JP3768226B2 - Method for producing 1α-hydroxyvitamin Ds - Google Patents

Description

  The present invention relates to a method for producing hydroxyvitamin D using microorganisms.

  Direct hydroxylation of 1α-position of vitamin D by organic chemical methods is considered to be extremely difficult, and in the literature, a method using a homogenate of an animal kidney such as a chicken or a method using a mitochondrial fraction (non-patented) Reference 1, Non-patent document 2, Non-patent document 3), and a method using a microorganism (Patent document 1, Patent document 2, Patent document 3, Patent document 4), etc. It is known that it can be introduced.

However, the above reports on 1α-position hydroxylation all convert 25-hydroxyvitamin D having a hydroxyl group at the 25-position to 1α, 25-dihydroxyvitamin D, and the 25-position is not a hydroxyl group but a hydrogen atom. The direct hydroxylation of the 1α-position of vitamin D in the case of having an enzyme has not yet been reported for successful enzymatic chemistry methods using animals and plants, conversion methods using microorganisms, and the like. On the other hand, 1α-hydroxyvitamin D ₃ is a pharmaceutical used for chronic renal failure, osteoporosis and the like, and its production is currently synthesized through complicated steps by an organic chemical method.

Japanese Patent Laid-Open No. 02-231089 Japanese Patent Laid-Open No. 04-166090 JP 04-356190 A Japanese Patent Publication No. 04-6478 "Nature", 230, 228, 1971 “Journal of Biological Chemistry” Vol. 247, p. 7528, 1972 “Biochemistry”, 25, 5512, 1986

  The synthesis of 1α-hydroxyvitamin Ds by organic chemistry techniques is complicated and difficult to prepare. An object of the present invention is to provide a novel method for producing 1α-hydroxyvitamin D by a simpler operation using microorganisms.

  The microorganism used in the present invention belongs to actinomycetes, and is a strain having the ability to newly convert the 1α-position hydrogen atom of vitamin D directly into a hydroxyl group. Specific examples thereof include Iriomote Island and Nagano, Okinawa Prefecture. It was found that a hydroxyl group can be directly introduced into the 1α-position of vitamin D by utilizing a specific microorganism newly isolated from the soil of Hase Village in the prefecture, and the present invention has been completed.

  The present invention adds actinomycetes belonging to the genus Amycolata that hydroxylates vitamin Ds, or a solution containing the enzyme produced by adding vitamin Ds having a hydrogen atom at the 1α position, This is a method for producing 1α-hydroxyvitamin D, characterized in that the hydrogen atom is converted into a hydroxyl group. That is, according to the present invention, vitamin D having a hydrogen atom at the 1α position is converted to a hydroxyl group.

The production method of the present invention is a method in which a hydroxyl group is directly introduced into the 1α-position of vitamin D in one step, and any side chain portion may have any substituent. Therefore, in the present invention, vitamin Ds are vitamin D ₂ and D ₃ compounds and have hydrogen atoms at the 1α and 25 positions. The hydrogen atom or hydroxyl group in the side chain may be substituted with a halogen atom such as fluorine, a lower alkyl group or the like.

  The strains involved in the present invention have the following mycological properties.

About Amycorata actinomycetes A2019 1) Morphotrophic mycelium develops well on synthetic and natural agar media and branches off irregularly. A break of 0.5 to 0.8 × 2.3 to 2.8 μm in diameter is observed. No sclerotia, sporangia, or flagellate spores are observed.

2) Growth condition on medium Table 1 shows the results of macroscopic observation when cultured in various mediums at 30 ° C for 7 days.

3) Physiological properties (1) Growth temperature range It grows well in the range of 20-40 ° C on various natural media. In addition, it does not grow in a temperature range of 5 ° C. or lower and 50 ° C. or higher.
(2) Biochemical properties a) Distinguishing between aerobic and anaerobic aerobic b) Liquefaction of gelatin Negative c) Coagulation of skim milk Negative d) Peptation of skim milk Positive e) Hydrolysis of starch False positive f) Melanin-like pigment formation Positive g) Nitrate reduction ability Positive (3) Utilization of carbon source As a result of examination with a Prideham-Godrieve agar medium, D-glucose, D-fructose, D-xylose, sucrose, mannitol, Inositol is used, L-arabinose is used slightly, and raffinose and L-rhamnose are not used.

  From the above bacteriological properties, it is clear that this strain belongs to actinomycetes. “Manual of Systematic Bacteriology” Vol. 2 written by Barsey, “Experimental Methods for Identification of Actinomycetes” edited by the Japanese Society for Actinomycetes As a result of comparing the above properties with the known bacterial species reported in "International Journal of Systematic Bacteriology", Volume 36, pages 29-37 (1986), the strain was identified as Actinomyces sp. It was identified, and it was judged to be a new strain because it was different from known bacteria, and was named Amycolata sp. A2019 (Amycolata sp. A2019). did.

  The method of the present invention is carried out by reacting substrate vitamin Ds in an aerobic condition in a solution containing actinomycetes or an enzyme produced therefrom.

  In order to obtain actinomycetes necessary for the reaction, the bacterium is cultured in a medium under aerobic conditions. As the medium, a liquid medium is mainly used, and glucose, maltose, sucrose, starch, arabinose or the like is used alone or in combination as a carbon source. As the nitrogen source, polypeptone, casamino acid, yeast extract, meat extract, corn steep liquor, soy bean meal and the like are used alone or in combination. In addition, organic substances and inorganic substances that help the growth of this strain and promote the production of 1α-hydroxyvitamin D can be added as necessary. As the culture method, aerobic culture such as shaking culture and aeration-agitation culture is suitable, and culture is performed at pH 6-8 for 2-8 days.

  The solution containing the cells obtained by this culture is used for the reaction for producing 1α-hydroxyvitamin Ds. That is, a culture solution containing cells in culture is used as it is, or a solution in which cells separated by centrifugation or filtration after the completion of culture is used. Alternatively, a solution obtained by crushing the microbial cells obtained after the culture and removing the microbial cells by centrifugation or the like can also be used. Furthermore, the microbial cells may be immobilized using various immobilizing agents such as polysaccharides such as κ-carrageenan and then added to the solution. Moreover, the lyophilized product of the cells can also be used in the same manner as described above.

  The reaction solution used in the present invention is a culture solution cultured using the above-mentioned medium, or Tris-acetic acid, Tris-hydrochloric acid, sodium succinate-succinic acid, potassium succinate-succinic acid, sodium citrate-citrate. Buffers such as acids, phosphates (potassium salts, sodium salts, etc.), sodium cacodylate-hydrochloric acid, imidazole-hydrochloric acid, boric acid-borax, etc. are used alone or in combination. In addition, surfactants, organic substances, and inorganic substances that promote the production of the desired vitamins can be added to the solution as necessary.

  In the present invention, an appropriate amount of vitamin D as a substrate is added to the solution containing the bacterial cells, and it is suitable to perform under aerobic conditions such as shaking operation and aeration stirring operation, and pH 6-9, 20-20. Approximately 24 to 96 hours are required while maintaining at 40 ° C. In the case of using a culture solution containing cells in culture, the substrate vitamin Ds are added, and the reaction is further performed by shaking for 24 to 96 hours under the same conditions.

  Isolation of 1α-hydroxyvitamin Ds produced by the above reaction is performed by extracting the reaction solution with an organic solvent, concentrating to dryness, re-dissolving it in an appropriate solvent such as acetone, and centrifuging or dissolving insoluble matter. After removal by filtration, etc., high performance liquid chromatography using a silica gel normal layer column (for example, Zorbax SIL: manufactured by DuPont, USA) or high performance liquid chromatography using a silica gel reverse layer column (for example, Biosil C18: manufactured by BioRad, USA). The target hydroxyvitamins can be isolated by subjecting to a graph.

  The target hydroxyvitamins are also isolated by gel filtration chromatography using Sephadex LH-20 (Sweden: Pharmacia) or silica gel chromatography using Rover columns (Switzerland: Merck). be able to.

  According to the method of the present invention, a hydroxyl group can be directly introduced into the 1α position of vitamin Ds. That is, in the method using the microorganism of the present invention, it takes less time to prepare a reaction solution and the like, and can be performed in a single step in a short time.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention is not limited by this.

Place 5 mL of liquid medium of glucose 1.5%, soy bean meal 1.5%, corn steep liquor 0.5%, sodium chloride 0.5%, calcium carbonate 0.2%, pH 7 in a test tube having a diameter of 24 mm. The pasteurization was performed at 20 ° C. for 20 minutes. To this, 1 platinum ear inoculum of Amycorrata actinomycetes A2019 was inoculated and cultured with shaking at 30 ° C. for 2 days. As prepared by dissolving vitamin _{D 3} of 1mg of ethanol 200μL To this was added a solution obtained by dissolving β- cyclodextrin 10mg of water 1 mL, was reacted further with shaking for 2 days at 30 ° C.. Equal amounts of methanol and dichloromethane were added to the culture broth after the reaction, and after stirring, the dichloromethane layer was recovered by centrifugation. This was concentrated to dryness under a nitrogen stream, dissolved in a mixed solvent of n-hexane: isopropanol = 22: 3, and after impurities were filtered off, it was subjected to Solvax SIL high performance liquid chromatography.
Eluent: n-hexane: isopropanol = 92: 8
Column size: φ4.6mm × 25cm
Elution rate: 2.0 mL / min
Detector: Photodiode array detector Waters 991J (manufactured by Nihon Millipore Limited)
Peak fractions with an elution time of about 7 minutes showing the ultraviolet absorption pattern of vitamin D were collected and concentrated, and then subjected to Biosil C18 high performance liquid chromatography.
Eluent: methanol: water = 90: 10
Column size: φ4.6mm × 15cm
Elution rate: 2.0 mL / min
Detector: Photodiode array detector Waters 991J (manufactured by Nihon Millipore Limited)
After elution, peak fractions with an elution time of about 10.5 minutes showing the ultraviolet absorption pattern of vitamin D were collected and concentrated under reduced pressure. As a result, 0.5 μg of purified 1α-hydroxyvitamin D ₃ was obtained. This is a complete sample of commercially available 1α-hydroxyvitamin D3 (manufactured by Wako Pure Chemical Industries, Ltd.) and various high performance liquid chromatography (Zorbax SIL, Biosil C18), UV absorption spectrum, and mass spectrum cleavage pattern. Matched.
Maximum ultraviolet absorption: λ _max = 265 nm (ethanol)
EI-MS (m / z) 400 (M +), 382 (M + -H20), 364, 287, 197, 174, 152, 134, 105, 95
Since the same ultraviolet absorption pattern was exhibited and the mass spectrum cleavage pattern was the same, it was identified as 1α-hydroxyvitamin D ₃ .

Claims (1)

Among the actinomycetes belonging to the genus Amycolata, Amycolata sp. A2019 (Amycolata sp. A2019 FERM P-14184) has a hydrogen atom at positions 1α and 25 in a solution containing the microbial cell or the enzyme produced therein. in addition vitamin D _3, 1.alpha.-producing method of hydroxyvitamin D _3, characterized in that the replacement of the hydrogen atom of the 1α-position to the hydroxyl group.