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WO2008013262A1 - L-leucine hydroxylase et adn codant pour l'enzyme - Google Patents

L-leucine hydroxylase et adn codant pour l'enzyme Download PDF

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Publication number
WO2008013262A1
WO2008013262A1 PCT/JP2007/064761 JP2007064761W WO2008013262A1 WO 2008013262 A1 WO2008013262 A1 WO 2008013262A1 JP 2007064761 W JP2007064761 W JP 2007064761W WO 2008013262 A1 WO2008013262 A1 WO 2008013262A1
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Prior art keywords
dna
protein
seq
culture
leucine
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PCT/JP2007/064761
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English (en)
Japanese (ja)
Inventor
Yasukatsu Ota
Shuichi Gomi
Koji Yanai
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Meiji Seika Kaisha, Ltd.
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Priority to JP2008526830A priority Critical patent/JPWO2008013262A1/ja
Publication of WO2008013262A1 publication Critical patent/WO2008013262A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine

Definitions

  • L bite isine hydroxylase and DNA encoding the enzyme
  • 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. .
  • (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.PerKin ⁇ rans.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!
  • (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.
  • 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.
  • the protein according to the present invention is selected from the group consisting of:
  • 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.
  • this DNA has the base sequence represented by SEQ ID NO: 2.
  • the DNA according to the present invention is selected from the group consisting of:
  • 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
  • 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.
  • the DNA according to the present invention is derived from a microorganism belonging to the cyanobacteria (Cvanobacteria).
  • the DNA derived from a microorganism belonging to the cyanobacteria is DNA derived from a microorganism belonging to the genus Nostock (N ⁇ l ⁇ ).
  • the microorganism belonging to the genus Nostock (N ⁇ l ⁇ ) is
  • 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.
  • the DNA according to the present invention is selected from the group consisting of:
  • 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.
  • the recombinant DNA according to the present invention is obtained by incorporating the DNA according to the present invention into a vector.
  • 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.
  • the host cell is preferably Escherichia coli, and more preferably, Streptomyces lividans.
  • the transformant according to the present invention preferably expresses L-mouth icine hydroxylase.
  • 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.
  • 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.
  • a culture of the transformant according to the present invention or a treated product of the culture is an enzyme source.
  • 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.
  • the present invention can also be as follows:
  • 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;
  • (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.
  • FIG. 1 shows a process for constructing an L-mouth icine hydroxylase expression plasmid pnos5A.
  • 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 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
  • 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.
  • 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.
  • 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.
  • 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.
  • non-polar (hydrophobic) amino acids such as alanine, norine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine.
  • polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine.
  • positively charged (basic) amino acids include arginine, histidine, and lysine.
  • negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • 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.
  • 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
  • R examples include a linear, branched or cyclic C1-6 alkyl group, a C6-12 aryl group, and the like.
  • 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.
  • 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.
  • “homology” may be used to mean “identity”.
  • any value of "homology” may be any value calculated using a homology search program known to those skilled in the art.
  • the homology algorithm BLAST Basic local alignment search tool
  • NCBI National Center for Biotechnology Information
  • 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.
  • DNA deoxyribonucleic acid
  • DNA may be used as a polynucleotide, if necessary.
  • 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.;!
  • SSC solution composition of 1 times SSC solution consists of 150mmol / L sodium chloride and 15mmol / L sodium quenate
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a genomic DNA library of microorganisms belonging to the genus Nostock is prepared by digesting genomic DNA with an appropriate restriction enzyme and ligating it with an appropriate vector.
  • the vector for example, various vectors such as a plasmid vector, a phage vector, a cosmid vector, and a BAC vector can be used.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • a DNA fragment of an appropriate length containing a portion encoding the protein is prepared.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • Any promoter can be used as long as it can function in a host cell.
  • trp promoter Ptrp
  • lac promoter Plac
  • T7 promoter PL promoter
  • PR promoter PR promoter
  • promoter derived from E. coli such as PSE promoter, phage, etc.
  • SP01 promoter SP02 promoter
  • penP promoter tipA promoter
  • tipA promoter J. Bacteriol., 171, 1459 (1989)]
  • erm E * promoter [Gene, 38, 215 (1985)] and the like.
  • Promors 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.
  • 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! /
  • 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 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 ⁇ ⁇ 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.
  • any method can be used as long as it is a method for introducing DNA into the host cell.
  • 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)].
  • YEpl 3 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, pHS15, etc. can be used as an expression vector.
  • Any promoter can be used as long as it functions in a yeast strain.
  • 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.
  • yeast strains belonging to the genus Saccharomyces, Schizosaccharomyces, Kluybe mouth mouth, Genus Trichosporon, Sisniomyces, Pichia, Candida, etc. are used. ⁇ , saccharomyces cervisiae, Shizosaccharomyces j) ombe, luvveromvces lactis. TricnosDoron pullulans ⁇ chwanniomvces alluvius., Pich ia pastoris, Candida utilis.
  • any method can be used as long as it introduces DNA into yeast.
  • the Elect Mouth Position Method [Methods Enzymol., 194, 182 (199 0)]
  • ferroplast method [? ⁇ Ji ⁇ & yoji & (1.5 (115, 81, 4889 (1984)]
  • lithium acetate method [J. Bacteriol., 153, 163 (1983)].
  • the method of culturing the transformant of the present invention can be carried out according to a usual method used for culturing a host.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • the pH is maintained at 3.0 ⁇ 9.0.
  • the pH is adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, etc.
  • antibiotics such as ampicillin, kanamycin, apramycin, thiostreptone, tetracycline, chloramphenicol, streptomycin and the like may be added to the medium as needed during the culture.
  • an inducer may be added to the medium as necessary.
  • an inducer may be added to the medium as necessary.
  • an inducer may be added to the medium.
  • an inducer may be added to the medium.
  • tistrepton may be added to the medium.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the derivative of the protein of the present invention or a modified sugar chain thereof 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.
  • the protein of the present invention 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.
  • 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.
  • 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.
  • 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.
  • a culture solution of a microorganism used as an enzyme source can be used as an aqueous medium.
  • 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)
  • 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
  • Surfactants are usually used at a concentration of 0.;!-50 g / l.
  • 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.
  • (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.
  • a carbon source such as glycerol or an inorganic salt such as MnCl can be added as necessary.
  • Quantification of (2S, 4S) -5-hydroxyleucine produced in an 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.
  • ATCC29133 Genomic DNA of Nostoc SD.
  • ATCC29133D 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.
  • 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.
  • 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
  • BB-4 SEQ ID NO: 13
  • primer-type (AA-1 and AA-2) 0 ⁇ ⁇ ⁇ ⁇ / ⁇ ⁇ —Plus-Polymerase (Toyobo Co., Ltd.) 1. Ounits, KOD—plus—X for polymerase 10 Slow impact 5 1, reaction containing deoxyNTP ⁇ 200 ⁇ mol, MgSO ImM
  • reaction solution 2.5 1 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 ⁇ mol each, MgSO ImM reaction ⁇ ⁇ 50 ⁇ 1
  • the process was repeated 15 times at 94 ° C for 30 seconds, 60 ° C for 30 seconds and 68 ° C for 1 minute.
  • 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.
  • the reaction solution obtained by PCR was incorporated into E. coli plasmid pTA2 using a TArget Clone TM -Plus- kit (manufactured by Toyobo Co., Ltd.). That is, 91 PCR reaction solution and 1 ⁇ l of 10 X A-attachment Mix (manufactured by Toyobo Co., Ltd.) were added and stirred and reacted at 60 ° C for 10 minutes.
  • 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.
  • 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 ⁇ 1, deo xyNTP ⁇ 200 i mol, MgSO ImM for 15 seconds at 94 ° C.
  • the process was repeated 25 times at 55 ° C for 30 seconds and 68 ° C for 1 minute.
  • the reaction solution obtained by PCR was incorporated into Escherichia coli plasmid pTA2 using a TArget Clone TM -Plus- kit (manufactured by Toyobo Co., Ltd.). That is, 91 PCR reaction solution and 1 ⁇ l of 10 X A-attachment Mix (manufactured by Toyobo Co., Ltd.) were added and stirred and reacted at 60 ° C for 10 minutes.
  • 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.
  • a recombinant plasmid that functions in mvces ikite was implemented by the following method.
  • 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.
  • 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.
  • an about 7.3 kb DNA fragment was recovered from an agarose gel.
  • 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. .
  • the obtained Escherichia coli recombinant (E.coli / pnos5A strain) wet microbial cell is composed of lOOmmol / 1 sodium phosphate buffer ⁇ ⁇ ( ⁇ 7.0), 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.
  • 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!
  • HPLC high performance liquid chromatography
  • 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.
  • 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.
  • 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.
  • 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!
  • HPLC high performance liquid chromatography
  • 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.
  • 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.
  • 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.
  • the S.lividans / pnos5E strain wet cells obtained above were used in a 20 mL reaction consisting of lOOmmol / 1 sodium phosphate buffer ( ⁇ 7.0), 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.

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Abstract

La présente invention concerne une nouvelle protéine présentant une activité L-leucine hydroxylase, c'est-à-dire une activité de conversion de la L-leucine en (2S,4S)-5-hydroxyleucine, ainsi qu'un ADN codant pour la protéine, un ADN recombinant contenant l'ADN, un transformant portant l'ADN recombinant, la protéine utilisant le transformant et un procédé permettant de produire la (2S,4S)-5-hydroxyleucine. Il devient possible d'élaborer un procédé permettant de produire la (2S,4S)-5-hydroxyleucine, une substance prévue pour être utilisée comme matériau de départ pour la production d'une substance utile telle qu'un agent pharmaceutique, en utilisant un organisme, une enzyme ou des éléments analogues. Le procédé permet de produire la (2S,4S)-5-hydroxyleucine avec une grande efficacité.
PCT/JP2007/064761 2006-07-28 2007-07-27 L-leucine hydroxylase et adn codant pour l'enzyme WO2008013262A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011021717A3 (fr) * 2009-08-21 2011-05-26 Ajinomoto Co.,Inc. Procédé de production d'acides aminés hydroxylés
CN109576234A (zh) * 2018-12-26 2019-04-05 天津科技大学 一种亮氨酸-5-羟化酶突变体及其应用

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [online] XP003020713, Database accession no. (NZ_AAAY02000011) *
HOFFMANN D. ET AL.: "Sequence analysis and biochemical characterization of the nostopeptolide A biosynthetic gene cluster from Nostoc sp. GSV224", GENE, vol. 311, 2003, pages 171 - 180, XP004437336 *
LARSSON M. ET AL.: "High-throughput protein expression of cDNA products as a tool in functional genomics", J. BIOTECHNOL., vol. 80, no. 2, 2000, pages 143 - 157, XP004213504 *
LUESCH H. ET AL.: "Biosynthesis of 4-Methylproline in Cyanobacteria: Cloning of nosE and nosF Genes and Biochemical Characterization of the Encoded Dehydrogenase and Reductase Activities", J. ORG. CHEM., vol. 68, no. 1, 2003, pages 83 - 91, XP003020714 *
MISAWA K. ET AL.: "A method to identify cDNAs based on localization of green fluorescent protein function products", PROC. NATL. ACAD. SCI. U.S.A., vol. 97, no. 7, 2000, pages 3062 - 3066, XP002290453 *
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011021717A3 (fr) * 2009-08-21 2011-05-26 Ajinomoto Co.,Inc. Procédé de production d'acides aminés hydroxylés
CN109576234A (zh) * 2018-12-26 2019-04-05 天津科技大学 一种亮氨酸-5-羟化酶突变体及其应用
CN109576234B (zh) * 2018-12-26 2021-05-07 天津科技大学 一种亮氨酸-5-羟化酶突变体及其应用

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