JPH08256785A - Preparation of d-dithiobiotin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently obtain d-dethiobiotin useful for a precursor for producing d-biotin by culturing microbes belonging to Kurthia capable of producing d- dethiobiotin under aerobic conditions in a culture medium and then by collecting the product from its fermentation broth.

SOLUTION: This d-dethiobiotin is efficiently obtained by inoculating a new microbe, Kurthia sp.536-6(DSM-9454) belonging to Kurthia capable of producing d-dethiobiotin, which was separated from a soil sample collected in the park of Maioka in Yokohama, into a liquid culture medium, by conducting the shaking culture of it at 28°C for one night, by inoculating the prepared culture solution containing spawns in a flask holding a culture medium, by conducting the shaking culture of it at 28°C for 72 h., by adjusting the culture solution so as to have pH 3.0 with 9N sulfuric acid, by subjecting the solution to centrifugation at 8,000 rpm, by treating the supernatant liquid with activated carbon to adsorb the product, and by eluting it, followed by purifying it with an ion-exchange resin.

COPYRIGHT: (C)1996,JPO

Description

Detailed Description of the Invention

The present invention relates to a method for producing d-dethiobiotin (hereinafter abbreviated as DTB) by fermentation.

[0002] DTB is one of so-called biotin vitamers and is generally considered to be a direct precursor of d-biotin in many microorganisms. Therefore, DT
B is a very important substance for biological production of d-biotin.

Studies on the production of biotin vitamers are known. Mucor, Rhizopus, Aspergillus
illus), Streptomyces (Streptomy)
ces), Escherichia,
It is known that various microorganisms belonging to genera such as Bacillus and Sporoboromyces produce biotin vitamers. However, Kurthia
It is not known that microorganisms belonging to the ia genus accumulate large amounts of DTB.

According to the present invention, a large amount of DTB can be produced. It has been found that a microorganism belonging to the genus Curtia can accumulate a large amount of DTB in a fermentation liquor, from which DTB accumulated with a desired purity can be collected.

The content of biotin vitamers containing d-biotin-d-sulfoxide, d-dethiobiotin (DTB), 7,8-diaminopelargonic acid, and 7-keto-8-aminopelargonic acid was determined to be Saccharomyces cerevisiae.
isiae) ATCC 7754 (J. Am. Chem. Soc., 6)
2, 175-178, 1940) for quantification by the paper disk plate method (J. Microbiological Methods, 6, 6.
237-246, 1987). In addition, each biotin vitamer in the sample is Saccharomyces cerevisiae AT
It can be identified by bioautograph method using CC7754 (Agr. Biol. Chem., 33, (12), 1730-
1736, 1969).

In carrying out the present invention, a microorganism belonging to the genus Curtia is cultured in a medium containing an assimilable carbon source, a digestible nitrogen source and other nutrients necessary for the growth of the microorganism. As the carbon source, for example, glucose, fructose, starch, lactose, maltose, galactose, sucrose, dextrin, glycerin or kibizely is used. As the nitrogen source, for example, peptone, soybean flour, corn steep liquor, meat extract, ammonium sulfate, ammonium nitrate, urea and the like are used alone or in combination. Further, as other trace elements, sulfates, hydrochlorides or phosphates of calcium, magnesium, zinc, manganese, cobalt and iron are used. Further, the medium may contain a biotin precursor such as pimelic acid. In addition, if necessary, a general nutritional factor such as animal oil, vegetable oil, mineral oil or an antifoaming agent may be supplementarily added. The pH of the medium used for culturing is about 5.0 to 9.0 and about 6.5.
Is preferably about 7.5, and the culture temperature is about 10 to 40 ° C, preferably about 26 to 30 ° C. The culture time is usually about 1 to 10 days, preferably about 2 to 5 days. In culture, aeration and agitation usually give favorable results. After culturing, the DTB produced in the culture broth is separated from the culture broth and purified. For this purpose, the methods normally used to extract the product from the fermentation liquor may be carried out utilizing the various properties of DTB. Thus, for example, cells are separated from the culture solution, the desired substance in the filtrate is adsorbed on activated carbon, then eluted, and purified with an ion exchange resin. Alternatively, the culture filtrate is applied directly to the ion exchange resin and after elution the desired product is recrystallized from a mixture of alcohol and water.

The microorganism used in the present invention is DTB.
It includes all strains belonging to the genus Curtia that have the ability to produce. Among the strains of the genus Kurthia, as a particularly preferable strain, Kurthia sp. 538 isolated from a soil sample collected in Maioka Park, Yokohama City is used.
-6 (hereinafter abbreviated as 538-6 bacterium),
On October 5, 1994, DSM (Deutsche Sammlung
von Mikroorganismen und Zellkulturen GmbH, Gotting
en, Germany) as DSM No. 9454. Classification of 538-6 bacteria is based on Bergey's Manual
of Systematic Bacteriology, 9th Edition (NR Krie
g and PHA Sneath, The Williams & Wilkins Co., B
altimore, 1984).

The morphological properties of 538-6 are summarized in Table I. The cell morphology is 0.87-1.2 μm × 1.5-5.2 μm of bacilli in a 24-hour culture (initial stage of culture), which are chained and have a length of 60 μm or more. In 96-hour culture (late stage of culturing), some bacilli were coccoid (C
occoid) cells are formed. Nutrient agar cultured at 30 ° C
r) Single colonies of 538-6 above are circular, regular, semi-lenticular, yellowish white-wax, matte and slightly wrinkled with no pigment production. It was The settlement in 24 hours had a diameter of 2 mm.
The bacillus was Gram-positive, non-motile and did not form spores. 53 in stab culture with nutrient agar in the presence or absence of liquid paraffin
Growth of No. 8-6 for 14 days was observed only near the agar surface in the absence of liquid paraffin. This result clearly shows that this bacterium is absolutely aerobic rather than facultative anaerobic. The above-mentioned property is that 538-6 strain is
Manual of Systematic Bacteriology Vol.2 Section 14: Regular, Non-Spalling Gram-Positive Rod (Regular, Nonspori
ng Gram-Positive Rods).

[0009]

[Table 1]

Bacterial No. 538-6 was positive for catalase, and the peptidoglycan in the cell wall contained lysine as a diamino acid. The major fatty acid types in the cell wall of No. 538-6 are saturated linear and anteiso- and iso-methyl branched forms, while the major fatty acid is 12-methyltetradecanoic acid (anteiso- C 15: 0). The major quinone in the cell wall was unsaturated menaquinone (MK-7) with 7 isoprene units. GC content is 41
% (Table II).

[0011]

[Table 2]

A: S, straight chain saturated fatty acid; A, anteiso-methyl-branched fatty acid; I, iso-methyl-branched fatty acid

These properties support that No. 538-6 belongs to the genus Curtia. The genus Kurthia has two species, the Kurthia Gibbsony (Kurthi).
agibsonii) and Kurthia Sophie (Ku
rthia zopfii). But one more species, the Kurthia Siblica (Kurth
ia sibirica) was reported by Berukoba et al. (Mikr
obiologiya, 55, 831-836, 1986). Kurthia species are distinguished by the following nine properties. Growth at 45 ° C, heat resistance, colonization color, assimilation of 4-amino-n-butyrate, acid production from ethanol and glycerin, deoxyribonuclease, ribonuclease, phosphatase and pantothenic acid requirement. Therefore, as shown in Table III, the nine properties of No. 538-6 were investigated.

[0014]

[Table 3]

Kurcia Gibbsony, Kurcia Sophie and Kurcia Sibilika's sources are Bergey's Manua
l of Systematic Bacteriology (9th Ed.) and Beruko
Quoted from ba et al. (1986). (): Proportion of strains showing positive among the species (%)

From these results, 538-6 strain differed from the above-mentioned three known Kurthia species in the following properties. Kurthia Sophie: Heat resistance, 4-amino-
Utilization of n-butyrate and requirement for pantothenic acid;
Kurtia Gibbsony: Growth at 45 ° C .; Kurtia Siblica: Color of the community and requirement for pantothenic acid. next,
As shown in Table IV, quantitative DN of 538-6 with three known species of Kurthia, Kurthiazophy (NCIB9878), Kurthia Gibbsony (NCIB9758) and Kurthia Siblica (BKM B-1549).
A-DNA hybridization was performed and
Homology values of 6, 19 and 4% were obtained. According to the concept of species independence in the taxonomy of bacteria, the homology value obtained between strains of the same species by DNA-DNA hybridization is generally 70% or more. From the homology values obtained with these four strains, they were determined to be independent species. Therefore, 538-6 was determined to be a new species of Kurthia, and for the time being, Kurthia sp.
a sp. ) 538-6.

[0017]

[Table 4]

Other taxonomic properties of No. 538-6 are shown in Table V.
In summary. No cell polymorphism was found and acid resistance was negative. The physiological properties were as follows.

Nitrate reduction: Positive; Denitrification reaction: Negative; Methyl red test: Negative; VP test: Negative; Indole formation: Negative; Hydrogen sulfide formation: Negative; Starch hydrolysis: Negative; Citrate Availability (Koser's): Positive;
Availability of ammonium chloride and potassium nitrate: positive;
Urease reaction: Positive; Oxidase reaction: Negative; Growth range: pH 5.7 to pH10 and growth at 15 ° C to 37 ° C; OF test (Hugh & Leifson): Negative; Acid production: D
-Glucose, D-fructose, D-mannitol,
And slightly positive for glycerin, negative for L-arabinose, D-xylose, D-mannose, D-galactose, maltose, sucrose, lactose, trehalose, D-sorbitol, inositol and starch; gas production: L-arabinose, D-xylose. , D-glucose, D-mannose, D-fructose, D-galactose, maltose, sucrose, lactose, trehalose, D-sorbitol, D-mannitol, inositol, glycerin and starch.

Other properties were as follows. Production of acetic acid, propionic acid and butyric acid from sugars: Negative; Oxidation of gluconic acid: Positive; Oxidation of alcohol: Negative; Dihydroxyacetone production: Negative; Esculin degradation: Slightly positive; Cellulose and hippuric acid degradation: Negative Acid assimilation: Negative; Arginine degradation: Negative; Lysine and ornithine decarboxylation: Negative; Phenylalanine deamination: Negative; Coagulase production: Negative; Horse blood hemolytic: Negative; 80 ° C Survival at 10 minutes: negative;
Resistance to sodium chloride: 0-7%; Resistance to potassium cyanide: negative; Pectinase production: negative;
Lipase production (Tween 80): positive; lecithinase (Egg yolk) production: negative; and vitamin requirement: negative.

[0021]

[Table 5]

[0022]

[Table 6]

[0023]

[Table 7]

[0024]

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

Example 1 Kurthia sp. 538-6 (DSM-945)
Cells of one platinum loop from the slanted solid culture medium of 4) were inoculated into a test tube containing 5 ml of a liquid medium containing 1% broth (manufactured by Kyokuto Pharmaceutical Co., Ltd.) and shake-cultured overnight at 28 ° C. 1 ml of the seed culture was added with 2.0% glycerin, 4% protease peptone (manufactured by Nippon Pharmaceutical Co., Ltd.), 0.2% yeast extract (manufactured by Difco), 0.1% monopotassium phosphate, 0.05%.
Magnesium sulfate heptahydrate, 0.001% ferrous sulfate 7
5 consisting of hydrate, 0.001% manganese sulfate 4-7 hydrate and 1 drop of antifoaming agent CA-115 (manufactured by NOF CORPORATION)
Inoculate into a 500 ml flask containing 0 ml of medium and inoculate at 28 ° C.
With shaking. After culturing for 72 hours, the culture solution was
The pH was adjusted to 3.0 with N-sulfuric acid and centrifuged at 8,000 rpm for 10 minutes. The content of biotin vitamer in the supernatant was determined by Saccharomyces cerevisiae ATCC
It was measured by the paper disk plate method using 7754. 15 μl of supernatant and biotin standard solution were soaked in a paper disc. After air-drying, the paper disk is Saccharomyces cerevisiae ATCC7
Place the assay plate containing 754 on agar plate at 28 ° C.
Culturing was carried out for 17 hours and the diameter of the growth zone was measured.
The content of biotin vitamer in the supernatant was calculated from the standard growth curve. As a result, the supernatant contained 18. mg / L of biotin vitamer.

Example 2 Biotin vitamer was collected from 1 L of the culture supernatant prepared in the same manner as described in Example 1. Saccharomyces biotin vitamers during all purification steps
Followed by the paper disc plate method using Cerevisia ATCC7754. The supernatant was passed through a 3.6 × 40 cm column packed with 370 ml of activated carbon (manufactured by Wako Pure Chemical Industries, Ltd.). After washing the column with 500 ml of deionized water, 400 ml of ethanol / ammonia mixture (379
The mixture was mixed with 50 ml of 50% ethanol and 21 ml of 25% ammonia (prepared by mixing) to collect 10 ml of each in a test tube. 1
7. Biotin equivalent to 1 mg biotin vitamer
The eluate of 30 test tubes containing vitamer activity was concentrated under reduced pressure to 50 ml. The pH of the solution was adjusted to 7.0 with 1N hydrochloric acid, and then the solution was packed in a column (2.6 × 38 cm) packed with 175 ml of Dowex 1 × 4 (formic acid type, 100-200 mesh, manufactured by Dow Chemical Co.). ). The column was washed with 200 ml deionized water and then eluted with 500 ml 0.25N formic acid,
10 ml of each was collected in a test tube. The eluate of 20 test tubes having biotin vitamer activity was collected and concentrated under reduced pressure to obtain 17.5 mg of white powder. The white powder was crystallized with a mixed solution of ethanol and water to obtain 10 mg of colorless needle crystals having a melting point of 156 ° C. The melting point, Rf value on thin layer chromatograph, infrared absorption spectrum, NMR spectrum and activity against Saccharomyces cerevisiae ATCC7754 of the sample were in good agreement with those of the DTB standard (manufactured by Sigma). In addition to DTB, a small amount of 7-keto-8-aminopelargonic acid, 7,8-diaminopelargonic acid and d-biotin were detected as products.

Example 3 Kurthia sp. 538-6 (DSM-945)
Cells of one platinum loop from the slanted solid culture medium of 4) were inoculated into a test tube containing 5 ml of a liquid medium containing 1% broth (manufactured by Kyokuto Pharmaceutical Co., Ltd.) and shake-cultured overnight at 28 ° C. Part 1
The seed culture solution of ml was 2.0% glucose, 4% protease peptone (manufactured by Nippon Pharmaceutical Co., Ltd.), 0.2% yeast extract (manufactured by Difco Co.), 0.1% monopotassium phosphate, 0.05%.
Magnesium sulfate heptahydrate, 0.001% ferrous sulfate 7
5 consisting of hydrate, 0.001% manganese sulfate 4-7 hydrate and 1 drop of antifoaming agent CA-115 (manufactured by NOF CORPORATION)
Inoculate into a 500 ml flask containing 0 ml of medium and inoculate at 28 ° C.
The cells were shake-cultured for 3 days. The cells were removed from the culture solution by centrifugation, and the content of biotin vitamer in the supernatant was determined by Saccharomyces cerevisiae ATCC.
It was measured by the paper disk plate method using 7754. The supernatant showed a biotin vitamer activity of 17.8 mg / L against Saccharomyces cerevisiae ATCC 7754.

Furthermore, the supernatant biotin vitamers were measured by the bioautograph method using Saccharomyces cerevisiae ATCC7754. 10 μl of the supernatant was added to a silica gel plate (10 × 20 cm, 0.25 mm,
Art. 5729, manufactured by Merck & Co., Inc., and the plate was developed with a mixed solution of ethyl acetate / methyl alcohol (10: 1). Biotin vitamers on the plates were detected on agar plates containing Saccharomyces cerevisiae ATCC 7754. Rf of sample growth zone
Values were compared to standards of 7-keto-8-aminopelargonic acid, 7,8-diaminopelargonic acid, DTB, d-biotin-d-sulfoxide or d-biotin. As a result, this biotin vitamer was identified as DTB.

Example 4 Kurthia sp. 538-6 (DSM-945)
The cells of one platinum loop from the slanted solid culture medium of 4) were inoculated into a test tube containing 5 ml of a liquid medium containing 1% broth (manufactured by Kyokuto Pharmaceutical Co., Inc.) and shake-cultured overnight at 28 ° C. 1 ml
Seed culture solution of 2.0% glucose, 4% protease peptone (manufactured by Nippon Pharmaceutical Co., Ltd.), 0.2% yeast extract (manufactured by Difco), 0.1% monopotassium phosphate, 0.05%
Magnesium sulfate heptahydrate, 0.001% ferrous sulfate 7
Hydrate, 0.001% manganese sulphate 4-7 hydrate, 0.001%.
A 500 ml flask containing 50 ml of a medium containing 05% pimelic acid and 1 drop of antifoaming agent CA-115 (manufactured by NOF CORPORATION) was inoculated and shake-cultured at 28 ° C for 3 days. The cells were removed from the culture medium by centrifugation, and the content of biotin vitamer in the supernatant was determined by Saccharomyces.
It was measured by the paper disc plate method using Cerevisia ATCC7754. The supernatant is 45 against Saccharomyces cerevisiae ATCC 7754.
The biotin vitamer activity of mg / L was shown.

The biotin vitamer in the supernatant was added to D
It was decided to be TB.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Hitoshi Kimura 882-24 Kotake, Odawara-shi, Kanagawa 8-13 Satsukigaoka (72) Inventor Masaaki Tazo, 3-6-405 Hinominami, Minami-ku, Yokohama-shi, Kanagawa

Claims (2)

[Claims] 1. A d-dethiobiotin characterized by culturing a microorganism belonging to the genus Kurthia capable of producing d-dethiobiotin in a medium under aerobic conditions and then collecting the produced d-dethiobiotin from a fermentation broth. Manufacturing method. 2. Kurtia S. 538-6 (DS
M-9454).