WO2008013262A1

WO2008013262A1 - L-leucine hydroxylase and dna encoding the enzyme

Info

Publication number: WO2008013262A1
Application number: PCT/JP2007/064761
Authority: WO
Inventors: Yasukatsu Ota; Shuichi Gomi; Koji Yanai
Original assignee: Meiji Seika Kaisha, Ltd.
Priority date: 2006-07-28
Filing date: 2007-07-27
Publication date: 2008-01-31
Also published as: JPWO2008013262A1

Abstract

Disclosed are: a novel protein having an L-leucine hydroxylase activity, i.e., an activity of converting L-leucine into (2S,4S)-5-hydroxyleucine; DNA encoding the protein; recombinant DNA containing the DNA; a transformant carrying the recombinant DNA; the protein using the transformant; and a method for producing (2S,4S)-5-hydroxyleucine. It becomes possible to provide a method for producing (2S,4S)-5-hydroxyleucine, which is a substance expected to be used as a starting material for the production of a useful substance such as a pharmaceutical agent, by utilizing an organism, an enzyme or the like. The method enables to produce (2S,4S)-5-hydroxyleucine at high efficiency.

Description

Specification

L bite isine hydroxylase and DNA encoding the enzyme

Reference to related applications

[0001] This application is based on Japanese Patent Application No. 2006-205755 (filing date: July 28, 2006), which is a prior Japanese patent application, and claims the benefit of its priority. The entire disclosure is hereby incorporated by reference.

Background of the Invention

[0002] Field of the Invention

The present invention relates to a protein having an activity to convert L-tipped isine hydroxylase, that is, L-tipped isine into (2S, 4S) -5-hydroxyleucine, DNA encoding the protein, recombinant DNA containing the DNA, The present invention relates to a transformant having recombinant DNA, a method for producing a protein having L-mouth isine hydroxylase activity using the transformant, and a method for producing (2S, 4S) -5-hydroxyleucine using the transformant. .

[0003] Minakei

Since (2S, 4S) -5-hydroxyleucine is an unnatural amino acid and an optically active isomer, it is expected to be used as a starting material in the production of various useful substances such as pharmaceuticals. As a method for producing this substance, only a chemical synthesis method has been reported, and hem.soc.PerKinΊrans.1, Organic and Bio-Organic uhemistry, 8, 2075 (1988); J. Org. Chem., 68, 83 (2003)), and as far as the present inventors know, methods for producing using organisms or enzymes have been reported so far!

[0004] (2S, 4S) — 5-Hydroxyleucine is produced in the biosynthetic pathway of polypeptides (nostop mark tolide A1 and nostop mark tolide A ² ) produced by Nostoc sp. GSV224, a kind of cyanobacteria. It is expected to exist as a synthetic intermediate (J. Org. Chem., 68, 83 (2 003); Gene, 311, 171 (2003)). However, no L-mouth isine hydroxylase gene has been identified or suggested in Nostoc SD. GSV224. In other organisms, as far as the present inventors have known, an enzyme having L-mouth icine hydroxylase activity and a gene encoding the enzyme have been reported so far. Not in.

[0005] The DNA sequence of Nostoc sp. ATCC29133, a kind of cyanobacteria, has been analyzed. However, the L-leucine hydroxylase gene has not been identified here, and its existence has been suggested so far!

Summary of the Invention

[0006] As a result of functional analysis using the DNA derived from microorganisms belonging to the cyanobacterium, specifically, the fine cattle (Nostoc SP. ATCC29133) belonging to the genus Nostoc, the present inventors have now been identified. Unexpectedly found a DNA encoding a novel L-tout isin hydroxylase that was not present and succeeded in obtaining the DNA. The present invention is based on strength and knowledge.

[0007] Therefore, the present invention provides a novel protein having L-mouth icine hydroxylase activity and DNA encoding the protein in order to obtain (2S, 4S) -5-hydroxyleucine, which can be used as a starting material in the production of various useful substances such as pharmaceuticals. And a recombinant DNA containing the DNA, a transformant carrying the recombinant DNA, the protein using the transformant, and a method for producing (2 S, 4S) -5-hydroxyleucine That is the purpose.

[0008] The protein according to the present invention is selected from the group consisting of:

(a) a protein having the amino acid sequence represented by SEQ ID NO: 1,

(b) a protein comprising an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted or added in the amino acid sequence of SEQ ID NO: 1 and having L-mouth isine hydroxylase activity; and

(c) A protein comprising an amino acid sequence having 60% or more homology with the amino acid sequence of SEQ ID NO: 1 and having L-leucine hydroxylase activity.

[0009] The DNA according to the present invention encodes the protein according to the present invention. Preferably, this DNA has the base sequence represented by SEQ ID NO: 2.

[0010] According to another embodiment, the DNA according to the present invention is selected from the group consisting of:

(0 having a base sequence represented by SEQ ID NO: 2, DNA,

(ii) DNA that has the nucleotide sequence represented by SEQ ID NO: 2 and high under stringent conditions DNA that encodes a protein that is brididized and has L-leucine hydroxylase activity,

(iii) a DNA comprising a nucleotide sequence in which one or more bases are deleted, substituted, inserted or added in SEQ ID NO: 2, and which encodes a protein having L-mouth isine hydroxylase activity; and

(iv) A DNA comprising a nucleotide sequence having 60% or more homology with the nucleotide sequence represented by SEQ ID NO: 2 and encoding a protein having L-mouth icine hydroxylase activity.

[0011] According to a preferred embodiment of the present invention, the DNA according to the present invention is derived from a microorganism belonging to the cyanobacteria (Cvanobacteria). According to a more preferred embodiment, the DNA derived from a microorganism belonging to the cyanobacteria is DNA derived from a microorganism belonging to the genus Nostock (N ^ l ^). According to a further preferred embodiment of the present invention, the microorganism belonging to the genus Nostock (N ^ l ^) is

[0012] According to another aspect of the present invention, the DNA according to the present invention encodes the protein according to the present invention, and has the base sequence represented by SEQ ID NO: 3.

[0013] According to yet another aspect of the present invention, the DNA according to the present invention is selected from the group consisting of:

(I) DNA having the base sequence represented by SEQ ID NO: 3,

(II) a DNA that hybridizes with a DNA having the base sequence represented by SEQ ID NO: 3 under a stringent condition and encodes a protein having an L-mouth isine hydroxylase activity;

(III) DNA encoding a protein consisting of a base sequence in which one or more bases have been deleted, substituted, inserted or added in SEQ ID NO: 3 and having L-mouth isine hydroxylase activity, and

(IV) A DNA comprising a nucleotide sequence having 60% or more homology with the nucleotide sequence represented by SEQ ID NO: 3 and encoding a protein having L-mouth icine hydroxylase activity.

[0014] The recombinant DNA according to the present invention is obtained by incorporating the DNA according to the present invention into a vector. [0015] The transformant according to the present invention is obtained by introducing the recombinant DNA according to the present invention into a host cell. It is. Here, the host cell is preferably Escherichia coli, and more preferably, Streptomyces lividans. Moreover, the transformant according to the present invention preferably expresses L-mouth icine hydroxylase.

[0016] The method for producing a protein having L-mouth icine hydroxylase activity according to the present invention comprises culturing the transformant according to the present invention using a medium, and adding a protein having L-portion isine hydroxylase activity in the culture. Producing, accumulating, and collecting the protein from the culture.

[0017] The method for producing (2S, 4S) -5-hydroxyleucine according to the present invention uses a culture of the transformant according to the present invention or a treated product of the culture as an enzyme source. In the aqueous medium to form and accumulate (2S, 4S) -5-hydroxyleucine in the aqueous medium, and (2S, 4S) -5-hydroxyleucine is removed from the aqueous medium. Collecting. Here, the treated product of the culture is preferably a culture concentrate, a dried product of the culture, a cell obtained by centrifuging the culture, a dried product of the cell, and a freeze-dried product of the cell. Product, surfactant-treated product of the microbial cell, ultrasonic treated product of the microbial cell, mechanically-ground product of the microbial cell, solvent-treated product of the microbial cell, enzyme-treated product of the microbial cell, the bacterium A protein fraction of the body, an immobilized product of the cell, or an enzyme preparation obtained by extraction from the cell.

[0018] The present invention can also be as follows:

(1) a protein having the amino acid sequence represented by SEQ ID NO: 1;

(2) The protein according to (1), which has L-sip Icin hydroxylase activity;

(3) a protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1 and having L-mouth icine hydroxylase activity;

(4) DNA encoding the protein according to any one of (1) to (3)!

(5) The DNA according to (4) having the base sequence represented by SEQ ID NO: 2;

(6) the DNA according to (4) having the base sequence represented by SEQ ID NO: 3; and

(7) A DNA that hybridizes with a DNA having the nucleotide sequence represented by SEQ ID NO: 2 or 3 under a stringent condition and encodes a protein having L-tissue isine hydroxylase activity. [0019] (2S, 4S) -5-Hydroxyleucine is an unnatural amino acid and an optically active isomer, and is expected to be used as a starting material in the production of various useful substances such as pharmaceuticals. 2S, 4S) — An efficient production method for 5-hydroxyleucine is strongly desired. According to the present invention, it is possible to obtain a large amount of L-sip Icin hydroxylase. By using the enzyme obtained by the present invention, (2S, 4S) -5-hydroxyleucine can be efficiently produced.

Brief Description of Drawings

[0020] FIG. 1 shows a process for constructing an L-mouth icine hydroxylase expression plasmid pnos5A.

In the figure of the present application, “Amp” is an ampicillin resistance gene, “Km” is a kanamycin resistance gene, “Apr” is an apramycin resistance gene, “nos5” is a leucine hydroxylase gene, “nos5-GC”. Represents the leucine hydroxylase gene (chemical synthesis) and “tipAp” represents the tipA promoter.

[FIG. 2] FIG. 2 shows the construction process of L-nose isine hydroxylase expression plasmid pnos5D constructed by constructing a chemically synthesized gene fragment.

FIG. 3 shows the construction process of L-mouth icine hydroxylase expression plasmid pnos5E. DETAILED DESCRIPTION OF THE INVENTION

[0021] L mouthful isine hydroxylase

The L-mouthed isine hydroxylase according to the present invention includes a protein consisting of the amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence substantially equivalent to the amino acid sequence represented by SEQ ID NO: 1, and It is possible to list proteins with hydroxylase activity.

[0022] Here, "substantially equivalent amino acid arrangement IJ" means the ability to modify one or more amino acid deletions, substitutions, additions and / or modifications due to insertions. An amino acid sequence that does not give The number of amino acid residues to be modified is preferably !!-40, more preferably 1-20, still more preferably 1-8, and most preferably 1-4.

[0023] Here, "activity of the polypeptide" means having L-mouth icine hydroxylase activity. “Having L-leucine hydroxylase activity” means having an activity capable of converting L-leucine into (2S, 4S) -5-hydroxyleucine. To this activity About it, it can measure according to a conventional method, for example, it can measure according to the method as described in the Example mentioned later.

[0024] Examples of! /, "Does not affect activity! /, Modification" in the present invention include conservative substitutions. “Conservative substitution” means the replacement of one or more amino acid residues with another chemically similar amino acid residue so as not to substantially alter the activity of the polypeptide. For example, when one hydrophobic amino acid residue is substituted with another hydrophobic amino acid residue, one polar amino acid residue is substituted with another polar amino acid residue having the same charge, and the like. Functionally similar amino acids capable of making such substitutions are known in the art for each amino acid. Specific examples include non-polar (hydrophobic) amino acids such as alanine, norine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Examples of polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Examples of positively charged (basic) amino acids include arginine, histidine, and lysine. Examples of negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0025] It should be noted that amino acid deletion, substitution, addition and / or modification by, for example, DNA coding for it, for example, site-directed mutagenesis (for example, Nucleic Acid Research, Vol.10, No.20, p.6487-6500, 1982). Other methods include treating the gene with a mutagen, and selectively cleaving the gene, then removing, appending, inserting and / or replacing selected nucleotides, and ligating at the next level. and so on.

[0026] The above "substantially equivalent amino acid arrangement IJ" includes those in which the N-terminus (amino terminus) and C-terminus (carboxyl terminus) are further altered or modified! /. Motole. For example, C-terminal carboxyl is carboxylate (—COO—), amide (—CONH

2

) Or an ester (one COOR). Here, examples of R include a linear, branched or cyclic C1-6 alkyl group, a C6-12 aryl group, and the like.

The “amino acid sequence substantially equivalent to the amino acid sequence represented by SEQ ID NO: 1” can include an amino acid sequence having 60% or more homology with the amino acid sequence of SEQ ID NO: 1. Here, the homology ratio is preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, even more preferably 95% or more, particularly preferably 98% or more, and most preferably 99%. % Or more. In the present specification, “homology” may be used to mean “identity”.

[0028] In the present specification, any value of "homology" may be any value calculated using a homology search program known to those skilled in the art. For example, the homology algorithm BLAST (Basic local alignment search tool) (available from the National Center for Biotechnology Information (NCBI) (available from http://www.ncbi.nlm.nih.gov/BLAST/)), using the default (initial setting) parameters That power S.

[0029] DNA encoding L-tissue isine hydroxylase

The DNA according to the present invention includes DNA encoding the protein according to the present invention, DNA consisting of the base sequence represented by SEQ ID NO: 2 or 3, or DNA consisting of the base sequence represented by SEQ ID NO: 2 or 3. And a DNA that hybridizes under stringent conditions and encodes a protein having L-mouth isine hydroxylase activity.

In the present specification, “DNA” (deoxyribonucleic acid) may be used as a polynucleotide, if necessary.

[0030] Here, "DNA that hybridizes under stringent conditions" refers to, for example, a DNA according to the present invention such as a DNA having the base sequence represented by SEQ ID NO: 2 or a partial DNA fragment thereof As used herein, it means DNA obtained by using a colony “nobly hybridization method, a plaque” nobly hybridization method, or the like. Specifically, as this DNA, using a filter on which DNA derived from colonies or plaques is immobilized, 0.7 to; 1. Hybridizer at 65 ° C. in the presence of Omol / L sodium chloride. After conducting the chilling, use 0.;! To 2 times SSC solution (composition of 1 times SSC solution consists of 150mmol / L sodium chloride and 15mmol / L sodium quenate) at 65 ° C Examples include DNA that can be identified by washing the filter under conditions. Hybridization includes molecular 'cloning 2nd edition, current'protocols' in 'molecular' neuron 1, DNA Cloning 1: Core echniques, A Practical Approach, second Editi. on, Oxford University (1995) and the like.

[0031] The hybridizable DNA is specifically a DNA having at least 60% homology to the base sequence represented by SEQ ID NO: 2, for example, when calculated based on the above algorithm BLAST, preferably Is a DNA having a homology of 70% or more, more preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, particularly preferably 98% or more, and most preferably 99% or more. be able to.

For this reason, the DNA according to the present invention includes, for example, a protein having a nucleotide sequence having 60% or more homology with the nucleotide sequence IJ represented by SEQ ID NO: 2 or 3, and having L-mouth isine hydroxylase activity. DNA encoding can be included.

[0032] The DNA according to the present invention also includes a protein having a base sequence in which one or a plurality of bases are deleted, substituted, inserted or added in SEQ ID NO: 2 or 3, and having L-mouth icin hydroxylase activity. DNA encoding can also be included. Here, the number of bases that may be deleted, substituted, inserted or added is preferably 1 to 30, more preferably;! To 20, more preferably 1 to 15; even more preferably 1 to 10; most preferably 1 to 5. The substitution mentioned here includes substitutions involving amino acid translation mutations and substitutions involving amino acid translation mutations! /, Substitutions! /, And deviations.

[0033] Identification of L-tout Icin hydroxylase

In Gene, 311,171 (2003), during the biosynthetic pathway of the polypeptides (nostop mark tolide A1 and nostop mark tolide A2) produced by Nostoc SD. GSV224, a rare cyanobacteria, (2S, 4S) — 5— Hydroxyleucine is expected to exist as a biosynthetic intermediate. However, reports and suggestions regarding the identification of the L-leucine hydroxylase gene have been reported in Nostoc sp. GSV224! /

[0034] On the basis of the possibility of via (2S, 4S) -5-hydroxyleucine in the biosynthetic pathway and the sequence information of Nostoc sp. GSV224, microorganisms belonging to the genus Nostoc Was analyzed to identify L-sip Icin hydroxylase. That is, the present invention was completed by predicting that an ORF whose function is unknown contributes to hydroxylation of leucine and isolating and obtaining a highly homologous DNA sequence in Nostoc sp. ATCC29133 strain.

[0035] ¾ ^ of DNA encoding L-leucine hydroxylase The DNA encoding the L-mouth icine hydroxylase of the present invention can be isolated from a microorganism belonging to the genus Nostoc, preferably Nostoc sp. ATCC29133, by the following method, for example. It can also be artificially synthesized chemically based on the disclosed sequence of Nostoc sp. ATCC29133.

[0036] A microorganism belonging to the genus Nostoc can be cultured by a known method [for example, Microbiology, 140, 3333 (1994)]. Furthermore, the chromosomal DNA of the microorganism can be isolated and purified from the cultured cells by a known method (for example, Current 'Protocols' Molecular' biology). It is also commercially available from Nostoc sp. ATCC 2913 Luchier Collection.

[0037] A genomic DNA library of microorganisms belonging to the genus Nostock (M ^ Sl ^) is prepared by digesting genomic DNA with an appropriate restriction enzyme and ligating it with an appropriate vector. As the vector, for example, various vectors such as a plasmid vector, a phage vector, a cosmid vector, and a BAC vector can be used.

[0038] Next, an appropriate probe is prepared based on the base sequence of the DNA encoding L-mouth isine hydroxylase disclosed in the present specification, and a desired kanamycin biosynthetic gene is generated by a hybrid DNA hybridization. The ability to isolate DNA fragments containing In addition, a primer for amplifying a desired gene was prepared based on the base sequence of the DNA encoding L-mouth isine hydroxylase disclosed in the present specification, and a microorganism belonging to the genus Nostock (H ^ £) was prepared. The ability to isolate the desired gene can be achieved by performing PCR using genomic DNA as a saddle and ligating the amplified DNA fragment with an appropriate vector. Furthermore, DNA encoding the L-tout isin hydroxylase according to the present invention is contained in plasmid pnos5 / pET24 and plasmid pnos5—GCl / pTA2 and pn os5—GC2 / pTA2. It can be used as In addition, a desired DNA fragment can be prepared from these plasmids using an appropriate restriction enzyme.

[0039] Deposit of microorganisms

Escherichia coli transformed with plasmid pnos5A (Escherichia coli BL 21 (DE3) / pnos5A) is the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (T305-8566, Tsukuba, Ibaraki, Japan) on June 16, 2006 (original deposit date). Deposited at East 1-chome, 1st Central 6). The accession number is FERM BP-10845. Escherichia coli transformed with the plasmid pnos5D (Escherichia coli BL 21 (DE3) / pnos5D) is an independent administrative institute for industrial technology research on June 16, 2006 (original deposit date). Deposited at the Institute for Patent Biological Deposits (T305-8566, Tsukuba Pass East 1-chome, 1st, 6th, Ibaraki, Japan). The accession number is FERM BP-10846. Streptomyces lividans (Streptomyces lividans / pnos5E), which has been transformed with the plasmid pnos5E, is an independent research institute for industrial technology as of June 16, 2006 (original deposit date). Deposited at the Institute for Patent Biological Deposits (T305-8566, Tsukuba Pass East 1-chome, 1st, 6th, Ibaraki, Japan). The accession number is FERM BP-10847.

[0040] Method for producing L-tissue isine hydroxylase

The L-tout isin hydroxylase according to the present invention is produced by the L-tout isin hydroxylase of the present invention according to the method described in Molecular 'Cloning 2nd Edition, Force Rent' Protocols 'In'Molecular' Biology etc. Can be produced by expressing the DNA encoding in a host cell.

[0041] Based on the DNA of the present invention, if necessary, a DNA fragment of an appropriate length containing a portion encoding the protein is prepared. In addition, the production rate of the protein can be improved by substituting the base sequence of the portion encoding the protein so that the codon is optimal for host expression.

[0042] Recombinant DNA is prepared by inserting the DNA fragment downstream of the promoter of an appropriate expression vector.

[0043] A transformant producing the protein of the present invention can be obtained by introducing the recombinant DNA into a host cell suitable for the expression vector.

[0044] As host cells, any bacteria, actinomycetes, yeast, animal cells, insect cells, plant cells, etc. can be used as long as they can express the target gene. Preferred are Streptomyces Strei3tomvces and Escherichia coli, and more preferred are Streptomyces eQtomv_ces. [0045] As the recombinant DNA, one that can autonomously replicate in the above host cell or can be integrated into a chromosome and contains a promoter at a position where the DNA of the present invention can be transcribed is used.

[0046] When a prokaryotic organism such as a bacterium is used as a host cell, the recombinant DNA containing the DNA encoding the protein of the present invention is capable of autonomous replication in the prokaryotic organism, and at the same time has a promoter and ribosome binding. It is preferably a recombinant DNA composed of the DNA of the present invention, a transcription termination sequence. In addition, the recombinant DNA contains a gene that controls the promoter.

[0047] Expression vectors include pTA2 (manufactured by Toyobo Co., Ltd.), Helixl (manufactured by Roche Diagnotex), PKK233-2 (manufactured by Amersham. Pharmacia Biotech), pSE280 (manufactured by Invitrogen), pGEMEX-1 (Promega), pQE-8 (Qiagen), pET24 (Novagen), pBluescript II SK), pBluescript II SK (-) (Stratagene), pUC19 [Gene, 33, 103 (1985) )], PSTV28 (manufactured by Takara Shuzo Co., Ltd.), pUC118 (manufactured by Takara Shuzo Co., Ltd.), pIJ6902 [Mol. Microbiol., 58, 1276 (2005)], pSETl 52 [J. Mol. Mic robiol. Biotechnol., 4, 417 (2002)]. PTA2, pET 24, pIJ6902, pUC118, pSET152 and the like are preferable, and (pTA2, pET 24, pIJ6902 and the like are more preferable.

[0048] Any promoter can be used as long as it can function in a host cell. For example, trp promoter (Ptrp), lac promoter (Plac), T7 promoter, PL promoter, PR promoter, promoter derived from E. coli such as PSE promoter, phage, etc., SP01 promoter, SP02 promoter, penP promoter, tipA promoter [ J. Bacteriol., 171, 1459 (1989)], erm E * promoter [Gene, 38, 215 (1985)] and the like. Artificially designed and modified promoters such as a promoter in which two Ptrps are connected in series (Ptrp X 2), tac promoter, lacT7 promoter, let I promoter, and the like can also be used. Preferred examples include lac promoter (Plac), ermE * promoter, T7 promoter, tipA promoter and the like, and more preferred examples include lac promoter (Plac) and ermE * promoter. [0049] In the present invention, a plasmid in which the distance between the Shine-Dalgamo sequence, which is a ribosome binding sequence, and the initiation codon is adjusted to an appropriate distance (for example, 6 to 18 bases) is used as a vector. I like it! /

[0050] In the recombinant DNA of the present invention, a transcription termination sequence is not necessarily required for the expression of the DNA of the present invention, but it is preferable to place the transcription termination sequence immediately below the structural gene.

Yes

[0051] Prokaryotes include microorganisms belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Streptomyces, etc., for example, Χ_ί, Escherichia coli XLl_Blue, Escherichia coli XL2_Blue, Escheric hia coli DH1, Escherichia coli MC1000, Escherichia coli KY3726, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli BL21 (DE 3), Escherichia coli Νο · ichi, 3 coli NY49, Serratia ficaria. Serratia fonticola. Serratia liauefaciens. Serratia marcescens. Bacillus subtili s Serratia amvloliquefaciens. This is That. Preferred examples include Escherichia coli BL21 (D E3), Escherichia coli JM109, Streptomvces lividans and the like, and more preferred are Escherichia coli JM109 and Streptomvces lividans.

[0052] As a method for introducing recombinant DNA, any method can be used as long as it is a method for introducing DNA into the host cell. For example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69 , 2110 (1972)], protoplast method (Japanese Patent Laid-Open No. 63-248394), electo-poration method [Nucleic Acids Res., 15, 6127 (1988)], Conjugation method [J. Bacteriol., 171, 3585 (1989)].

[0053] Here, a case where a yeast strain is used as a host cell will be described as an example.

When yeast strains are used as host cells, YEpl 3 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, pHS15, etc. can be used as an expression vector. [0054] Any promoter can be used as long as it functions in a yeast strain. For example, PH05 promoter, PKG promoter, GAP promoter, ADH promoter, gal 1 promoter, gal 10 promoter, heat shock The ability to list promoters such as polypeptide promoters, MF a 1 promoters, CUP 1 promoters, etc.

[0055] As the host cell, yeast strains belonging to the genus Saccharomyces, Schizosaccharomyces, Kluybe mouth mouth, Genus Trichosporon, Sisniomyces, Pichia, Candida, etc. are used.ュ, saccharomyces cervisiae, Shizosaccharomyces j) ombe, luvveromvces lactis. TricnosDoron pullulans ^^ chwanniomvces alluvius., Pich ia pastoris, Candida utilis.

[0056] As a method for introducing recombinant DNA, any method can be used as long as it introduces DNA into yeast. For example, the Elect Mouth Position Method [Methods Enzymol., 194, 182 (199 0)], It is possible to cite the ferroplast method [? ^ Ji ^ & yoji & (1.5 (115, 81, 4889 (1984)]), lithium acetate method [J. Bacteriol., 153, 163 (1983)].

[0057] The method of culturing the transformant of the present invention can be carried out according to a usual method used for culturing a host.

[0058] As a medium for cultivating transformants obtained by using prokaryotes such as E. coli 'actinomycetes or eukaryotes such as yeast as a host, carbon sources, nitrogen sources, inorganic salts, etc. that can be assimilated by the organism Any of a natural medium and a synthetic medium may be used as long as the medium can contain the yeast and can efficiently culture the transformant.

[0059] As the carbon source, as long as the organism can be assimilated, glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, acetic acid, propionic acid, etc. Organic acids, alcohols such as ethanol, propanol and the like can be used.

[0060] Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, and other ammonium-containing organic salts, other nitrogen-containing compounds, peptone, meat extract, yeast Extract, corn steep liquor, casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermentation cells, and their digestion A thing etc. can be used.

[0061] As the inorganic salt, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like can be used.

[0062] The culture is preferably performed under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is preferably 15 to 40 ° C, and the culture time is usually 5 hours to 7 days. During the cultivation, the pH is maintained at 3.0 · 9.0. The pH is adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, etc.

[0063] In addition, antibiotics such as ampicillin, kanamycin, apramycin, thiostreptone, tetracycline, chloramphenicol, streptomycin and the like may be added to the medium as needed during the culture.

[0064] When cultivating a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when cultivating a microorganism transformed with an expression vector using the lac promoter, use isopropyl 13D thiogalatatopyranoside or the like when culturing a microorganism transformed with an expression vector using the trp promoter. When cultivating a microorganism transformed with an expression vector using the tip A promoter, for example, tistrepton may be added to the medium.

[0065] As described above, a transformant derived from a microorganism having a recombinant DNA incorporating a DNA encoding the protein of the present invention is cultured according to a normal culture method, and the protein is produced and accumulated, and the culture By collecting the protein, it is possible to produce the protein.

[0066] As a method for producing the protein of the present invention, there are a method of producing in the host cell, a method of secreting it outside the host cell, and a method of producing it on the host cell outer membrane.

[0067] As a method for isolating and purifying the protein produced by the transformant of the present invention, usual enzyme isolation and purification methods can be used.

[0068] For example, when the protein of the present invention is produced in a dissolved state in cells, the cells are collected by centrifugation after culturing, suspended in an aqueous buffer solution, ultrasonically disrupted, French Cells are disrupted with a loess, mantongaurine homogenizer, dynomill, etc. to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, an ordinary enzyme purification method, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, a jetylamino Anion exchange chromatography using resin such as ethyl (DEAE) -cephalose, DIAION HPA_75 (manufactured by Mitsubishi Kasei Co., Ltd.), cation exchange chromatography using resin such as SS sign arose FF (manufactured by Pharmacia) Methods such as hydrophobic chromatography using resin such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, isoelectric focusing, etc. Use a technique such as electrophoresis, alone or in combination, to obtain a purified product.

[0069] When the protein is produced as an insoluble substance in the cell, the protein is recovered by a usual method from the precipitate fraction obtained by centrifugation after disrupting the cell in the same manner as described above. The protein insoluble matter is solubilized with a protein denaturant. The solubilized solution is diluted or dialyzed into a solution that does not contain a protein denaturing agent or is so thin that the concentration of the protein denaturing agent does not denature the protein, and the protein is formed into a normal three-dimensional structure. A purified sample can be obtained by the same isolation and purification method.

[0070] When a derivative of the protein of the present invention or a modified sugar chain thereof is secreted and produced extracellularly, the derivative of the protein or a sugar chain adduct can be recovered in the culture supernatant. . That is, a culture supernatant is obtained by treating the culture by a method such as centrifugation as described above, and a purified standard is obtained from the culture supernatant by using the same isolation and purification method as described above. Power to get goods S.

[0071] In addition, when the protein of the present invention is produced in a dissolved state in cells and the enzyme activity cannot be obtained with the cell-free extract, the cells are collected by centrifugation after the completion of the culture, After suspending cells in an aqueous buffer solution to produce quiescent cells, the solution of quiescent cells can be directly subjected to enzyme reaction.

[0072] (2S. 4S) — Method for Producing 5 Hydroxyleucine

Using the culture of the transformant according to the present invention and the treated product of the culture as an enzyme source, the enzyme source and L-sip Icin are present in an aqueous medium, and the (2S, 4S) Production of 5-hydroxyleucine and accumulation to produce (2S, 4S) — 5-hydroxyleucine.

[0073] Here, the treated product of the culture includes, for example, a concentrate of the culture, a dried product of the culture, a cell obtained by centrifuging the culture, a dried product of the cell, Lyophilized product of body, surfactant-treated product of the cell, ultrasonically treated product of the cell, mechanically ground product of the cell, solvent-treated product of the cell, enzyme treatment of the cell Products, protein fractions of the cells, immobilized products of the cells, enzyme preparations obtained by extraction from the cells, and the like.

[0074] (2S, 4S) — Aqueous media that can be used to produce 5-hydroxyleucine include buffers such as water, phosphates, carbonates, acetates, borates, kenates, tris, Examples thereof include alcohols such as methanol and ethanol, esters such as ethyl acetate, ketones such as acetone, and amides such as acetate amide. In addition, a culture solution of a microorganism used as an enzyme source can be used as an aqueous medium.

[0075] In the production of (2S, 4S) -5-hydroxyleucine, a surfactant or an organic solvent may be added as necessary. Surfactants include nonionic surfactants such as polyoxyethylene 'octadecylamine (for example, Naimine S-215, manufactured by Nippon Oil & Fats Co., Ltd.), cetyl trimethyl ammonium' bromide and alkyldimethyl 'benzyl ammonium chloride (for example, Cationic surfactants such as cation F2-40E (manufactured by NOF Corporation), anionic surfactants such as lauroyl sarcosinate, and tertiary amines such as alkyldimethylamine (eg, tertiary amine FB, NOF Corporation) As long as it can promote the production of (2S, 4S) -5-hydroxyleucine, one kind or a mixture of several kinds may be used. Surfactants are usually used at a concentration of 0.;!-50 g / l. Examples of the organic solvent include xylene, toluene, fatty acid alcohol, acetone, ethyl acetate and the like, and they are usually used at a concentration of 0.;! To 50 ml / l.

[0076] (2S, 4S) -5-Hydroxyleucine is formed in an aqueous medium at pH 5 to 10, preferably pH 6 to 8 and 20 to 50 ° C for 1 to 96 hours. In the production reaction, a carbon source such as glycerol or an inorganic salt such as MnCl can be added as necessary.

2

[0077] Quantification of (2S, 4S) -5-hydroxyleucine produced in an aqueous medium can be performed using high performance liquid chromatography (HPLC) or the like. [0078] Isolation and purification of (2S, 4S) -5-hydroxyleucine produced in the reaction solution can be carried out by a usual method using activated carbon, ion exchange resin, preparative HPLC or the like. Example

[0079] Hereinafter, the present invention will be described in detail with reference to the following examples. The present invention is not limited to these examples.

[0080] ¾i: Nosutsk ろろ牛物物一イシンシンシンシンシン

Genomic DNA of Nostoc SD. ATCC29133 was obtained from ATCC (American 'Type' Culture 'Collection) as a commercial product (ATCC29133D). Primers consisting of the nucleotide sequences represented by SEQ ID NOs: 4 and 5 were synthesized, and PCR was performed using the genomic DNA as a saddle. PCR is chromosomal DNAO. 1〃 g, each primer 0 · δ, πιοΐ / ΐ, LA Taq DNA polymerase (Takara Shuzo Co., Ltd.) 2.5 units, X 10 buffer for LA Taq DNA polymerase 5 a 1, deoxyNTP 250 μm each The reaction was carried out 30 times using 95 µC for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 1 minute using 50 µl of the reaction solution containing mol and MgC122.5 mM. 1/10 volume of the reaction mixture was subjected to agarose electrophoresis, and after confirming that the DNA fragments were amplified, the remaining reaction mixture was purified using the High Pure PCR Product Purification Kit (Roche) kit. .

[0081] Using the obtained purified DNA solution 101, the DNA was cleaved with restriction enzymes Ndel and BamHI, and 1.7 kb DNA was separated by agarose electrophoresis. Then, the DNA was recovered from the agarose gel according to a conventional method.

[0082] About 0.2 g of pET24 DNA (manufactured by Novagen) was cut with restriction enzymes Ndel and BamHI, and the DNA fragment was separated by agarose electrophoresis to recover the 5.3 kb DNA fragment.

[0083] Using a ligation kit (Takara Shuzo Co., Ltd.), the fragments of 1.7 kb and 5.3 kb were subjected to ligation reaction at 16 ° C for 16 hours. Escherichia coli JM109 strain (manufactured by Takara Shuzo Co., Ltd.) was transformed using the ligation reaction solution according to the above-mentioned known method, and the transformant was transformed into LB agar medium containing 30 g / ml kanamycin [Batatryptone (Difco). And 10 g / l fermented mother extract (Difco), 10 g / l sodium chloride, 5 g / l sodium chloride, 15 g / l agarose, and then cultured at 37 ° C. The grown colonies are purified according to the aforementioned known method. And pn osA, a recombinant plasmid containing the leucine hydroxylase gene nos5, was obtained. The construction procedure and structure of the plasmid are shown in FIG.

[0084] Example 2: Chemical synthesis of L-tout Icin hydroxylase gene

AA-1 (SEQ ID NO: 6), AA-2 (SEQ ID NO: 7), AA—3 (SEQ ID NO: 8), AA—4 (SEQ ID NO: 9), BB—1 (SEQ ID NO: 10), BB—2 (sequence) No. 11), BB-3 (SEQ ID NO: 12), and BB-4 (SEQ ID NO: 13) were synthesized as eight primers. Of these, two were selected and PCR was performed using the primer set as a vertical DNA.

[0085] First, primer-type (AA-1 and AA-2) 0 · δ ^ ΐηοΙ / Ι ΚΟϋ —Plus-Polymerase (Toyobo Co., Ltd.) 1. Ounits, KOD—plus—X for polymerase 10 Slow impact 5 1, reaction containing deoxyNTP ^ 200 μmol, MgSO ImM

Four

The process was repeated 15 times at 30 ° C for 30 seconds, 60 ° C for 30 seconds and 68 ° C for 1 minute. For other primer sets {(AA-3, AA-4), (BB-1, l, BB-2), (BB-3, BB-4)}, PCR was performed in the same manner, and a total of four PCR reaction solutions were used. Got.

[0086] Next, the PCR reaction was performed again using the obtained reaction solution 2.5 1 as a shared primer set and cage. The reaction conditions are the above reaction solution 2.51 for the primer-and-cum-type (AA-1 and AA-2), the above reaction solution 2 · 51 and the one for the primer-and-cum-type (AA-3 and AA-4), KOD — Plus—Polymerase (manufactured by Toyobo Co., Ltd.) 1.0 units, KOD—plus—Polymerase X 10 Slow night 5 a 1, deoxyNTP 200 μmol each, MgSO ImM reaction ί 夜 50 μ 1

Four

The process was repeated 15 times at 94 ° C for 30 seconds, 60 ° C for 30 seconds and 68 ° C for 1 minute. In the same way, change the primer / combination type to the above reaction solution of (BB-1, BB-2) and the above reaction solution of (BB-3, BB-4) and perform the PCR reaction for a total of two types of reactions. A liquid was obtained.

[0087] The reaction solution obtained by PCR was incorporated into E. coli plasmid pTA2 using a TArget Clone ™ -Plus- kit (manufactured by Toyobo Co., Ltd.). That is, 91 PCR reaction solution and 1 μl of 10 X A-attachment Mix (manufactured by Toyobo Co., Ltd.) were added and stirred and reacted at 60 ° C for 10 minutes. Add the reaction mixture 2.5 1 and 51¾ / 1 1-8 vector (Toyobo Co., Ltd.) 1 1 and use the Ligation Kit (Takara Shuzo Co., Ltd.) to make the final reaction volume ΙΟ Ι The ligation reaction was carried out at 16 ° C for 16 hours. Using the ligation reaction solution, E. coli J M109 (Takara Shuzo Co., Ltd.) was transformed, and the transformant was applied to an LB agar medium containing ampicillin 50 [Ig / ml] and cultured at 37 ° C. A plasmid was extracted from the grown colonies according to a conventional method. From the PCR fragments derived from AA-1, AA-2, AA-3, AA-4 (SEQ ID NOs: 6, 7, 8, 9), plasmid pA-8 was transformed into BB-1, BB-2, BB-3, Plasmid pB-5 was obtained from a PCR fragment derived from BB-4 (SEQ ID NOs: 10, 11, 12, and 13). These plasmids were digested with the restriction enzyme Mrol and subsequently ligated with ligation to obtain a plasmid (pnos5B) containing a DNA fragment (nos 5—GC) consisting of the base sequence shown in SEQ ID NO: 3.

[0088] Next, by carrying out PCR using the plasmid pnos5B obtained above as a cage and using the synthetic DNA (SEQ ID NOs: 14 and 15) as a primer set, Ndel and Bglll restriction enzyme sites were used. A DNA fragment having a terminal was amplified. That is, PCR is about 0.1 g of rod-shaped DNA (plasma pnos5B), each primer 0 · S ^ mol / l, KOD —Plus—Polymerase (Toyobo Co., Ltd.) 1 · 0 units, KOD—plus—polymerase Use reaction solution 50 1 containing X 10 buffer 5 μ1, deo xyNTP ^ 200 _i mol, MgSO ImM for 15 seconds at 94 ° C.

Four

The process was repeated 25 times at 55 ° C for 30 seconds and 68 ° C for 1 minute.

[0089] The reaction solution obtained by PCR was incorporated into Escherichia coli plasmid pTA2 using a TArget Clone ™ -Plus- kit (manufactured by Toyobo Co., Ltd.). That is, 91 PCR reaction solution and 1 μl of 10 X A-attachment Mix (manufactured by Toyobo Co., Ltd.) were added and stirred and reacted at 60 ° C for 10 minutes. Add the reaction liquid 2.5 1 and 51¾ / 1 Ding Hachi 2 Vector (Toyobo Co., Ltd.) 1 11 1 and use Ligation Kit (Takara Shuzo) to make the final reaction volume 10 1 The ligation reaction was carried out at 16 ° C for 16 hours. Subsequently, Escherichia coli JM109 (manufactured by Takara Shuzo Co., Ltd.) was transformed, and the transformant was applied to an LB agar medium containing 50 g / ml of ampicillin and cultured at 37 ° C. overnight. A plasmid was extracted from the grown colonies according to a conventional method, and a recombinant plasmid pnos5C was obtained. The construction method and structure of the plasmid are shown in FIG.

[0090] The obtained recombinant plasmid pnos5C was digested with restriction enzymes Ndel and Bglll, and 0.8 kb of DNA was separated by agarose gel electrophoresis. Then, the DNA was recovered from the agarose gel according to a conventional method. [0091] About 0 · of pET24 DNA (manufactured by Novagen) was cut with restriction enzymes Ndel and BamHI, and the DNA fragments were separated by agarose gel electrophoresis to recover the 5.3 kb DNA fragment.

[0092] Using the ligation kit (Takara Shuzo Co., Ltd.), the above 0/8 kb and 5.3 kb fragments were subjected to ligation reaction at 16 ° C for 16 hours. Escherichia coli BL21 (D E3) strain was transformed with the ligation reaction solution, and the transformant was applied to an LB agar medium containing 30 g / ml of kanamycin and cultured at 37 ° C. overnight. Plasmids were extracted from the grown colonies according to the above-mentioned known method to obtain pnos5D, a recombinant plasmid containing the leucine hydroxylase gene nos5. The construction procedure and structure of the plasmid are shown in FIG.

[0093] Example 3: Construction of a plasmid mos5E

A recombinant plasmid that functions in mvces ikite was implemented by the following method.

[0094] The recombinant plasmid pnos5C obtained in Example 2 was cleaved with restriction enzymes Ndel and Bglll, and the DNA was recovered from an agarose gel according to a conventional method. In addition, about 0.2 g of plj 6902 DNA was cleaved with restriction enzymes Ndel and BamHI, and DNA fragments were separated by agarose gel electrophoresis. Similarly, an about 7.3 kb DNA fragment was recovered from an agarose gel.

[0095] Using the ligation kit (Takara Shuzo Co., Ltd.), the fragments of 0.8 kb and about 7.3 kb were subjected to ligation reaction at 16 ° C for 16 hours. Escherichia coli JM109 (Takara Shuzo Co., Ltd.) was transformed with the ligation reaction solution, and the transformant was applied to an LB agar medium containing abramycin lO ^ g / ml, and then incubated at 37 ° C. Cultured. A plasmid was extracted from the grown colony according to a conventional method to obtain pnos5E, a recombinant plasmid containing the leucine hydroxylase gene nos5. The construction procedure and structure of the plasmid are shown in FIG.

Example 4: Production of (2S, 4S) -5-hydroxyleucine by Escherichia coli transformant (E. coli / ipnos5A strain)

The recombinant plasmid pnos5A containing the leucine hydroxylase gene nos5 obtained in Example 1 was transformed into Escherichia coli BL21 (DE3) (manufactured by Novagen) and assembled. A modified Escherichia coli (E. coli / pn _OS 5A strain) was obtained. The resulting recombinant Escherichia coli was inoculated into an Erlenmeyer flask containing lOOmL of LB medium containing 30 g / ml of kanamycin and cultured at 37 ° C for 18 hours. The culture broth was inoculated at 1% into a conical flask containing lOOmL of LB medium containing 30 g / ml of kanamycin, and cultured at 37 ° C for 6 hours. Then, the culture broth lOOmL was centrifuged to obtain wet cells. .

[0097] The obtained Escherichia coli recombinant (E.coli / pnos5A strain) wet microbial cell is composed of lOOmmol / 1 sodium phosphate buffer ί 夜 (ρΗ7.0), 10% glyceronole, 10mmol / l L-leucine 20 The suspension was suspended in mL of the reaction solution, and the reaction solution was transferred to an Erlenmeyer flask and reacted at 28 ° C for 72 hours.

[0098] After completion of the reaction, the reaction product was analyzed using high performance liquid chromatography (HPLC) (manufactured by Shimadzu Corporation) under the following analytical conditions, and about 3 mmol / l (2S , 4S)-5-Hydroxyleucine was confirmed to be generated and accumulated!

Analysis conditions:

Kahum: L— column

Mobile phase: 0.1% formic acid aqueous solution

Column temperature: 40 ° C

Detection method: For the eluate from the column, the OPA reaction solution (0.3 mmol borate buffer (pH 10.5), orthophthalaldehyde (OPA) O. 4 g / l, ethanol 16 ml / l, 2-mercapto Ethanol (2 ml / l) was allowed to act at 40 ° C to make the primary amino acid OPA derivatized, and the absorbance at 340 nm was measured.

[0099] 300 ml of the reaction solution obtained above was applied to 400 ml of cation exchange resin Dowex-50W (H +). Subsequently, the resin was washed with 1200 ml of deionized water, and the fraction containing (2S, 4S) -5-hydroxyleucine was eluted with 1600 ml of 0.1 N aqueous water. Concentrate the fraction containing (2 S, 4S) -5-hydroxyleucine and use (2S, 4S) -5-hydroxyleucine under the following conditions using high performance liquid chromatography (manufactured by Shimadzu Corporation). Preparative purification. The fraction obtained by preparative purification was freeze-dried to obtain 53 mg of white powder. The obtained white powder was confirmed to be (2S, 4S) -5-hydroxyleucine by ¹ H-NMR, ¹³ C-NMR, LC / MS, and specific rotation measurement. Each instrumental analysis is a separate organic synthesis. (2S, 4S) -5-hydroxyleucine standard product [J. Org. Chem., 54, 1859 (198 9)] obtained in the , 4S) -5-Hydroxyleucine and the standard (2S, 4S) -5-hydroxyleucine were confirmed to be in perfect agreement.

Preparative HPLC conditions:

Column: Inertsil ODS—3

Eluent: 0.1% formic acid aqueous solution

Column temperature: 40 ° C

Detection method: Each fraction obtained by preparative analysis was analyzed by the OPA post-label derivatization HPLC described in Example 3, and the purified fraction of (2S, 4S) -5-hydroxyleucine was identified. did.

Example 5: (2S. 4S) —5-hydroxyleucine produced in cattle by E. coli transformed plant (E. coli nos5D strain)

The recombinant plasmid pnos5D containing the leucine hydroxylase gene nos5 obtained in Example 2 was transformed into Escherichia coli BL21 (DE3) (manufactured by Novagen) and recombinant E. coli / pn _OS 5D. Obtained). The obtained recombinant Escherichia coli was inoculated into an Erlenmeyer flask containing LB medium lOOmL containing 30 kg / ml of kanamycin and cultured at 37 ° C for 18 hours. The culture broth was inoculated 1% into an Erlenmeyer flask containing LB medium lOOmL containing 30 g / ml kanamycin and cultured at 37 ° C. for 6 hours, and then the culture broth lOOmL was centrifuged to obtain wet cells.

[0101] Recombinant Escherichia coli (E.coli / pnos5D strain) wet cells obtained above were transferred to 20 mL of lOOmmol / 1 sodium phosphate buffer (ρΗ7.0), 10% glycerol, 10 mmol / l L-leucine. The reaction solution was suspended in the reaction solution, transferred to an Erlenmeyer flask, and reacted at 28 ° C for 72 hours.

[0102] After completion of the reaction, the reaction product was analyzed using high performance liquid chromatography (HPLC) (manufactured by Shimadzu Corporation) under the following analytical conditions, and about 1 mmol / 1 (2S , 4S)-5-Hydroxyleucine was confirmed to be generated and accumulated!

The analysis conditions were the same as those described in Example 4. [0103] About 800 ml of the reaction solution obtained above was applied to 400 ml of cation exchange resin 1 ^ 0 6 50 \ (^ 1 +). Subsequently, the resin was washed with 2000 ml of deionized water, and the fraction containing (2S, 4S) -5-hydroxyleucine was eluted with 1600 ml of 0.1 N aqueous water. Concentrate the fraction containing (2S, 4S) -5-hydroxyleucine and use (2S, 4S) -5-hydroxyleucine to separate it using high performance liquid chromatography (manufactured by Shimadzu Corporation) under the following conditions. It was collected and purified. The fraction obtained by preparative purification was freeze-dried to obtain about 47 mg of white powder. The obtained white powder was confirmed to be (2S, 4S) -5 hydroxyleucine by 1H-NMR and LC / MS. Each instrumental analysis was carried out by comparison with (2S, 4S) 5 hydroxyleucine standard (J. Org. Chem., 54, 1859 (1989)) obtained separately by organic synthesis, and obtained by enzymatic reaction. It was confirmed that the instrumental analysis characteristic values of (2S, 4S) -5-hydroxyleucine and the standard (2S, 4S) -5-hydroxyleucine were in perfect agreement.

Preparative HPLC conditions were the same as described in Example 4.

[0104] Example 6: Actinomycete-shaped transformed body (S. lividans nos5E strain) (2S. 4S) — 5 Hydroxyleucine from cattle

Recombinant plasmid Onos 5E containing the leucine hydroxylase gene nos5 obtained in Example 3 was introduced into the actinomycetes StreiDtomvces lividans by the conjugative transfer method, and MS agar medium containing apramycin 10 g / ml [mannitol (Kanto Chemical) 20g / l, defatted soybean (Ajinomoto Oil Co., Ltd.) 10g / l, agarose 20g / l], and then incubated at 28 ° C, so that the zygote (S. lividans / nos5E) I guarded.

[0105] The obtained conjugation transmitter was treated with a modified YEME medium containing 5 g / ml of apramycin [Batato Peptone (Difco) 5 g / l, Yeast Extract (Difco) 3 g / l, Malto Extratote (Oxoid) 3g / l, Gnore course 10g / l. Sucrose 30 g / l] was inoculated into an Erlenmeyer flask containing 50 mL and cultured at 30 ° C for 18 hours. Inoculate 2% of Erlenmeyer flask containing 50 mL of modified YEME medium containing apramycin 5; ^ / ml and thiostrepton 5 μg / ml, and incubate for 24 hours at 30 ° C. Centrifuge 50 mL of the culture As a result, wet cells were obtained.

[0106] The S.lividans / pnos5E strain wet cells obtained above were used in a 20 mL reaction consisting of lOOmmol / 1 sodium phosphate buffer (ρΗ7.0), 10% glyceronole, 10 mmol / l L leucine. The reaction solution was suspended in a liquid, transferred to an Erlenmeyer flask, and reacted at 30 ° C for 24 hours.

After completion of the reaction, the reactive organism was analyzed using high performance liquid chromatography (HPLC) (manufactured by Shimadzu Corporation) under the following analytical conditions, and 0.2 mmol / l (2S, 4 It was confirmed that S) -5-hydroxyleucine was generated and accumulated!

The analysis conditions were the same as those described in Example 4.

搿 on paper (Ru ¾: child data is urine book does not form part of country ¾ output ^, number of papers from international 1: 1 does not enter)

Claims

The scope of the claims

[1] A protein selected from the group consisting of:

(a) a protein having the amino acid sequence represented by SEQ ID NO: 1,

(b) in the amino acid sequence of SEQ ID NO: 1, a protein having one or more amino acid residues deleted, substituted, inserted or added, and having L-leucine hydroxylase activity, and

(c) A protein having an amino acid sequence having 60% or more homology with the amino acid sequence of SEQ ID NO: 1 and having L-mouth icin hydroxylase activity.

[2] DNA encoding the protein according to claim 1.

[3] The DNA according to claim 2, which has the base sequence represented by SEQ ID NO: 2.

[4] DNA selected from the group consisting of:

(i) DNA having the base sequence represented by SEQ ID NO: 2,

(ii) DNA that hybridizes under stringent conditions with the DNA having the base sequence represented by SEQ ID NO: 2 and encodes a protein having L-sip Icin hydroxylase activity,

(iii) In SEQ ID NO: 2, a DNA having a nucleotide sequence that has one or more bases deleted, substituted, inserted or added, and which codes for a protein having a single leucine hydroxylase activity, and DNA,

(iv) A DNA encoding a protein having L-leucine hydroxylase activity, having a nucleotide sequence ability of 60% or more with the nucleotide sequence represented by SEQ ID NO: 2.

[5] The DNA according to claim 3, wherein the DNA is derived from a microorganism belonging to the cyanobacteria (Cvanobacteria).

4. DNA according to 4.

[6] DNA strength S derived from microorganisms belonging to cyanobacteria S, derived from microorganisms belonging to the genus Hototo

6. DNA according to claim 5, which is DNA.

[7] The DNA according to claim 6, wherein the microorganism belonging to the genus Nostoc is an ostoc awakening cti forme.

[8] The DNA according to claim 2, which has the base sequence represented by SEQ ID NO: 3.

[9] DNA selected from the group also with:

Replacement paper (Rule 26) (I) DNA having the base sequence represented by SEQ ID NO: 3,

(II) DNA that encodes a protein having a no-bridging condition under stringent conditions with DNA having the base sequence represented by SEQ ID NO: 3 and having L-leucine hydroxylase activity,

(III) In SEQ ID NO: 3, one or a plurality of bases are deleted, substituted, inserted or added, resulting in a base sequence ability, and a DNA that codes for a protein having L-mouth icine hydroxylase activity, DNA, and

(IV) A DNA comprising a nucleotide sequence having at least 60% homology with the nucleotide sequence represented by SEQ ID NO: 3 and encoding a protein having L-leucine hydroxylase activity.

[10] Recombinant DNA obtained by incorporating the DNA according to any one of claims 2 to 9 into a vector ₀

[11] A transformant obtained by introducing the recombinant DNA according to claim 10 into a host cell.

[12] The transformant according to claim 11, wherein the host cell is Escherichia coli.

[13] The transformant according to claim 11, wherein the host cell is Streptomvces lividans.

[14] The transformant according to any one of claims 11 to 13, which expresses L-leucine hydroxylase.

[15] The transformant according to any one of claims 11 to 14, wherein the transformant is cultured using a medium, and a protein having L-leucine hydroxylase activity is produced and accumulated in the culture. A method for producing a protein having L-leucine hydroxylase activity, which comprises collecting the protein from the culture force.

[16] The transformant culture according to any one of claims 11 to 14, or the processed product of the culture, as an enzyme source, and the enzyme source and L-leucine in an aqueous medium Producing and accumulating (2S, 4S) -5-hydroxyleucine in the aqueous medium, and collecting (2S, 4S) -5-hydroxyleucine from the aqueous medium, 2S, 4S)-5 A process for producing monohydroxyleucine.

[17] Processed cultures are prepared by centrifuging the culture concentrate, the culture dry product, and the culture.

Replacement paper (Rule 26) 08/013262 Country B p _{CT /} jP20 ₀ Back 3. 2007

28

Cells obtained, dried products of the cells, freeze-dried products of the cells, surfactant-treated products of the cells, ultrasonically treated products of the cells, mechanical polishing products of the cells, A solvent-treated product of the bacterial cell, an enzyme-treated product of the bacterial cell, a protein fraction of the bacterial cell, an immobilized product of the bacterial cell, or an enzyme preparation obtained by extraction from the bacterial cell. 16. The production method according to 16.

'Replacement form (Rule 26)