JPWO2008013262A1 - L-leucine hydroxylase and DNA encoding the enzyme - Google Patents

Abstract

The present invention relates to a novel protein having an activity of converting L-leucine hydroxylase, that is, L-leucine into (2S, 4S) -5-hydroxyleucine, a DNA encoding the protein, and a recombinant containing the DNA The present invention relates to DNA, a transformant carrying the recombinant DNA, the protein using the transformant, and a method for producing (2S, 4S) -5-hydroxyleucine. ADVANTAGE OF THE INVENTION According to this invention, the manufacturing method using the organism or enzyme etc. of (2S, 4S) -5-hydroxyleucine which can be anticipated as a starting material in manufacture of various useful substances such as pharmaceuticals is provided. According to the present invention, (2S, 4S) -5-hydroxyleucine can be efficiently produced.

Description

Reference to related applications

  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 entirety is hereby incorporated by reference.

Background of the Invention

FIELD OF THE INVENTION The present invention relates to an L-leucine hydroxylase, that is, a protein having an activity of converting L-leucine into (2S, 4S) -5-hydroxyleucine, a DNA encoding the protein, and a recombinant containing the DNA. DNA, a transformant carrying the recombinant DNA, a method for producing a protein having L-leucine hydroxylase activity using the transformant, and (2S, 4S) -5-hydroxyleucine using the transformant It relates to the manufacturing method.

Background Art (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. As a method for producing this substance, only a method by chemical synthesis has been reported (J. Chem. Soc. Perkin Trans. 1, Organic and Bio-Organic Chemistry, 8, 2075 (1988); J. Org. Chem., 68, 83 (2003)), production methods using organisms or enzymes have not been reported so far as the present inventors know.

(2S, 4S) -5-hydroxyleucine is expected to exist as a biosynthetic intermediate in the biosynthetic pathway of polypeptides (nostopeptolide A1 and nostopeptolide A2) produced by Nostoc sp. GSV224, a kind of cyanobacteria . (J. Org. Chem., 68, 83 (2003); Gene, 311, 171 (2003)). However, no L-leucine hydroxylase gene has been identified in Nostoc sp. GSV224, and no such suggestion has been made. In addition, as far as the present inventors have known, other organisms have not yet reported an enzyme having L-leucine hydroxylase activity and a gene encoding the enzyme.

Nostoc sp. ATCC29133, a kind of cyanobacteria, has been analyzed for its nucleotide sequence. However, the L-leucine hydroxylase gene has not been identified here, and its existence has not been suggested so far.

Summary of the Invention

As a result of functional analysis using a DNA derived from a microorganism belonging to the genus Cyanobacteria, specifically a microorganism belonging to the genus Nostoc ( Nostoc sp. ATCC29133), the present inventors have recently identified a novel L that has not been identified so far. -Unexpectedly found DNA encoding leucine hydroxylase and succeeded in obtaining the DNA. The present invention is based on such knowledge.

  Therefore, in order to obtain (2S, 4S) -5-hydroxyleucine which can be a starting material in the production of various useful substances such as pharmaceuticals, the present invention provides a novel protein having L-leucine hydroxylase activity, a DNA encoding the protein, and the DNA A recombinant DNA containing the recombinant DNA, a transformant having the recombinant DNA, the protein using the transformant, and a method for producing (2S, 4S) -5-hydroxyleucine And

The protein according to the 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-leucine 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.

  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.

According to another aspect, 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: 2,
(ii) a DNA that hybridizes with a DNA having the base sequence represented by SEQ ID NO: 2 under a stringent condition and encodes a protein having L-leucine hydroxylase activity,
(iii) DNA comprising a nucleotide sequence in which one or more bases are deleted, substituted, inserted or added in SEQ ID NO: 2, and encoding a protein having L-leucine hydroxylase activity, and
(iv) A DNA comprising a base sequence having 60% or more homology with the base sequence represented by SEQ ID NO: 2 and encoding a protein having L-leucine hydroxylase activity.

According to a preferred embodiment of the present invention, DNA according to the invention are those derived from a microorganism belonging to the blue-green algae (Cyanobacteria). According to a more preferred embodiment, the DNA derived from a microorganism belonging to the cyanobacterium is DNA derived from a microorganism belonging to the genus Nostoc . According to a further preferred embodiment of the present invention, the microorganism belonging to the genus Nostoc is Nostoc punctiforme .

  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.

According to yet another aspect of the invention, the DNA according to the 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 L-leucine hydroxylase activity,
(III) DNA comprising a nucleotide sequence in which one or more bases are deleted, substituted, inserted or added in SEQ ID NO: 3, and encoding a protein having L-leucine 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-leucine hydroxylase activity.

  The recombinant DNA according to the present invention can be obtained by incorporating the DNA according to the present invention into a vector.

The transformant according to the present invention can be obtained by introducing the recombinant DNA according to the present invention into a host cell. Here, the host cell is preferably Escherichia coli , and more preferably Streptomyces lividans . The transformant according to the present invention preferably expresses L-leucine hydroxylase.

  In the method for producing a protein having L-leucine hydroxylase activity according to the present invention, the transformant according to the present invention is cultured using a medium to produce and accumulate a protein having L-leucine hydroxylase activity in the culture. And collecting the protein from the culture.

  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, and the enzyme source and L-leucine are aqueous. Including in the medium to produce and accumulate (2S, 4S) -5-hydroxyleucine in the aqueous medium and collecting (2S, 4S) -5-hydroxyleucine from the aqueous medium. Become. 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, ultrasonically 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.

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-leucine hydroxylase activity;
(3) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1 and having L-leucine hydroxylase activity;
(4) DNA encoding the protein according to any one of (1) to (3);
(5) The DNA according to (4) having a 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) The DNA having the base sequence represented by SEQ ID NO: 2 or 3 is hybridized under stringent conditions. And a DNA encoding a protein having L-leucine hydroxylase activity.

  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. (2S, 4S There is a strong desire for an efficient process for the production of) -5-hydroxyleucine. According to the present invention, a large amount of L-leucine hydroxylase can be obtained. By using the enzyme obtained by the present invention, (2S, 4S) -5-hydroxyleucine can be efficiently produced.

The figure shows the construction process of L-leucine 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. The figure shows the construction process of an L-leucine hydroxylase expression plasmid pnos5D constructed by constructing a chemically synthesized gene fragment. The figure shows the construction process of L-leucine hydroxylase expression plasmid pnos5E.

Detailed description of the invention

L-leucine hydroxylase L-leucine hydroxylase according to the present invention comprises 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 a protein having L-leucine hydroxylase activity.

  Here, the “substantially equivalent amino acid sequence” means an amino acid sequence that has a modification by deletion, substitution, addition and / or insertion of one or more amino acids, but does not affect the activity of the polypeptide. Means. The number of amino acid residues to be modified is preferably 1 to 40, more preferably 1 to 20, further preferably 1 to 8, and most preferably 1 to 4.

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

  Examples of “modifications that do not affect activity” as used in the present invention include conservative substitutions. By “conservative substitution” is meant replacing one or more amino acid residues with another chemically similar amino acid residue such that the activity of the polypeptide is not substantially altered. For example, when a certain hydrophobic amino acid residue is substituted with another hydrophobic amino acid residue, a certain polar amino acid residue is substituted with another polar amino acid residue having the same charge, and the like. Functionally similar amino acids that can make such substitutions are known in the art for each amino acid. Specifically, examples of nonpolar (hydrophobic) amino acids include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine, and the like. Examples of polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, cysteine and the like. 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.

  For example, modification by amino acid deletion, substitution, addition and / or insertion may be performed on, for example, a DNA encoding the same by site-directed mutagenesis (for example, Nucleic Acid Research, Vol. 10, No. 20), which is a well-known technique. , p. 6487-6500, 1982). Other methods include treating the gene with a mutagen, and selectively cleaving the gene, then removing, adding, inserting and / or replacing selected nucleotides, and then ligating.

The aforementioned “substantially equivalent amino acid sequences” may further include those in which the N-terminus (amino terminus) and C-terminus (carboxyl terminus) are altered or modified. For example, the C-terminal carboxyl may be carboxylate (—COO—), amide (—CONH ₂ ), or ester (—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, still more preferably 90% or more, even more preferably 95% or more, particularly preferably 98% or more, and most preferably 99% or more. It is. In the present specification, “homology” may mean “identity”.

  In the present specification, any numerical value of “homology” may be a numerical value calculated using a homology search program known to those skilled in the art. For example, a homology algorithm BLAST (Basic local alignment search tool) ( It can be calculated by using default (initial setting) parameters at the National Center for Biotechnology Information (NCBI) (available from http://www.ncbi.nlm.nih.gov/BLAST/).

DNA encoding L-leucine hydroxylase
The DNA according to the present invention includes a DNA encoding the protein according to the present invention, a DNA consisting of the base sequence represented by SEQ ID NO: 2 or 3, or a DNA comprising the base sequence represented by SEQ ID NO: 2 or 3, Examples thereof include DNA that hybridizes under conditions, and that encodes a protein having L-leucine hydroxylase activity.
In the present specification, “DNA” (deoxyribonucleic acid) may be used instead of a polynucleotide as necessary.

  Here, “DNA that hybridizes under stringent conditions” means, for example, a colony using, as a probe, the DNA according to the present invention, such as the DNA having the base sequence represented by SEQ ID NO: 2, or a partial DNA fragment thereof -It means DNA obtained by using a hybridization method, plaque hybridization method or the like. Specifically, as the DNA, hybridization was performed at 65 ° C. in the presence of 0.7 to 1.0 mol / L sodium chloride using a filter on which colony or plaque-derived DNA was immobilized. By washing the filter under a condition of 65 ° C. using a 0.1 to 2 times concentrated SSC solution (the composition of the 1 times concentrated SSC solution consists of 150 mmol / L sodium chloride and 15 mmol / L sodium citrate). Mention may be made of DNA that can be identified. Hybridization is performed according to the method described in Molecular Cloning 2nd Edition, Current Protocols in Molecular Biology, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University (1995), etc. It can be done according to this.

Specifically, the hybridizable DNA is, for example, DNA having at least 60% homology to the base sequence represented by SEQ ID NO: 2 when calculated based on the above algorithm BLAST, preferably 70% or more More preferred is a DNA having a homology of 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.
For this reason, the DNA according to the present invention encodes a protein having a base sequence having 60% or more homology with the base sequence represented by SEQ ID NO: 2 or 3, and having L-leucine hydroxylase activity, for example. DNA can be included.

  The DNA according to the present invention also encodes a protein having a base sequence in which one or more bases are deleted, substituted, inserted or added in SEQ ID NO: 2 or 3, and having L-leucine hydroxylase activity. DNA can also be included. Here, the number of bases that may be deleted, substituted, inserted or added is preferably 1 to 30, more preferably 1 to 20, still more preferably 1 to 15, and even more preferably 1 to 10. And most preferably 1-5. The substitution referred to here includes both substitution involving amino acid translation mutation and substitution not involving amino acid translation mutation.

Identification of L-leucine hydroxylase
In Gene, 311, 171 (2003), (2S, 4S) -5-hydroxyleucine is in the middle of biosynthesis in the biosynthetic pathway of polypeptides (nostopeptolide A1 and nostopeptolide A2) produced by Nostoc sp. GSV224 , a kind of cyanobacteria . It is expected to exist as a body. However, there are no reports or suggestions regarding the identification of the L-leucine hydroxylase gene in Nostoc sp. GSV224.

Based on the possibility of via (2S, 4S) -5-hydroxyleucine in the biosynthetic pathway and the sequence information of Nostoc sp. GSV224, a microorganism belonging to the genus Nostoc was analyzed and analyzed. -Leucine hydroxylase was identified. That is, it was predicted that the ORF whose function is unknown contributes to hydroxylation of leucine, and the present invention was completed by isolating and obtaining a highly homologous DNA sequence in Nostoc sp. ATCC29133 strain.

Obtaining DNA encoding L-leucine hydroxylase DNA encoding L-leucine hydroxylase of the present invention can be obtained , for example, from a microorganism belonging to the genus Nostoc , preferably Nostoc sp. ATCC29133, by the following method . Can be separated. It is also possible to artificially chemically synthesize based on the disclosed sequence of Nostoc sp. ATCC29133.

A microorganism belonging to the genus Nostoc can be cultured by a known method [for example, Microbiology, 140, 3233 (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). The Nostoc sp. No. ATCC29133 strain is commercially available from ATCC (American Type Culture Collection) as ATCC29133D.

A genomic DNA library of microorganisms belonging to the genus Nostoc is prepared by digesting the genomic DNA with an appropriate restriction enzyme and then ligating it with an appropriate vector. As a vector, various things, such as a plasmid vector, a phage vector, a cosmid vector, a BAC vector, can be used, for example.

Next, an appropriate probe is prepared based on the base sequence of the DNA encoding L-leucine hydroxylase disclosed in the present specification, and a DNA fragment containing a desired kanamycin biosynthetic gene is hybridized from the genomic DNA library. It can be isolated. Further, a primer for amplifying a desired gene is prepared based on the base sequence of DNA encoding L-leucine hydroxylase disclosed in the present specification, and a genomic DNA of a microorganism belonging to the genus Nostoc is used as a template. The desired gene can be isolated by performing PCR and ligating the amplified DNA fragment with an appropriate vector. Furthermore, since the DNA encoding L-leucine hydroxylase according to the present invention is contained in the plasmid pnos5 / pET24 and the plasmids pnos5-GC1 / pTA2 and pnos5-GC2 / pTA2, these are used as template DNA for PCR. It is possible. Moreover, a desired DNA fragment can be prepared from these plasmids with an appropriate restriction enzyme.

Escherichia coli transformed with the microorganism deposit plasmid pnos5A (Escherichia coli BL21 (DE3) / pnos5A) is an independent administrative agency, National Institute of Advanced Industrial Science and Technology as of June 16, 2006 (original deposit date). Deposited at the Institute's Patent Biological Deposit Center (Central 6th, 1-chome Higashi 1-chome, Tsukuba, Ibaraki, Japan 305-8586). The accession number is FERM BP-10845.
Escherichia coli transformed with plasmid pnos5D (Escherichia coli BL21 (DE3) / pnos5D) is a patent issued by the National Institute of Advanced Industrial Science and Technology as of June 16, 2006 (original deposit date). Deposited at the Biological Depositary Center (Central 6th, East 1-chome, Tsukuba City, Ibaraki, Japan 305-8565). The accession number is FERM BP-10846.
Streptomyces lividans (Streptomyces lividans / pnos5E) transformed with plasmid pnos5E is a patent biological deposit center of the National Institute of Advanced Industrial Science and Technology on June 16, 2006 (original deposit date). It was deposited at (Central 6th, 1-chome Higashi 1-chome, Tsukuba City, Ibaraki Prefecture 305-8565, Japan). The accession number is FERM BP-10847.

Method for Producing L-Leucine Hydroxylase L-leucine hydroxylase according to the present invention can be produced according to the method described in Molecular Cloning 2nd edition, Current Protocols in Molecular Biology, etc. -It can be produced by expressing DNA encoding leucine hydroxylase in a host cell.

  Based on the DNA according to the present invention, if necessary, a DNA fragment having 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 in the base sequence encoding the protein so that the codon is optimal for host expression.

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

  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.

Any host cell can be used as long as it can express the target gene, such as bacteria, actinomycetes, yeast, animal cells, insect cells, and plant cells. Preferably, actinomycetes Streptomyces , Escherichia coli Escherichia coli, etc. are mentioned, More preferably, actinomycetes Streptomyces etc. are mentioned.

  As the recombinant DNA, one that can be autonomously replicated or integrated into a chromosome in the host cell and contains a promoter at a position where the DNA of the present invention can be transcribed is used.

  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 can be autonomously replicated in the prokaryotic organism, and at the same time a promoter, a ribosome binding sequence, It is preferably a recombinant DNA composed of the DNA of the invention and a transcription termination sequence. Further, the recombinant DNA may contain a gene that controls the promoter.

  As expression vectors, pTA2 (manufactured by Toyobo Co., Ltd.), Helix1 (manufactured by Roche Diagnostics), pKK233-2 (manufactured by Amersham Pharmacia Biotech), pSE280 (manufactured by Invitrogen), pGEMEX-1 (Promega) PQE-8 (manufactured by Qiagen), pET24 (manufactured by Novagen), pBluescript II SK (+), pBluescript II SK (-) (manufactured by Stratagene), pUC19 [Gene, 33, 103 (1985)], pSTV28 (Takara Shuzo Co., Ltd.), pUC118 (Takara Shuzo Co., Ltd.), pIJ6902 [Mol. Microbiol., 58, 1276 (2005)], pSET152 [J. Mol. Microbiol. Biotechnol., 4, 417 (2002)], etc. Can be illustrated. Preferably, pTA2, pET24, pIJ6902, pUC118, pSET152, etc. are mentioned, More preferably, pTA2, pET24, pIJ6902 etc. are mentioned.

  The promoter is not particularly limited as long as it can function in a host cell, and any promoter can be used. 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)], ermE * promoter [Gene, 38, 215 (1985)] and the like. In addition, artificially designed and modified promoters such as a promoter in which two Ptrps are connected in series (Ptrp × 2), a tac promoter, a lacT7 promoter, and a let I promoter can 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.

  In the present invention, it is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence, which is a ribosome binding sequence, and the initiation codon is adjusted to an appropriate distance (for example, 6 to 18 bases).

  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.

Prokaryotes include microorganisms belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Streptomyces, etc., for example, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli KY3726, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli BL21 (DE3), Escherichia coli No.49, Escherichia coli W3110, Escherichia coli NY49, Serratia ficaria, Serratia fonticola, Serratia liquefaciens , Serratia marcescens, Bacillus subtilis, Serratia amyloliquefaciens, Brevibacterium immariophilum, B revibacterium saccharolyticum, Corynebacterium ammoniagenes, Corynebacterium glutamicum, Corynebacterium acetoacidophilum, Microbacterium ammoniaphilum, Streptomyces lividans, ani a Streptomyces coelicolor, etc. Rukoto can. Preferably, Escherichia coli BL21 (DE3), Escherichia coli JM109, Streptomyces lividans, etc. are mentioned, More preferably, Escherichia coli JM109, Streptomyces lividans etc. are mentioned.

  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 (JP-A 63-248394), electroporation method [Nucleic Acids Res., 15, 6127 (1988)], conjugation method [J. Bacteriol., 171, 3585 (1989)], etc. Can be mentioned.

Here, a case where a yeast strain is used as a host cell will be described as an example.
When using a yeast strain as a host cell, YEp13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, pHS15 etc. can be used as an expression vector, for example.

  Any promoter may be used as long as it functions in yeast strains. For example, PH05 promoter, PKG promoter, GAP promoter, ADH promoter, gal 1 promoter, gal 10 promoter, heat shock polypeptide promoter And promoters such as MFα1 promoter and CUP 1 promoter.

Examples of host cells include yeast strains belonging to the genus Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Siwaniomyces, Pichia, Candida, etc., specifically, Saccharomyces cervisiae , Shizosaccharomyces pombe, Kluyveromyces lactis, may be mentioned Trichosporon pullulans, Schwanniomyces alluvius, Pichi a pastoris, the Candida utilis and the like.

  As a method for introducing recombinant DNA, any method for introducing DNA into yeast can be used. For example, electroporation method [Methods Enzymol., 194, 182 (1990)], spheroplast method [Proc. Natl. Acad. Sci. USA, 81, 4889 (1984)], lithium acetate method [J. Bacteriol., 153, 163 (1983)] and the like.

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

  As a medium for culturing a transformant obtained by using a prokaryote such as E. coli / actinomycetes or a eukaryote such as yeast as a host, it contains a carbon source, a nitrogen source, inorganic salts, etc. Either a natural medium or a synthetic medium may be used as long as the medium can efficiently culture the transformant.

  The carbon source may be anything that can be assimilated by the organism, such as glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, organic acids such as acetic acid and propionic acid, ethanol, Alcohols such as propanol can be used.

  Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate and other ammonium salts, other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep liquor, casein A hydrolyzate, soybean meal, soybean meal hydrolyzate, various fermented cells, digests thereof, and the like can be used.

  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.

  The culture is preferably carried out 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 culture, the pH is maintained at 3.0 to 9.0. The pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, or the like.

Moreover, you may add antibiotics, such as ampicillin, kanamycin, apramycin, thiostrepton, tetracycline, chloramphenicol, streptomycin, to a culture medium as needed during culture | cultivation.

  When culturing 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, when culturing a microorganism transformed with isopropyl-β-D-thiogalactopyranoside or the like with an expression vector using the trp promoter. When culturing microorganisms transformed with indole acrylic acid or the like with an expression vector using the tipA promoter, thiostrepton or the like may be added to the medium.

  As described above, a transformant derived from a microorganism having a recombinant DNA incorporating the 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 protein is produced from the culture. By collecting the protein, the protein can be produced.

  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.

  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.

  For example, when the protein of the present invention is produced in a state of being dissolved in the cells, the cells are collected by centrifugation after culturing, suspended in an aqueous buffer solution, ultrasonically disrupted, French press, Manton Gaurin homogenizer. Then, the cells are disrupted with dynomill or the like 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, diethylaminoethyl ( DEAE) -cephalose, anion exchange chromatography using resin such as DIAION HPA-75 (manufactured by Mitsubishi Kasei Co., Ltd.), cation exchange chromatography using resin such as S-Separose FF (manufactured by Pharmacia), Hydrophobic chromatography methods using resins such as butyl sepharose and phenyl sepharose, gel filtration methods using molecular sieves, affinity chromatography methods, chromatofocusing methods, electrophoresis methods such as isoelectric focusing etc. Used in combination, a purified preparation can be obtained.

  In addition, when the protein is produced as an insoluble substance in the cell, the protein is recovered by a conventional method from the precipitate fraction obtained by centrifugation after disrupting the cell in the same manner as described above. 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 diluted 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.

  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, the culture supernatant is obtained by treating the culture by a technique such as centrifugation as described above, and a purified sample is obtained from the culture supernatant by using the same isolation and purification method as described above. Obtainable.

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

(2S, 4S) -5-Hydroxyleucine Production Method Using the transformant culture according to the present invention and the treated product of the culture as an enzyme source, the enzyme source and L-leucine are present in an aqueous medium. (2S, 4S) -5-hydroxyleucine can be produced and accumulated in the aqueous medium to produce (2S, 4S) -5-hydroxyleucine.

  Here, the treated product of the culture includes, for example, a concentrate of the culture, a dried product of the culture, a microbial cell obtained by centrifuging the culture, a dried product of the microbial cell, and a lyophilization of the microbial cell. Product, surfactant-treated product of the microbial cell, sonicated 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 Examples include protein fractions of the body, immobilized products of the cells, and enzyme preparations obtained by extraction from the cells.

  Examples of aqueous media that can be used for the production of (2S, 4S) -5-hydroxyleucine include buffers such as water, phosphate, carbonate, acetate, borate, citrate, and tris, methanol, and ethanol. And alcohols such as ethyl acetate, esters such as ethyl acetate, ketones such as acetone, and amides such as acetamide. Moreover, the culture solution of the microorganism used as an enzyme source can be used as an aqueous medium.

  In the production of (2S, 4S) -5-hydroxyleucine, a surfactant or an organic solvent may be added as necessary. Examples of the surfactant include nonionic surfactants such as polyoxyethylene / octadecylamine (for example, Nimine S-215, manufactured by NOF Corporation), cetyltrimethylammonium / bromide and alkyldimethyl / benzylammonium chloride (for example, cation F2). -40E, manufactured by NOF Corporation), anionic surfactants such as lauroyl / sarcosinate, etc., and tertiary amines such as alkyldimethylamine (eg, tertiary amine FB, manufactured by NOF Corporation). Any one can be used as long as it can promote the production of (2S, 4S) -5-hydroxyleucine, and one kind or a mixture of several kinds can be used. The surfactant is usually used at a concentration of 0.1 to 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 0.1 to 50 ml / l.

The formation reaction of (2S, 4S) -5-hydroxyleucine is carried out in an aqueous medium at pH 5 to 10, preferably pH 6 to 8, 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 ₂ can be added as necessary.

  Quantification of (2S, 4S) -5-hydroxyleucine produced in the aqueous medium can be performed using high performance liquid chromatography (HPLC) or the like.

  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.

  Hereinafter, the present invention will be described in detail with reference to the following examples, but the present invention is not limited thereto.

Example 1: Isolation of an L-leucine hydroxylase gene derived from a microorganism belonging to the genus Nostock
Genomic DNA of Nostoc sp. ATCC29133 was obtained from ATCC (American Type Culture Collection) as a commercial product (ATCC 29133D). Primers having the nucleotide sequences represented by SEQ ID NOs: 4 and 5 were synthesized, and PCR was performed using the genomic DNA as a template. PCR is a reaction containing 0.1 μg of chromosomal DNA, each primer 0.5 μmol / l, LA Taq DNA polymerase (Takara Shuzo Co., Ltd.) 2.5 units, LA Taq DNA polymerase x10 buffer 5 μl, deoxyNTP 250 μmol each, MgCl 22.5 mM Using 50 μl of the solution, the process of 95 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 1 minute was repeated 30 times. 1/10 volume of the reaction solution was subjected to agarose electrophoresis, and after confirming that the DNA fragment was amplified, DNA was purified from the remaining reaction solution using a kit of High Pure PCR Product Purification Kit (Roche). .

  Using 10 μl of the obtained purified DNA solution, the DNA was cleaved with restriction enzymes NdeI and BamHI, and 1.7 kb of DNA was separated by agarose electrophoresis. Then, the DNA was recovered from the agarose gel according to a conventional method.

  About 0.2 μg of pET24 DNA (manufactured by Novagen) was cleaved with restriction enzymes NdeI and BamHI, and DNA fragments were separated by agarose electrophoresis to recover a 5.3 kb DNA fragment.

  Using a ligation kit (Takara Shuzo Co., Ltd.), the fragments of 1.7 kb and 5.3 kb were subjected to a ligation reaction at 16 ° C. for 16 hours. Escherichia coli JM109 strain (Takara Shuzo Co., Ltd.) was transformed using the ligation reaction solution according to the above-mentioned known method, and the transformant was LB agar medium containing 30 μg / ml kanamycin [Bacttryptone (Difco) 10 g / l, yeast extract (manufactured by Difco) 10 g / l, sodium chloride 5 g / l, agarose 15 g / l], and then cultured at 37 ° C. overnight. From the grown colonies, a plasmid was extracted according to the above-described known method to obtain pnosA, which is a recombinant plasmid containing the leucine hydroxylase gene nos5. The construction procedure and structure of the plasmid are shown in FIG.

Example 2: Chemical synthesis of L-leucine 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 -8 (SEQ ID NO: 10), BB-2 (SEQ ID NO: 11), BB-3 (SEQ ID NO: 12), and BB-4 (SEQ ID NO: 13) were synthesized. Of these, two were selected and PCR was performed using the primer set as the template DNA.

First, each primer and template (AA-1, AA-2) 0.5 μmol / l, KOD-Plus-polymerase (manufactured by Toyobo Co., Ltd.) 1.0 units, KOD-plus-polymer 10 × 5 buffer 5 μl, deoxyNTP Using 50 μl of a reaction solution containing 200 μmol each and 1 mM MgSO ₄ , the process of 94 ° C. for 30 seconds, 60 ° C. for 30 seconds, and 68 ° C. for 1 minute was repeated 15 times. PCR was similarly performed for the other primer sets {(AA-3, AA-4), (BB-1, BB-2), (BB-3, BB-4)}, and a total of four types of PCR reaction solutions were used. Got.

Next, the PCR reaction was performed again using 2.5 μl of the obtained reaction solution as a shared primer set and template. The reaction conditions were 2.5 μl of the above reaction solution for primer / template (AA-1, AA-2), 2.5 μl of the reaction solution for primer / template (AA-3, AA-4), KOD-Plus-polymerase ( Toyobo Co., Ltd.) 1.0 units, KOD-plus-polymerase x10 buffer 5 μl, deoxyNTP 200 μmol each, 50 μl of a reaction solution containing 1 mM MgSO ₄ , 94 ° C. for 30 seconds, 60 ° C. for 30 seconds, 68 This was carried out by repeating the process for 1 minute at 15 ° C. 15 times. Similarly, the primer / template combination is changed to the above reaction solution of (BB-1, BB-2) and the above reaction solution of (BB-3, BB-4), and PCR reaction is carried out. Got.

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, 9 μl of the PCR reaction solution and 1 μl of 10 × A-attachment Mix (manufactured by Toyobo Co., Ltd.) were added and reacted at 60 ° C. for 10 minutes after stirring. 2.5 μl of the reaction solution and 1 μl of 5 ng / μl of pTA2 vector (manufactured by Toyobo Co., Ltd.) were added, and ligation kit (manufactured by Takara Shuzo Co., Ltd.) was used. A ligation reaction was performed. 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 50 μg / ml of ampicillin and cultured at 37 ° C. overnight. 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 MroI and then ligated by ligation to obtain a plasmid (pnos5B) containing a DNA fragment (nos5-GC) consisting of the base sequence shown in SEQ ID NO: 3.

Next, by performing PCR using the plasmid pnos5B obtained above as a template and the synthetic DNA (SEQ ID NOs: 14 and 15) as a primer set, a DNA fragment having NdeI and BglII restriction enzyme sites added to the ends. Was amplified. That is, PCR is about 0.1 μg of template body DNA (plasmid pnos5B), each primer is 0.3 μmol / l, KOD-Plus-polymerase (manufactured by Toyobo Co., Ltd.) 1.0 units, KOD-plus-polymerase × 10 buffer 5 μl, Using 50 μl of a reaction solution containing 200 μmol each of deoxyNTPs and 1 mM MgSO ₄ , the process of 94 ° C. for 15 seconds, 55 ° C. for 30 seconds, and 68 ° C. for 1 minute was repeated 25 times.

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, 9 μl of the PCR reaction solution and 1 μl of 10 × A-attachment Mix (manufactured by Toyobo Co., Ltd.) were added and reacted at 60 ° C. for 10 minutes after stirring. 2.5 μl of the reaction solution and 1 μl of 5 ng / μl pTA2 vector (Toyobo Co., Ltd.) were added, and ligation kit (Takara Shuzo Co., Ltd.) was used for ligation at 16 ° C. for 16 hours at a final reaction solution volume of 10 μl. Reaction was performed. Subsequently, E. coli JM109 (Takara Shuzo Co., Ltd.) was transformed, and the transformant was applied to an LB agar medium containing 50 μg / ml of ampicillin and then cultured at 37 ° C. overnight. A plasmid was extracted from the grown colonies according to a conventional method to obtain a recombinant plasmid, pnos5C. The construction method and structure of the plasmid are shown in FIG.

  The obtained recombinant plasmid pnos5C was cleaved with restriction enzymes NdeI and BglII, and 0.8 kb DNA was separated by agarose gel electrophoresis. Then, the DNA was collected from the agarose gel according to a conventional method.

  About 0.2 μg of pET24 DNA (manufactured by Novagen) was cleaved with restriction enzymes NdeI and BamHI, and DNA fragments were separated by agarose gel electrophoresis to recover a 5.3 kb DNA fragment.

  The 0.8 kb and 5.3 kb fragments were subjected to a ligation reaction at 16 ° C. for 16 hours using a ligation kit (Takara Shuzo Co., Ltd.). Escherichia coli BL21 (DE3) strain was transformed with the ligation reaction solution, and the transformant was applied to an LB agar medium containing 30 μg / ml of kanamycin and cultured at 37 ° C. overnight. From the grown colonies, a plasmid was extracted according to the above-mentioned known method to obtain pnos5D, which is a recombinant plasmid containing the leucine hydroxylase gene nos5. The construction procedure and structure of the plasmid are shown in FIG.

Example 3 Construction of Recombinant Plasmid pnos5E A recombinant plasmid containing the leucine hydroxylase gene chemically synthesized in Example 2 and functioning in Streptomyces lividans was implemented by the following method.

  The recombinant plasmid pnos5C obtained in Example 2 was cleaved with restriction enzymes NdeI and BglII, and the DNA was recovered from an agarose gel according to a conventional method. Further, about 0.2 μg of pIJ6902 DNA was cleaved with restriction enzymes NdeI and BamHI, and DNA fragments were separated by agarose gel electrophoresis. Similarly, an about 7.3 kb DNA fragment was recovered from the agarose gel.

  Using a ligation kit (manufactured by Takara Shuzo Co., Ltd.), the above-mentioned 0.8 kb and about 7.3 kb fragments 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 10 μg / ml of apramycin and cultured at 37 ° C. overnight. A plasmid was extracted from the grown colony according to a conventional method to obtain pnos5E, which is 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 an E. coli transformant (E. coli / pnos5A strain) A recombinant plasmid pnos5A obtained in Example 1 and containing the leucine hydroxylase gene nos5 was transformed into E. coli. Escherichia coli BL21 (DE3) (manufactured by Novagen) was transformed to obtain recombinant E. coli (E. coli / pnos5A strain). The obtained recombinant Escherichia coli was inoculated into an Erlenmeyer flask containing 100 mL of LB medium containing 30 μg / ml of kanamycin and cultured at 37 ° C. for 18 hours. The culture solution was inoculated 1% into an Erlenmeyer flask containing 100 mL of LB medium containing 30 μg / ml of kanamycin and cultured at 37 ° C. for 6 hours, and then 100 mL of the culture solution was centrifuged to obtain wet cells.

  The obtained Escherichia coli recombinant (E.coli / pnos5A strain) wet cells were placed in a 20 mL reaction solution consisting of 100 mmol / l sodium phosphate buffer (pH 7.0), 10% glycerol, 10 mmol / l L-leucine. After suspending, the reaction solution was transferred to an Erlenmeyer flask and reacted at 28 ° C. for 72 hours.

After completion of the reaction, the reaction product was analyzed under the following analytical conditions using high performance liquid chromatography (HPLC) (manufactured by Shimadzu Corporation), and about 3 mmol / l (2S, 4S) -5 was added to the reaction solution. -It was confirmed that hydroxyleucine was produced and accumulated.
Analysis conditions:
Column: L-column
Mobile phase: 0.1% formic acid aqueous solution Column temperature: 40 ° C
Detection method: For the eluate from the column, OPA reaction solution (0.3 mmol borate buffer (pH 10.5), orthophthalaldehyde (OPA) 0.4 g / l, ethanol 16 ml / l, 2-mercaptoethanol 2 ml / L) was allowed to act at 40 ° C. to make the primary amino acid OPA derivatized, and the absorption at 340 nm was measured.

300 ml of the reaction solution obtained above was applied to 400 ml of cation exchange resin Dowex-50W (H +). Next, the resin was washed with 1200 ml of deionized water, and the fraction containing (2S, 4S) -5-hydroxyleucine was eluted with 1600 ml of 0.1N aqueous ammonia. The fraction containing (2S, 4S) -5-hydroxyleucine is concentrated, and (2S, 4S) -5-hydroxyleucine is fractionated under the following conditions using high performance liquid chromatography (manufactured by Shimadzu Corporation). Purified. 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 was carried out by comparison with (2S, 4S) -5-hydroxyleucine standard product [J.Org.Chem., 54,1859 (1989)] obtained separately by organic synthesis. It was confirmed that the instrumental analysis characteristic values of (2S, 4S) -5-hydroxyleucine obtained in this way and (2S, 4S) -5-hydroxyleucine of the standard product completely coincided.
Preparative HPLC conditions:
Column: Inertsil ODS-3
Eluent: 0.1% formic acid aqueous solution Column temperature: 40 ° C
Detection method: Fractions obtained by fractionation were analyzed by the OPA post-label derivatization HPLC described in Example 3 to identify purified fractions of (2S, 4S) -5-hydroxyleucine. did.

Example 5 Production of (2S, 4S) -5-hydroxyleucine by E. coli transformant (E. coli / pnos5D strain) The recombinant plasmid pnos5D containing the leucine hydroxylase gene nos5 obtained in Example 2 was transformed into E. coli. Escherichia coli BL21 (DE3) (manufactured by Novagen) was transformed to obtain recombinant E. coli (E. coli / pnos5D strain). The obtained recombinant Escherichia coli was inoculated into an Erlenmeyer flask containing 100 mL of LB medium containing 30 μg / ml of kanamycin and cultured at 37 ° C. for 18 hours. The culture solution was inoculated 1% into an Erlenmeyer flask containing 100 mL of LB medium containing 30 μg / ml of kanamycin and cultured at 37 ° C. for 6 hours, and then 100 mL of the culture solution was centrifuged to obtain wet cells.

  Recombinant Escherichia coli (E.coli / pnos5D strain) wet cells obtained above were placed in a 20 mL reaction solution consisting of 100 mmol / l sodium phosphate buffer (pH 7.0), 10% glycerol, 10 mmol / l L-leucine. The reaction solution was transferred to an Erlenmeyer flask and reacted at 28 ° C. for 72 hours.

After completion of the reaction, the reaction product was analyzed under the following analysis conditions using high performance liquid chromatography (HPLC) (manufactured by Shimadzu Corporation), and about 1 mmol / l (2S, 4S) -5 was added to the reaction solution. -It was confirmed that hydroxyleucine was produced and accumulated.
The analysis conditions were the same as those described in Example 4.

About 800 ml of the reaction solution obtained above was applied to 400 ml of cation exchange resin Dowex-50W (H +). Next, the resin was washed with 2000 ml of deionized water, and the fraction containing (2S, 4S) -5-hydroxyleucine was eluted with 1600 ml of 0.1N aqueous ammonia. The fraction containing (2S, 4S) -5-hydroxyleucine is concentrated, and (2S, 4S) -5-hydroxyleucine is fractionated under the following conditions using high performance liquid chromatography (manufactured by Shimadzu Corporation). 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 product [J.Org.Chem., 54,1859 (1989)] obtained separately by organic synthesis. It was confirmed that the instrumental analysis characteristic values of (2S, 4S) -5-hydroxyleucine obtained in this way and (2S, 4S) -5-hydroxyleucine of the standard product completely coincided.
Preparative HPLC conditions were the same as described in Example 4.

Example 6: Production of (2S, 4S) -5-hydroxyleucine by actinomycetes transformant (S. lividans / pnos5E strain) Recombinant plasmid pnos5E containing leucine hydroxylase gene nos5 obtained in Example 3 was activated. was introduced in conjugative transfer method fungus Streptomyces lividans, MS agar medium containing apramycin 10 [mu] g / ml [mannitol (manufactured by Kanto Chemical Co., Inc.) 20 g / l, (manufactured by Ajinomoto oil Co.) defatted soybean 10 g / l, agarose 20 g / l] After application, the culture was carried out at 28 ° C. to obtain a conjugative transmitter (S. lividans / pnos5E).

  The obtained conjugation transmitter was modified YEME medium containing 5 μg / ml of apramycin [Bactopepton (Difco) 5 g / l, Yeast extract (Difco) 3 g / l, Malto extract (Oxoid) 3 g / L, glucose 10 g / l. Sucrose 30 g / l] Inoculated into an Erlenmeyer flask containing 50 mL and cultured at 30 ° C. for 18 hours. The culture broth was inoculated 2% into an Erlenmeyer flask containing 50 mL of a modified YEME medium containing 5 μg / ml apramycin and 5 μg / ml thiostrepton, and cultured at 30 ° C. for 24 hours. Bacteria were obtained.

  The S. lividans / pnos5E strain wet cells obtained above were suspended in a 20 mL reaction solution consisting of 100 mmol / l sodium phosphate buffer (pH 7.0), 10% glycerol, 10 mmol / l L-leucine, and reacted. The liquid was 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 analysis conditions, and 0.2 mmol / l (2S, 4S)- It was confirmed that 5-hydroxyleucine was produced and accumulated.
The analysis conditions were the same as those described in Example 4.

Claims (17)

A protein 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-leucine 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.   DNA encoding the protein according to claim 1.   The DNA according to claim 2, which has the base sequence represented by SEQ ID NO: 2. DNA selected from the group consisting of:
(i) DNA having the base sequence represented by SEQ ID NO: 2,
(ii) a DNA that hybridizes with a DNA having the base sequence represented by SEQ ID NO: 2 under a stringent condition and encodes a protein having L-leucine hydroxylase activity,
(iii) DNA comprising a nucleotide sequence in which one or more bases are deleted, substituted, inserted or added in SEQ ID NO: 2, and encoding a protein having L-leucine hydroxylase activity, and
(iv) A DNA comprising a base sequence having 60% or more homology with the base sequence represented by SEQ ID NO: 2 and encoding a protein having L-leucine hydroxylase activity. DNA is a DNA derived from a microorganism belonging to the blue-green algae (Cyanobacteria), DNA of claim 3 or 4. The DNA according to claim 5, wherein the DNA derived from a microorganism belonging to the cyanobacteria is a DNA derived from a microorganism belonging to the genus Nostoc . The DNA according to claim 6, wherein the microorganism belonging to the genus Nostoc is Nostoc punctiforme .   The DNA according to claim 2, which has the base sequence represented by SEQ ID NO: 3. DNA 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 L-leucine hydroxylase activity,
(III) DNA comprising a nucleotide sequence in which one or more bases are deleted, substituted, inserted or added in SEQ ID NO: 3, and encoding a protein having L-leucine 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-leucine hydroxylase activity.   A recombinant DNA obtained by incorporating the DNA according to any one of claims 2 to 9 into a vector.   A transformant obtained by introducing the recombinant DNA according to claim 10 into a host cell. The transformant of Claim 11 whose host cell is Escherichia coli ( Escherichia coli ). The transformant of Claim 11 whose host cell is Streptomyces lividans ( Streptomyces lividans ).   The transformant according to any one of claims 11 to 13, which expresses L-leucine hydroxylase.   The transformant according to any one of claims 11 to 14 is cultured using a medium to produce and accumulate a protein having L-leucine hydroxylase activity in the culture. A method for producing a protein having L-leucine hydroxylase activity, which comprises collecting the protein.   A culture of the transformant according to any one of claims 11 to 14 or a processed product of the culture is used as an enzyme source, the enzyme source and L-leucine are present in an aqueous medium, Producing and accumulating (2S, 4S) -5-hydroxyleucine in an aqueous medium and collecting (2S, 4S) -5-hydroxyleucine from the aqueous medium, comprising (2S, 4S)- A process for producing 5-hydroxyleucine.   A processed product of the culture is a concentrate of the culture, a dried product of the culture, a cell obtained by centrifuging the culture, a dried product of the cell, a freeze-dried product of the cell, Surfactant treated product, ultrasonic treated product of the bacterial cell, mechanically ground treated product of the bacterial cell, solvent treated product of the bacterial cell, enzyme treated product of the bacterial cell, protein fraction of the bacterial cell The production method according to claim 16, which is an immobilized product of the microbial cell or an enzyme preparation obtained by extraction from the microbial cell.

Images