence identity with the full length amino acid sequence shown in SEQ ID NO: 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134, and having R-selective L-lysine 5-position hydroxylation activity; (F-2) A polypeptide having a motif of a consecutive amino acid sequence "HGWHWDD"; and a motif of any consecutive amino acid sequence selected from the group consisting of "HETMDXLW", "HETMEXLW", "HETNDXLF" and "HETNNXLF"; and having R-selective L-lysine 5-position hydroxylation activity (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V). 　前記細胞が、前記Ｌ－リジン５位水酸化酵素をコードするＤＮＡで形質転換された細胞である、請求項１８に記載の（２Ｓ，５Ｒ）－５－ヒドロキシ－Ｌ－リジンの製造方法。 The method for producing (2S,5R)-5-hydroxy-L-lysine according to claim 18, wherein the cell is a cell transformed with DNA encoding the L-lysine 5-hydroxylase. 　前記ＤＮＡが、以下の（Ｇ－２）、（Ｈ－２）、（Ｊ－２）、（Ｋ－２）又は（Ｌ－２）である、請求項１９に記載の（２Ｓ，５Ｒ）－５－ヒドロキシ－Ｌ－リジンの製造方法：
（Ｇ－２）配列番号３、２３、２５、２７、２９、３１、３３、３５、３７、３９、４１、４３、４５、４７、４９、５１、５５、６７、６９、７１、７３、７５、７９、８１、８３、８５、８７、８９、９１、９３、９５、９７、９９、１０１、１０３、１０５、１０７、１０９、１１１、１１３、１１５、１１９、１２１、１２３、１２５、１２７、１２９、１３１又は１３３に示す塩基配列を含むＤＮＡ；
（Ｈ－２）配列番号３、２３、２５、２７、２９、３１、３３、３５、３７、３９、４１、４３、４５、４７、４９、５１、５５、６７、６９、７１、７３、７５、７９、８１、８３、８５、８７、８９、９１、９３、９５、９７、９９、１０１、１０３、１０５、１０７、１０９、１１１、１１３、１１５、１１９、１２１、１２３、１２５、１２７、１２９、１３１又は１３３に示す塩基配列において、１又は複数個の塩基が置換、欠失、及び／又は付加された塩基配列を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ；
（Ｊ－２）配列番号２３、２５、２７、２９、３１、３３、３５、３７、３９、４１、４３、４５、４７、４９、５１、５５、６７、６９、７１、７３、７５、７９、８１、８３、８５、８７、８９、９１、９３、９５、９７、９９、１０１、１０３、１０５、１０７又は１０９に示す塩基配列がコードするポリペプチドと５０％以上の配列同一性を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ；
（Ｋ－２）配列番号１１１、１１３、１１５、１１９、１２１、１２３、１２５、１２７、１２９、１３１又は１３３に示す塩基配列がコードするポリペプチドと５８％以上の配列同一性を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ；
（Ｌ－２）連続したアミノ酸配列のモチーフである「ＨＧＷＨＷＤＤ」；と、「ＨＥＴＭＤＸＬＷ」、「ＨＥＴＭＥＸＬＷ」、「ＨＥＴＮＤＸＬＦ」及び「ＨＥＴＮＮＸＬＦ」よりなる群から選ばれるいずれかの連続したアミノ酸配列のモチーフ；を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチドをコードするＤＮＡ（ここで、Ｘは、Ａ、Ｒ、Ｎ、Ｄ、Ｃ、Ｑ、Ｅ、Ｇ、Ｈ、Ｉ、Ｌ、Ｋ、Ｍ、Ｆ、Ｐ、Ｓ、Ｔ、Ｗ、Ｙ又はＶのいずれかのアミノ酸を示す）。 The method for producing (2S,5R)-5-hydroxy-L-lysine according to claim 19, wherein the DNA is the following (G-2), (H-2), (J-2), (K-2) or (L-2):
(G-2) DNA comprising the base sequence shown in SEQ ID NO: 3, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133;
(H-2) DNA encoding a polypeptide having an R-selective L-lysine 5-hydroxylation activity, comprising a nucleotide sequence represented by SEQ ID NO: 3, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133, in which one or more nucleotides have been substituted, deleted and/or added;
(J-2) a DNA encoding a polypeptide having 50% or more sequence identity to a polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107 or 109, and having R-selective L-lysine 5-hydroxylation activity;
(K-2) a DNA having a sequence identity of 58% or more to a polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133, and encoding a polypeptide having R-selective L-lysine 5-hydroxylation activity;
(L-2) A DNA encoding a polypeptide having a consecutive amino acid sequence motif of "HGWHWDD" and any consecutive amino acid sequence motif selected from the group consisting of "HETMDXLW", "HETMEXLW", "HETNDXLF" and "HETNNXLF" and having R-selective L-lysine 5-hydroxylation activity (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V). 　以下の（Ａ－２）、（Ｂ－２）、（Ｄ－２）、（Ｅ－２）又は（Ｆ－２）に示すポリペプチド又はそれを含む細胞、前記細胞の調製物若しくは前記細胞を培養して得られた培養液を含み、Ｒ選択的Ｌ－リジン５位水酸化活性を有する酵素剤組成物：
（Ａ－２）配列番号４、２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８、１１０、１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列を有するポリペプチド；
（Ｂ－２）配列番号４、２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８、１１０、１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換及び／又は付加されたアミノ酸配列を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチド；
（Ｄ－２）配列番号２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５２、５６、６８、７０、７２、７４、７６、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、１０４、１０６、１０８又は１１０に示すアミノ酸配列全長と５０％以上の配列同一性を有するアミノ酸配列を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチド；
（Ｅ－２）配列番号１１２、１１４、１１６、１２０、１２２、１２４、１２６、１２８、１３０、１３２又は１３４に示すアミノ酸配列全長と５８％以上の配列同一性を有するアミノ酸配列を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を有するポリペプチド；（Ｆ－２）連続したアミノ酸配列のモチーフである「ＨＧＷＨＷＤＤ」と；「ＨＥＴＭＤＸＬＷ」、「ＨＥＴＭＥＸＬＷ」、「ＨＥＴＮＤＸＬＦ」及び「ＨＥＴＮＮＸＬＦ」よりなる群から選ばれるいずれかの連続したアミノ酸配列のモチーフと；を有し、かつ、Ｒ選択的Ｌ－リジン５位水酸化活性を保持するポリペプチド（ここで、Ｘは、Ａ、Ｒ、Ｎ、Ｄ、Ｃ、Ｑ、Ｅ、Ｇ、Ｈ、Ｉ、Ｌ、Ｋ、Ｍ、Ｆ、Ｐ、Ｓ、Ｔ、Ｗ、Ｙ又はＶのいずれかのアミノ酸を示す）。 An enzyme composition comprising a polypeptide shown in the following (A-2), (B-2), (D-2), (E-2) or (F-2), or a cell containing the same, a preparation of the cell, or a culture medium obtained by culturing the cell, and having R-selective L-lysine 5-hydroxylation activity:
(A-2) a polypeptide having an amino acid sequence shown in SEQ ID NO: 4, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134;
(B-2) a polypeptide having an amino acid sequence represented by SEQ ID NO: 4, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134 in which one or more amino acids have been deleted, substituted, and/or added, and having R-selective L-lysine 5-hydroxylation activity;
(D-2) a polypeptide having an amino acid sequence having 50% or more sequence identity to the full length amino acid sequence shown in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110, and having R-selective L-lysine 5-hydroxylation activity;
(E-2) A polypeptide having an amino acid sequence having 58% or more sequence identity with the full length amino acid sequence shown in SEQ ID NO: 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134, and having R-selective L-lysine 5-position hydroxylation activity; (F-2) A polypeptide having a consecutive amino acid sequence motif "HGWHWDD" and any consecutive amino acid sequence motif selected from the group consisting of "HETMDXLW", "HETMEXLW", "HETNDXLF" and "HETNNXLF" and having R-selective L-lysine 5-position hydroxylation activity (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V).
PCT/JP2024/035265 2023-12-25 2024-10-02 L-lysine position-5 hydroxylase, and method for producing 5-hydroxy-l-lysine using same Pending WO2025142009A1 (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004537296A (en) * 2001-06-08 2004-12-16 ロディア・シミ Stereoselective production of cyclic L-amino acids
WO2014129459A1 (en) * 2013-02-19 2014-08-28 株式会社エーピーアイ コーポレーション Production method for l-lysine hydroxylase and hydroxy-l-lysine using same, and production method for hydroxy-l-pipecolic acid

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004537296A (en) * 2001-06-08 2004-12-16 ロディア・シミ Stereoselective production of cyclic L-amino acids
WO2014129459A1 (en) * 2013-02-19 2014-08-28 株式会社エーピーアイ コーポレーション Production method for l-lysine hydroxylase and hydroxy-l-lysine using same, and production method for hydroxy-l-pipecolic acid

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KAWAI SEIJI, SUGAYA YUKO, HAGIHARA RYOTA, TOMITA HIROYA, KATSUYAMA YOHEI, OHNISHI YASUO: "Complete Biosynthetic Pathway of Alazopeptin, a Tripeptide Consisting of Two Molecules of 6-Diazo-5-oxo-L-norleucine and One Molecule of Alanine", ANGEWANDTE CHEMIE, VERLAG CHEMIE, HOBOKEN, USA, vol. 60, no. 18, 26 April 2021 (2021-04-26), Hoboken, USA, pages 10319 - 10325, XP093329852, ISSN: 1433-7851, DOI: 10.1002/anie.202100462 *

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