JP2003325175A - Method for producing 25-hydroxy vitamin d - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for efficiently producing a 25-hydroxyvitamin D by directly introducing a hydroxy group into the 25-position of a vitamin D. <P>SOLUTION: The cell body of a bacterium which belongs to the genus Bacillus and hydrates the vitamin D or the cell body of a transformant obtained by isolating a hydroxylase gene of the 25-position of the vitamin D from the bacterium, inserting the gene into a vector and transferring the vector into which the gene is inserted to a host or a treated material thereof is brought into contact with the vitamin D in an aqueous medium. The 25-hydroxyvitamin D formed and accumulated is collected from the aqueous medium. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing 25-hydroxyvitamin Ds, and more specifically, biologically producing 25-hydroxyvitamin Ds from vitamin Ds by using bacteria belonging to the genus Bacillus. It relates to a method of manufacturing.

[0002]

2. Description of the Related Art Vitamin Ds are important vitamins related to absorption of calcium in the body, promotion of calcium metabolism in bones, induction of cell differentiation, and immunoregulation, and their activity is 2 in the liver.
It is known that it is expressed by hydroxylation at position 5 and 1α in the kidney (HF Delka and HK Jones: Annual Review of Biochemistry No. 52).
Vol. 411 (1983)). However, 25-hydroxyvitamin D ₃ is in patients with hepatic dysfunction are insufficient biosynthesis amount in vivo. Thus, the administration of 25-hydroxyvitamin D ₃ such to the patient is effective from the viewpoint of compensating for the shortage in vivo. Again 1
α, 25-dihydroxyvitamin D ₃ has a particularly strong activity and is particularly effective for renal failure patients in the same meaning as above. Therefore, 25-hydroxyvitamin D ₃ and 1α, 25-
An inexpensive method for producing dihydroxyvitamin D ₃ is extremely useful in industry.

No organic chemical method for directly introducing a hydroxyl group at the 1α and / or 25-position of vitamin Ds is known. On the other hand, it has been conventionally known that a hydroxyl group is directly introduced into the 1α and / or 25-position of the starting vitamin Ds by an enzymatic method using an animal organ, but it is an inefficient and industrially practical method. Was not. In recent years, 1α-position of vitamin Ds and / or by enzymatic method using microorganisms
Alternatively, a method of using actinomycetes has been disclosed as a method of hydroxylating the 25th position (Japanese Patent Laid-Open No. 2-469 and Japanese Patent Laid-Open No. 2-469).
231089).

[0004]

However, the production of 25-hydroxylated vitamin Ds and 1α, 25-dihydroxyvitamin Ds from vitamin Ds by an enzymatic chemical method using the above-mentioned microorganisms has been disclosed. JP-A-2-
According to Japanese Patent Application Laid-Open No. 469 and Japanese Patent Laid-Open No. 2-231809),
For example, the hydroxylation activity from the substrate vitamin D ₃ to 25-hydroxyvitamin D ₃ or vitamin D ₃ to 1α, 25-dihydroxyvitamin D ₃ is very weak, the substrate specificity is low, and it is suitable for the preparation of microorganisms and reaction solutions. There are still problems to be solved as an industrial manufacturing method such as labor-intensive, and a new microorganism that is a more suitable enzyme source for gene recombination, with a view to mass expression of enzymes by gene recombination. A search for sources was desired.

The present invention has been made for the purpose of providing a method for efficiently producing 25-hydroxyvitamin Ds from vitamin Ds by a biological method using a microorganism or an enzyme derived from the microorganism, which has not been reported so far. It is a thing.

[0006]

Means for Solving the Problems In the conversion of vitamin Ds to 25-hydroxyvitamin Ds using a microorganism, the present inventors have conducted extensive studies for the purpose of searching a new microbial source, and as a result, The present invention has been completed by finding that it is possible to efficiently introduce a hydroxyl group directly into the 25-position of vitamin Ds by utilizing Bacillus bacterium.

[0007] That is according to the present invention, Bacillus (Bacill
us ) contacting the bacterial cells of the bacterium belonging to the genus and capable of hydroxylating vitamin Ds or a treated product thereof with vitamin Ds in an aqueous medium to produce and accumulate 25-hydroxyvitamin Ds, There is provided a method for producing 25-hydroxyvitamin Ds, which comprises collecting the aqueous medium from the medium.

According to another aspect of the present invention, the vitamin Ds are converted to 25-hydroxyvitamin Ds from bacteria belonging to the genus Bacillus and having the ability to hydroxylate the 25th position of the vitamin Ds. Ability to hydroxylate the 25-position of vitamin Ds, characterized by isolating a gene encoding an enzyme, inserting the obtained gene into a vector, and introducing the vector into which the gene has been inserted into a host. A method for producing a transformant having the following: a transformant obtained by the method; and contacting the transformant or a treated product thereof with vitamin Ds in an aqueous medium to produce 25-hydroxyvitamin Ds There is provided a method for producing 25-hydroxyvitamin Ds, which comprises accumulating and collecting this from the aqueous medium.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The microorganism used in the present invention belongs to the genus Bacillus, oxidizes vitamin Ds, that is, converts the hydrogen atom at the 25th position of vitamin Ds into a hydroxyl group, and converts 25-hydroxyvitamin Ds into Any bacteria may be used as long as they have the ability to generate. Specifically, Bacillus megaterium IFO1
2108, Bacillus megaterium (Bacillus megater
ium ) IFO15308, Bacillus megaterium ( Baci
llus megaterium IAM1111, Bacillus megaterium IAM1166, Bacillus megaterium IAM124
5 and the like. Among these, those having an IFO number are conserved strains of the Institute for Fermentation Research Institute, and those having an IAM number are preserved strains of the Institute for Molecular Cell Biology, The University of Tokyo, and they can be easily obtained. The mycological properties have already been clarified.

The method of the present invention is carried out by reacting the substrate vitamin Ds under aerobic conditions in a solution containing cells of Bacillus bacteria or a treated product thereof. Specifically, the bacterial cells obtained by culturing the above-mentioned microorganism as they are,
Alternatively, a treated product such as a microbial cell obtained by culturing, treated with an organic solvent, lyophilized, or crushed physically or enzymatically can be used. Further, it is also possible to take out an enzyme fraction having a capability of acting on vitamin Ds to hydroxylate the hydrogen atom at the 25th position from these bacterial cells or treated products as a crudely purified product or a purified product and use it. is there. Further, it has a recombinant DNA obtained by incorporating a single or multiple genes specified based on the purified enzyme into a vector and using Escherichia coli, Bacillus subtilis, coryneform bacterium, yeast, etc. as a host. Transformants can also be used. Furthermore, it is also possible to use the thus obtained bacterial cells, treated products, enzyme fractions, transformants or the like immobilized on a carrier such as polyacrylamide gel or carrageenan gel. Therefore,
In the present specification, the term “bacterial body or treated product thereof” is used as a concept including all of the above-mentioned bacterial bodies, processed products, enzyme fractions, immobilized products thereof and the like.

The transformant having the ability to hydroxylate the 25-position of vitamin Ds of the present invention is the above-mentioned Bacillus ( Bacill).
us ) was isolated from a bacterium belonging to the genus and having the ability to hydroxylate the 25-position of vitamin Ds, and the gene encoding the enzyme that converts vitamin Ds to 25-hydroxyvitamin Ds was isolated. It can be obtained by inserting it into a vector and introducing the vector into which the gene has been inserted into a host. This transformant can be prepared by a commonly used method known per se, for example, the following method.

First, from a bacterium belonging to the genus Bacillus and having the ability to hydroxylate the 25-position of vitamin Ds, an enzyme for converting vitamin Ds to 25-hydroxyvitamin Ds (hereinafter referred to as "vitamin"). Isolation of a gene encoding the "Ds 25-position hydroxylase" may be abbreviated. (Hereinafter, this may be abbreviated as "vitamin Ds 25-position hydroxylase gene.") Can be done by the method.

The vitamin D 25-position hydroxylase gene is
The enzyme protein was purified from a bacterium belonging to the above genus Bacillus and capable of hydroxylating the 25-position of vitamin Ds, and a DNA probe was synthesized based on the amino acid sequence of the bacterium, and the genus Bacillus was genus. , Which has the ability to hydroxylate the 25-position of vitamin Ds. Microorganisms that are the source of this belong to the genus Bacillus, oxidize vitamin Ds, and
Any bacteria may be used as long as they have the ability to purify 5-hydroxyvitamin Ds. Specifically, the above Bacillus megateriu
m) IFO12108, Bacillus megaterium (Bacil
lus megaterium IFO15308, Bacillus megaterium IAM1111, Bacillus megaterium IAM11
66, Bacillus megaterium
IAM1245 etc. are mentioned.

The vitamin D 25-position hydroxylase gene is
Usually, it is present on the chromosome of the above-mentioned source microorganism and can be isolated and obtained from the DNA bank derived from the chromosome by the hybridization method described below. (1) Purification of vitamin D 25-hydroxylase and partial determination of its amino acid sequence and preparation of a DNA probe based on it: Purification of vitamin D 25-hydroxylase itself or bacterial cells such as the above strains A known method can be used in which bacterial cells are destroyed and the enzyme is purified from the disrupted product.

Examples of the method for destroying the cells include ultrasonic disruption; mechanical disruption using a French press, homogenizer, etc .; enzymatic disruption using lysozyme. The vitamin D 25-position hydroxylase can be obtained as a crude enzyme solution by separating the soluble fraction from the disrupted product of the cells. The method for purifying the vitamin D 25-position hydroxylase from the obtained crude enzyme solution is usually (a) separation by a precipitation method, for example, ammonium sulfate precipitation method,
(B) Separation methods by chromatography, such as ion exchange chromatography, affinity adsorption chromatography, gel filtration chromatography, etc., and (c) separation methods by electrophoresis can be combined.

The detection of vitamin D 25-hydroxylase in the above-mentioned purification process is a general method of detecting vitamin D 25-hydroxylase activity from the blood by the Bligh & Dyer method ( Can. J. Biochem., 37, 911
(1959)), it can be carried out by quantifying vitamin Ds and 25-hydroxyvitamin Ds in the reaction solution.

Purified vitamin D 25-hydroxylase was purified by the Davis method [BJDavis, Ann.NYAcad.Sci., Vol.12].
1, p.404 (1964)] or Laemmli method [UK Laemmli, Natu
Re, Vol.227, p.680 (1970)] and subjected to polyacrylamide gel electrophoresis, and Coomassie blue dye solution [composition:
0.2% (w / v) Coomassie Brilliant Blue R25
0, 40% (v / v) methanol, 10% (v / v) acetic acid], or purified enzyme and vitamin Ds 25 by staining the enzyme protein using a silver staining kit (manufactured by Wako Pure Chemical Industries, Ltd.) It is possible to know the molecular weight of the position hydroxylase.

The partial determination of the amino acid sequence of the purified vitamin D 25-hydroxylase is carried out by separating the enzyme into its constituent subunits, and then the amino acid sequence from the N-terminal of each subunit, or an appropriate amino acid sequence. It is carried out by determining the amino acid sequence from the N terminus of the peptide fragment obtained by digesting each subunit with a protease, using an amino acid sequencer by the Edman degradation method (Applied Biosystems, 473A type). You can

Next, a probe having a nucleotide sequence predicted from the obtained amino acid sequence is synthesized using a DNA synthesizer (Applied Biosystems 394 type). The probe DNA can be designed based on the amino acid sequence of the purified enzyme as described above, but it is also designed from the known nucleotide sequence of the vitamin D 25-position hydroxylase gene (WO94 / 00576). You can also (2) Isolation of a chromosomal DNA fragment containing the vitamin D 25-position hydroxylase gene: A method for isolating a chromosomal DNA containing the vitamin D 25-position hydroxylase gene from bacterial cells such as the above-mentioned strain is a gene Any known method for the isolation of An example will be described below.

(A) Preparation of chromosomal DNA library:
When extracting the chromosomal DNA from the above strain, cells of the strain cultured in an appropriate medium can be used,
It is also possible to use a preserved sample obtained by cryopreserving the cultured bacterial cells after collecting the bacterial cells.

The obtained chromosomal DNA is treated with an appropriate restriction enzyme,
For example, XhoI, Sau3AI, TaqI and the like are partially digested, and a chromosomal DNA library is prepared using a host-vector system such as Escherichia coli. As a vector that can be specifically used,
For example, lambda phage vector such as λFIXII (manufactured by Toyobo Co., Ltd.), pUC118 (manufactured by Takara Shuzo), pBR32
2 (manufactured by Takara Shuzo) and plasmid vectors such as cosmid pWE15 (manufactured by Stratagene).

Various DNAs obtained by the above partial decomposition
Insertion of the fragment into the above vector, for example, the phage vector λFIXII (manufactured by Toyobo Co., Ltd.) is carried out by using T4 DNA obtained by cleaving the vector and the partially digested DNA fragment with a suitable restriction enzyme such as XhoI, Sau3AI, TaqI.
It can be performed by ligation using ligase. A chromosomal DNA library is thus obtained.

(B) Selection of vector containing vitamin D 25-hydroxylase gene: To select vector containing vitamin D 25-hydroxylase gene from the chromosomal DNA library prepared in the above (A) Is this chromosome D
The NA library is used to transduce or transform a host microorganism, for example, Escherichia coli, and the vitamin D 25-position hydroxylase gene is obtained from the resulting transductant or transformant by an appropriate means. It suffices to select a clone that retains.

[0024] Specifically, the above-mentioned phage vector is prepared by using Escherichia coli such as P239.
2 strains [Ausubel et al., Nucleic Acid Res. Vol.7, p.
1513 (1979)], and plaques are formed by overlaying this on an agar medium. The phage DNA in this plaque is then transferred to a nitrocellulose membrane,
This phage DNA was immobilized on the nitrocellulose membrane,
Plaque hybridization [Molecular Cloning, Cold Spring Harbor Laboratory] using the probe prepared in the above (1), for example, a synthetic oligonucleotide.
Press (1989)].

The probe DNA used for hybridization can be designed based on the amino acid sequence of the purified enzyme as described above, but it is also known that the nucleotide sequence of the known vitamin D 25-position hydroxylase gene (WO94 / 005
It can also be designed from 76). In this way, it is possible to detect and select a transductant containing a phage vector having a vitamin D 25-position hydroxylase gene derived from the chromosomal DNA of the target strain. Alternatively, when a chromosomal DNA library is prepared using the above plasmid vector, Escherichia coli JM109 (Takara Shuzo) is transformed with this library DNA, and the obtained transformant is used to transform the Colony hybridization method [R. Bruce Wallace, et al., Nuc.Acid.Res., Vol.9,
p.879 (1981)].

Furthermore, phage DNA or plasmid DNA is extracted from the transformants selected as described above, and the inserted fragment is excised from the vector with an appropriate restriction enzyme to obtain a DNA fragment containing the DNA of the present invention. can do. For the DNA fragment cut out by the above operation, Southern hybridization [EM Southern, J. Mol. Bio was performed using the probe prepared in (1) above.
, Vol. 98, p. 503 (1975)], it can be reconfirmed that the vitamin D 25-position hydroxylase gene is present in the inserted DNA fragment.

(3) Determination of nucleotide sequence: The entire nucleotide sequence of the DNA fragment obtained in the above item (2) can be obtained, for example, from plasmid p.
The dideoxynucleotide enzyme method [dideoxy chain termin using the Bluescript vector (Stratagene), the same pUC118 vector (Takara Shuzo), etc.
ation method Sanger, F.et.al., Proc.Natl.Acad.Sci.USA, V
ol.74, P.5463 (1977)].

From the base sequence of the above DNA fragment thus determined, an open reading frame (O
The presence of the vitamin D 25-position hydroxylase gene of each strain can be estimated by searching for the presence of RF). In addition, by searching the obtained base sequence for the presence of a sequence corresponding to the expected base sequence from the amino acid sequence of the enzyme protein determined in (1) above, the gene is found in the obtained DNA fragment. You can confirm that it is included.

Vitamin Ds 2 including the above base sequence
The 5-position hydroxylase gene is not limited to that isolated from natural chromosomal DNA of bacteria, but may be a commonly used DNA synthesizer, such as Applied Biosystems (Applied Biosystems).
It may be synthesized using a 394 DNA / RNA synthesizer manufactured by Biosystems). Bacillus ( Ba
From the bacterium belonging to the genus Cillus ) and having the ability to hydroxylate the 25-position of vitamin Ds, the vitamin D 25-position hydroxylase gene can be incorporated into an appropriate vector and used as a recombinant vector.

The vector can be selected in consideration of the combination with the host, and is preferably capable of autonomous replication in the host cell or integration into the chromosome and contains a promoter at a position where the gene of the present invention can be transcribed. The existing one is used. When the bacterium of the present invention is used as a host cell, the expression vector for expressing DNA is capable of autonomous replication in the bacterium, and at the same time, a promoter,
A recombinant vector composed of a ribosome binding sequence, the above DNA and a transcription termination sequence is preferable. A gene that controls the promoter may be included.

Examples of expression vectors for bacteria include, for example:
pBTrP2, pBTac1, pBTac2 (all commercially available from Behringer Mannheim), pKK233-2 (Pharmacia), pSE280 (I
nvitrogen), pGEMEX-1 (Promega), pQE-8 (QIAGE
N company), pQE-30 (QIAGEN company), pKYP10 (JP-A-58-11060)
0), pKYP200 [Agrc.Biol.Chem., 48, 669 (1984)], PLS
A1 (Agrc. Blo1. Chem., 53, 277 (1989)), pGEL1 (Pro
c. Natl. Acad. Sci. USA, 82, 4306 (1985)], pBlues
crlpt II SK (+), pBluescript II SK (-) (Stratagene), pTrS30 (FERMBP-5407), pTrS32 (FERM BP-5408), pG
EX (Pharmacia), pRSET, pTrcHis, pTrcHis2 (all Invitrogen), pET-3 (Novagen), pTerm2 (US46)
86191, US4939094, US5160735), pSupex, pUB110, pTP
5, pC194, pUC18 (Gene, 33, 103 (1985)), pUC19 (Gen
e, 33, 103 (1985)], pSTV28 (Takara Shuzo), pSTV29 (Takara Shuzo), pUC118 (Takara Shuzo), pPA1 (JP-A-63-23379)
8), pEG400 [J. Bacterio 1., 172, 2392 (1990)] and the like. As a promoter for bacteria,
For example, trp promoter (P trp), lac promoter (P trp)
lac), trc promoter (P trc), T7 promoter (PT
7), a promoter derived from Escherichia coli or phage, such as PL promoter, PR promoter, PSE promoter,
Examples thereof include SP01 promoter, SP02 promoter, penP promoter and the like.

As an expression vector for yeast, for example, pY
ES2 (manufactured by Invitrogen), pESC, pESP (all manufactured by Stratagene), pAUR (manufactured by Takarasha), YEp13 (ATCC3711)
5), YEp24 (ATCC37051), Ycp5O (ATCC37419), pHS19, pHS
15 etc. can be illustrated. Examples of the promoter for yeast include PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal1 promoter, gal10 promoter, heat shock protein promoter, MFα1 promoter, CUP1 promoter and the like.

Examples of expression vectors for animal cells include pcDNAI, pcDM8 (commercially available from Funakoshi), pAGE107.
[JP-A-3-22979; Cytotechnology, 3, 133, (1990)],
pAS3-3 (JP-A-2-227075), pCDM8 (Nature, 329, 840, (1
987)], pcDNAI / AmP (manufactured by Invitrogen), pREP4 (Invit
rogen), pAGE103 (J. Blochem., 101, 1307 (198
7)], pAGE210 and the like. Examples of promoters for animal cells include, for example, IE (immediate early) gene promoter of cytomegalovirus (human CMV), SV40 early promoter, retrovirus promoter, metallothionein promoter, heat shock promoter, SRα promoter and the like. You can

As an expression vector for plant cells, for example, pIG121-Hm [Plant Cell Report, 15, 809-814 (199
5)], pBI121 [EMBO J. 6, 3901-3907 (1987)] and the like can be exemplified, and as a promoter for plant cells,
Examples include cauliflower mosaic virus 35S promoter [Mol. Gen. Genet (1990) 220, 389-392] and the like.

From a bacterium belonging to the genus Bacillus and having the ability to hydroxylate the 25-position of vitamin Ds, a transformant having the 25-hydroxylase gene of vitamin Ds can be prepared by using the above recombinant vector. It can be prepared by introducing it into a host. Specific examples of bacterial host cells include Escherichia, Corynebacterium, Brevibacte.
genus rium, genus Bacillus, genus Microbacterium, genus Serratia,
Pseudomonas, Agrobacterium, Alicyclobacillus
Genus, Anabaena, Anacystis, Arthrobacter, Azoba
cter, Chromatium, Erwinia, Methylobacterium
Genus, Phormidium, Rhodobacter, Rhodopseudomonas
Genus, Rhodospiri11um, Scenedesmun, Streptomyces
Examples thereof include microorganisms belonging to the genus, Synnecoccus genus, Zymomonas genus, and the like. Examples of the method for introducing the recombinant vector into the bacterial host include a method using calcium ions, an electroporation method, a protoplast method, and the like.

Specific examples of yeast hosts include Saccharomyces cerevisae, Schizosaccharomyces pombe, Kluyveromyces lactis,
Trichosporon pu11ulan
s), Schwanniomyces a1
1 uvius) and the like.

As a method for introducing a recombinant vector into a yeast host, any method can be used as long as it is a method for introducing DNA into yeast, and examples thereof include electroporation method, spheroblast method and lithium acetate method. be able to. Examples of animal cell hosts include Hela cells, Namalwa cells, COS1 cells, COS7 cells, CHO cells and the like.

As a method for introducing the recombinant vector into animal cells, any method capable of introducing DNA into animal cells can be used, and for example, the electroporation method, calcium phosphate method, lipofection method and the like can be used. In the case of transforming a plant cell, the host can be arbitrarily selected according to the purpose of use of the transformant. The type of plant into which the 25-hydroxylase gene of vitamin Ds can be introduced is not particularly limited, but examples thereof include oilseed crops such as rapeseed, soybean, sunflower and palm palm; and cereals such as rice, corn and wheat. Examples thereof include various vegetables such as cabbage and lettuce; trees and flowers.

In the present specification, examples of the transformed plant source include seeds, seedlings, seedlings, calli, cultured cells, plants and the like. For example, in the case of rapeseed, seedlings or protoplasts; in the case of soybean, seedlings , Callus or cultured cells; seedlings for sunflower; callus or cultured cells for palm palm; seedlings, callus, cultured cells or protoplasts for rice; seedlings, callus, culture for corn Cells or protoplasts; in wheat, sprout, callus or cultured cells; in cabbage, sprout, callus, cultured cells or protoplasts; in lettuce, sprout,
As in the case of callus, cultured cells, protoplasts, etc., it may be carried out by appropriately selecting a preferable site depending on the target plant, as is usually done by those skilled in the art.

The method for transforming a plant can be carried out by a conventional method. For example, a method using an Agrobacterium method, an electroporation method, a DEAE dextran method, a calcium phosphate method, a polyethylene glycol method, a particle gun method or the like can be used. DNA direct introduction method can be mentioned. The expression cassette to be incorporated can be prepared by a conventional method using a known plasmid.

As described above, the "transformant" in the present invention means a bacterium, yeast, animal cell, plant cell, plant or the like into which the vitamin D 25-position hydroxylase gene has been introduced. In the method for producing 25-hydroxyvitamin Ds of the present invention, the cells or transformant necessary for the reaction are usually obtained by culturing. Culture of the bacterial cells can be carried out by a commonly used method known per se. The medium used for the culture is mainly a liquid medium, and the carbon source is glucose, maltose, fructose,
Sucrose, glycerin, etc. are used alone or as a mixture. As the nitrogen source, organic nitrogen sources such as polypeptone, peptone, casamino acid, yeast extract, meat extract, and corn steep liquor and inorganic nitrogen sources such as ammonium sulfate and sodium nitrate are used alone or in combination. In addition to these components, organic substances and inorganic salts that help the growth of the present microorganism and promote the production of vitamin Ds having a hydroxyl group introduced at the 25-position can be added as necessary. For the culture, the pH of the medium is adjusted to a range of 4 to 10 and the temperature is 20 to
It is preferable to carry out at 45 ° C. for 1 to 10 days until the maximum activity is reached.

The bacterial cells obtained by this culturing or a treated product thereof and vitamin Ds are brought into contact with each other in an aqueous medium to react with each other to obtain 25-hydroxyvitamin Ds as a reaction product. Examples of the aqueous medium used here include water, a buffer solution, a culture solution, and the like, and this aqueous medium may appropriately contain a water-soluble organic solvent or a fat-soluble organic solvent. In the reaction, glucose is added to the reaction solution,
It is also possible to add a carbon source such as sucrose or fructose as an energy source and, if the yield is improved, to add those carbon sources appropriately. If necessary, cyclodextrins, a surfactant, an organic substance, an inorganic salt or the like that promotes the production of desired 25-hydroxyvitamin Ds can be added to the solution.

Cyclodextrins include α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, methyl-β-cyclodextrin and β-cyclodextrin.
Dimethyl cyclodextrin, β-trimethyl cyclodextrin, partially methylated cyclodextrin, branched methylated cyclodextrin and the like can be mentioned. As the surfactant, a polyoxyethylene sorbitan fatty acid ester (for example, Tween80 manufactured by Sigma), a sorbitan fatty acid ester (for example Span85 manufactured by Sigma) as a nonionic surfactant, and a bridge which is another polyoxyethylene derivative ( Brij / manufactured by Sigma),
Triton (Triton / manufactured by Sigma), Nonidet P45, and anionic surfactants such as Direx (manufactured by NOF Corporation) and Trax (manufactured by NOF Corporation) can be used.

The concentration of cyclodextrins to be added is 0.1 to 15% by weight, preferably 0.1 to 2% by weight, in the medium or reaction solution. The concentration of the surfactant to be added is 0.01 to 5% by weight in the medium or the reaction solution, preferably 0.05 to 0.5.
Weight percent is suitable. The production method of the present invention is preferably carried out under aerobic conditions in a solution containing the cells and / or a treated product thereof by shaking, aeration and stirring, and the like, and the pH is 4 to 10, 20. Shake with shaking at -40 ° C for 5 minutes to 1 week. It can also react under an oxygen stream. Vitamin D as a substrate is added in an appropriate amount at the start of stirring and shaking, but can be added sequentially during the reaction if necessary. When using a culture solution containing the cells in culture, after adding the substrate vitamin Ds, 24 to 16 under the same conditions.
Incubate for 8 hours to carry out hydroxylation reaction. Vitamin D when added
In addition to being added as it is, it is also possible to add the one dissolved in advance in a water-soluble low-molecular organic compound such as methanol or ethanol.

The concentration of the substrate vitamin D to be added is 10 to 1000 μg / ml, preferably 50 to 5
It is desirable to add in the range of 00 μg / ml. The production method of the present invention is a method of directly introducing a hydroxyl group into the 25-position of vitamin Ds in one step. As the vitamin D used as a substrate, any vitamin D having a hydrogen atom at the 25-position may be used.
Other than the 25-position of the class, those having a halogen atom such as a fluorine atom, a hydroxyl group or a lower alkyl group may be used. Specifically, for example, vitamin D ₂ -based and vitamin D ₃ -based compounds, such as vitamin D ₂ , vitamin D ₃ ,
1α-hydroxyvitamin D ₃ , 1α, 24-dihydroxyvitamin D _{3 and the} like can be mentioned.

The reaction time can be determined by confirming the product by HPLC described in Examples below. To isolate the vitamin Ds produced by these reactions, Bl is a general method for detecting vitamin Ds in blood.
The method of igh & Dyer (Can. J. Biochem., 37, 911 (1959))
You can follow the procedure in. For example, after the reaction is completed, the bacterial cells or treated products thereof are removed by centrifugation or the like, and then the reaction solution is extracted with an organic solvent such as ethyl acetate, methylene chloride, chloroform or the like and concentrated. After dissolving this in a suitable solvent and removing insolubles by centrifugation, the target hydroxyvitamin Ds can be isolated by using a known method such as distillation, column chromatography, high performance liquid chromatography and the like. .

The above-mentioned production method has been described mainly for the case of using bacterial cells or treated products thereof. However, even if transformants such as yeast, animal cells, plant cells and plants are used, they are known per se. The method and 25-hydroxyvitamin Ds can be produced according to the above method. The method of the present invention has made it possible to efficiently introduce a hydroxyl group directly into the 25-position of vitamin Ds. That is, for example, in the method using Bacillus bacterium, preparation of microbial cells, reaction solution, etc. does not require any labor, and it is extremely easy and efficient to perform 25
-Hydroxyvitamin Ds can be produced. Further, by using a transformant obtained by a method such as gene recombination, it is possible to dramatically improve the productivity of 25-hydroxyvitamin Ds.

[0048]

The present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples. Example 1 Glucose 0.5%, yeast extract 0.5%, nutrient broth (manufactured by Difco) 0.8%, calcium carbonate 0.2%, pH 7.0 sterile liquid medium (hereinafter referred to as "A medium") One 21 φ test tube containing 8 ml was inoculated with 1 platinum loop of Bacillus megaterium IFO12108 strain, and shake culture was performed at 30 ° C. for 20 hours. After completion of the culture, the culture solution was centrifuged to remove 6 ml of the medium aseptically and resuspended. Sterilized in this concentrated cell culture medium 4
Add 0.1 ml of 0% glucose solution, and add 20μ
Substrate vitamin D ₃ 0.4 mg dissolved in 1 L of ethanol
Was added and the culture was continued at 30 ° C. for 96 hours. The product was confirmed by thin layer chromatography on silica gel (Kieselgel 60F254, manufactured by Merck) (developing solvent; dichloromethane: methanol = 15: 1, spots detected by ultraviolet absorption). Commercially available 25-hydroxyvitamin D ₃ (reagent name calcifediol, manufactured by Wako Pure Chemical Industries, Ltd.) was used as a standard compound.

The reaction solution was extracted with 5 ml of ethyl acetate,
The mixture was concentrated to dryness while purging with nitrogen gas at room temperature, immediately dissolved in 100 μl of a mixed solvent of hexane / ethanol = 95/5, and analyzed by normal phase HPLC [column; Cosmocil 5SL-II.
(Manufactured by Nacalai Tesque), eluent: hexane / ethanol = 95/5, flow rate: 1 ml / min, UV: 265 nm, column temperature: 30 ° C.]. Vitamin D ₃ metabolites were identified by comparison with retention times of standard compounds. As a result, the accumulated concentration of 25-hydroxyvitamin D ₃ was 0.6.
It was μg / ml.

Example 2 Bacillus megaterium IFO12108 strain was grown at 30 ° C. in the A medium (8 ml / 21φ test tube) used in Example 1.
Shaking culture was performed for 20 hours. After the culture was completed, the culture solution was centrifuged to collect the bacterial cells. The cells were suspended in 8 ml of 50 mM potassium phosphate buffer solution (hereinafter abbreviated as "buffer solution A") having a pH of 7 and containing 2% of glucose, and centrifuged again to collect the cells. This Bacillus megaterium IFO12108
Add 2 ml of Buffer A to the washed cells of the strain, and add 20μ
Substrate vitamin D ₃ 0.4 mg dissolved in 1 L of ethanol
Was added and reacted at 30 ° C. for 96 hours. After completion of the production reaction, the reaction solution was extracted with 5 ml of ethyl acetate, concentrated to dryness at room temperature while purging with nitrogen gas, and the accumulated concentration of 25-hydroxyvitamin D ₃ was confirmed by the method described in Example 1. A value of 1.1 μg / ml was obtained.

Example 3 30 ml of Bacillus megaterium IFO12108 strain was added to the A medium (10 ml / 21φ test tube) used in Example 1.
Shaking culture was performed at 20 ° C. for 20 hours, and 1 ml of a 5% methyl-β-cyclodextrin solution and Tween were added to the culture solution.
20 mg of n80 and 0.5 ml of 40% glucose solution
2 mg of the substrate vitamin D ₃ dissolved in 100 μl of ethanol was added, and the culture was continued at 30 ° C. for 72 hours. The reaction solution was extracted with 25 ml of ethyl acetate,
The mixture was concentrated to dryness at room temperature while substituting with nitrogen gas, and the accumulated concentration of 25-hydroxyvitamin D ₃ was confirmed by the method described in Example 1, and a value of 3.5 μg / ml was obtained.

Example 4 As a strain to be used, the Bacillus megaterium strain shown in Table 1 was used, and the medium A (10 ml / 2) used in Example 1 was used.
Shake culture at 30 ° C for 20 hours in a 1φ test tube)
To this culture medium, 1 ml of 5% methyl-β-cyclodextrin solution, 20 mg of Tween 80 and 0.5 ml of 40% glucose solution were added, and 2 mg of substrate vitamin D ₃ dissolved in 100 μl of ethanol was added to give 3
Culture was continued at 0 ° C. for 96 hours. The reaction solution was extracted with 25 ml of ethyl acetate, concentrated to dryness at room temperature while purging with nitrogen gas, and the accumulated concentration of 25-hydroxyvitamin D ₃ confirmed by the method described in Example 1 is shown in Table 1 together with the strains used. Show.

[0053]

Strain No. 25- Hydroxyvitamin D ₃ Accumulation amount (μg / ml) IFO12108 9.5 IFO15308 4.1 IAM1111 2.0 IAM1166 7.3 IAM1245 9.5 As is clear from the results in Table 1, vitamin D By contacting ₃ with a Bacillus megaterium strain,
It can be seen that hydroxyvitamin D ₃ is efficiently produced.

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Claims (6)

[Claims] 1. A bacterial cell belonging to the genus Bacillus and having the ability to hydroxylate the 25-position of vitamin Ds, or a treated product thereof, and vitamin Ds are contacted in an aqueous medium to give 25 -A method for producing 25-hydroxyvitamin Ds, characterized in that hydroxyvitamin Ds are produced and accumulated and collected from the aqueous medium. 2. The bacterium is Bacillus megaterium ( Bacill
The method according to claim 1, which is us megaterium ). 3. The vitamin D is vitamin D ₃ and 2
The method according to claim 1, wherein the 5-hydroxyvitamin Ds is 25-hydroxyvitamin D ₃ . 4. A bacterium belonging to the genus Bacillus and having the ability to hydroxylate the 25th position of vitamin Ds,
A method for isolating a gene encoding an enzyme for converting vitamin Ds into 25-hydroxyvitamin Ds, inserting the obtained gene into a vector, and introducing the vector into which the gene has been inserted into a host, A method for producing a transformant having the ability to hydroxylate the 25th position of vitamin Ds. 5. A transformant obtained by the method according to claim 4. 6. The method according to claim 5, wherein the transformant or a treated product thereof is contacted with vitamin Ds in an aqueous medium,
A method for producing 25-hydroxyvitamin Ds, which comprises collecting and accumulating 5-hydroxyvitamin Ds, and collecting the 5-hydroxyvitamin Ds from the aqueous medium.