JPH0716404B2 - Kyandeida Boydini AP-15 shares - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a Kyandeida voidini AP-15 strain having a high content of nicotinamide adenine dinucleotide (hereinafter abbreviated as NAD).

(Prior Art) NAD has been known for a long time as a coenzyme for various redox enzymes in the living body, energy metabolism in the living body, and a coenzyme for various substances. Further, in recent years, NAD is an expensive substance, although a wide range of applications have expanded to biochemistry and medical fields as clinical reagents and biochemical reagents, and the demand for them has increased significantly.

Conventionally, NAD has been produced by culturing yeast, bacteria, etc., and extracting from cultured cells. In recent years, various efforts have been made in the culture method in order to improve the production amount. For example, a method of adding a NAD precursor or a surfactant together with the NAD precursor to the culture process to accumulate NAD in the cells or in the culture medium (Japanese Patent Publication No. 47-19748, Japanese Patent Publication No. 47-19749, JP-B-45-29436 and JP-A-48-56891), or after culturing, cells are once separated from the culture solution and treated with a surfactant, and then in a solution containing a NAD precursor. Further culturing methods (see JP-A-54-80493 and JP-A-54-80494) have been proposed.

However, the microorganisms used in the conventional methods as described above are limited to those that grow with carbohydrate as a carbon source and an energy source, and as raw materials for industrial scale production, they are common raw materials of the present fermentation industry. It is inevitable to use waste molasses or starch saccharified liquid, and the supply of these raw materials is unstable from an industrial point of view, and it is difficult to control the culture because a uniform product cannot be obtained. It is costly and has problems such as difficulty in isolating and purifying the target substance, which in turn is a major factor in increasing the price of NAD.

In order to solve this problem, the present inventors have previously set up
-A is cultured in a medium containing methanol as a main carbon source,
We proposed a method for producing NAD that collects NAD from cultures (see Japanese Society of Agricultural Chemistry, 1986 Annual Meeting Abstracts P505).

(Problems to be Solved by the Invention) However, the Kyandeida 25- used in the above
In A, the NAD content was not so high, so the NAD production was not yet satisfactory. Because of this, NAD is still an expensive substance.

(Means for Solving Problems) The inventors of the present invention have conducted extensive studies for the purpose of solving the above problems and providing a strain having a high NAD content. By carrying out a mutation treatment with A, a mutant strain having resistance to 3-acetylpyridine was discovered, and it was found that the strain had a high NAD content, and the present invention was completed.

That is, the present invention is resistant to 3-acetylpyridine and has a high NAD content.
AP-15 strain is the gist.

Next, the mycological properties of the present invention are shown below. Among these examinations of mycological properties, the following medium containing methanol as a carbon source was used for examination of 3-acetylpyridine resistance and NAD content.

Unless otherwise specified,% means% by weight (the same applies hereinafter).

Methanol 2% by volume, ammonium chloride 0.5%, potassium dihydrogen phosphate 0.1%, potassium monohydrogen phosphate 0.1%, magnesium sulfate 0.05%, ferric chloride 30 μg / ml, calcium chloride 10 μg / ml, manganese sulfate 10 μg / ml , Zinc sulfate 10 μg /
ml, thiamine hydrochloride 2 μg / ml, biotin 0.02 μg / ml in methanol medium (pH 6.0).

For other studies, classification and identification of yeast [Hiroshi Iizuka, Shoji Goto, (1969)], revised classification and identification of microorganisms (above)
[Takeshi Hasegawa, (1984)], The Yeasts A taxonomics
tudy) [Edited by Lodder, (1970) and The Yeasts Taxonomy Study (The Yeasts
a taxonomic study) [edited by Rij, (1984)]
Followed the method described in.

(A) Growth state in each medium MY liquid medium, 28 ° C., 3-5 days culture Cell size: 1.5-3.5 μ × 2.0-12.0 μ Cell morphology: oval or cylindrical, not alone 5-30
Form agglomerates with cells Propagation type: Multipolar budding Medium turbidity due to growth: Slightly cloudy Precipitate: Powdery precipitate Film formation: Wrinkle film on the surface MY agar medium, 28 ℃, 3 Cultivation for 5 days Growth level: Good Peripheral shape: Wavy Raised state: Semi-lenticular Surface shape: Rough surface Surface gloss: Dull light Surface property: Buttery surface color tone: Milky white Slide with Valeisyo glucose agar Culture Pseudohyphae: Formed spores, chlamydospores: Not formed (b) Ascospore formation: None (c) Ejected spore formation: None (d) Physiological properties Optimal growth conditions: pH 4.0- 7.0,27-30 ℃ Growth range: pH2.5-9.5,18-33 ℃ Nitrate assimilation: Yes Fat decomposition: No Urea decomposition: No Gelatin liquefaction: No Osmotic resistance: At 10% sodium chloride or more Formation of non-viable carotenoids: None Remarkable organic Generation: None Starch-like substance formation: None Vitamin requirement: Grows weakly even in a medium containing no vitamin, but growth is increased by addition of biotin. Physiological characteristic of the strain 3-Acetylpyridine resistance: Yes NAD content in methanol medium containing 0.3% quinolinic acid: 22 mg / g dry cells (44 mg / medium) (e) Fermentability and assimilation Fermentability carbon source: D-glucose Non-fermentation carbon source: D- Galactose, maltose, sucrose, lactose, raffinose Carbon sources having assimilating properties: D-ribose, D-xylose, D-glucose, D-mannose, D-fructose, ethanol, adonit, erythritol, D-mannitol, D-sorbit , Glycerin Carbon source with weak assimilation: L-arabinose, D-galactose, maltose, D-gluconate, DL-lactate, succinate Sex without carbon sources: D-arabinose, L- rhamnose, L- sorbose, sucrose, lactose, melibiose, cellobiose, trehalose, raffinose, D- melezitose, alpha-methyl -D-
Glucoside, arbutin, dextrin, soluble starch,
Inulin, Inosit, Drusit, 2-Keto-D-Gluconate, Citrate Based on the above-mentioned mycological properties, from Lodder's "THE Yeasts Taxonomy Study (THE YEASTS
A taxonomic study) "(1970) and Rigi (Ri
As a result of searching based on the search table of “The Yeasts a taxonomic study” (1984) of j), this strain was identified as Cyandeida (Candida).
This strain was found to belong to the genus Candida boidaini (Candida boi) and was found to belong to the genus (Candida bod)
It was judged appropriate to identify it as idinii).

The parent strain, Kyandeida 25-A, was not resistant to 3-acetylpyridine, whereas this strain became resistant to 3-acetylpyridine, thus showing a significant difference between the two. . Moreover, the content of NAD increased to about 2.0 times that of the parent strain, Kyandeida 25-A.

In this way, this strain is clearly different in bacteriological properties from the existing strains, so it was judged as a new strain, and Kyandeida.
It was named as Candida boidinii AP-15 and deposited on September 25, 1986, at the Institute for Microbial Technology, Ministry of International Trade and Industry, Ministry of International Trade and Industry [FERM P-897].
Four)〕.

In order to obtain the strain of the present invention, for example, the methanol-assimilating yeast Candida 25-A, which has already been deposited as a parent strain with MIC, is MICRO LAB 8725 (FERM).
P-8725) and ultraviolet rays as a mutagenesis source. At this time, as the mutagenesis source, a physical factor or a drug may be used in addition to ultraviolet rays. Examples of such substances include radiation, high heat treatment, 5-bromouracil, ethyl methane sulfonate, mustard gas,
Nitrosoguanidine can be mentioned.

The medium used for culturing the strain of the present invention may be a medium normally used for yeast belonging to the genus Canydadea except that methanol is used as a carbon source and quinolinic acid is added as a precursor. As the medium composition at this time, the concentration of methanol is about 0.5 to 3% by volume, and the concentration of quinolinic acid is about 0.1 to 0.5%. Other medium compositions include, for example, peptone, urea, ammonium sulfate, ammonium salt, ammonium nitrate, and sodium nitrate. And a nitrogen source such as sodium dihydrogen phosphate, potassium dihydrogen phosphate, potassium monohydrogen phosphate and the like are used. Further, as a growth promoting factor, a small amount of biotin, thiamine, yeast extract, or a metal salt of iron, calcium, manganese, zinc or the like can be added.

Using these media, the strain of the present invention was treated at pH 4 to 7, temperature 25
The culture may be performed with aeration and stirring at ~ 33 ° C. At this time, the culture may be completed at any time after the growth of the bacteria, but preferably after the methanol in the culture solution is completely consumed, NAD may be accumulated more.

In the present invention, to isolate and purify NAD, for example, first, the bacterial cells are collected from the culture by centrifugation or filtration, and then the collected bacterial cells are subjected to a conventional method, that is, after crushing the bacterial cells. It can be carried out by a method such as fractionating and purifying by adding an organic solvent or various salts to the supernatant obtained by centrifugation, or adsorbing on a carrier for purification.

(Examples) Hereinafter, the present invention will be described more specifically with reference to Examples.

In the examples,% means% by weight unless otherwise specified.

Example 1 and Comparative Example 1 First, in order to examine the sensitivity of the Kyandeida 25-A (Microtechnical Laboratory No. 8725) used as a parent strain to ultraviolet rays, 1% by volume of methanol, 0.5% of ammonium chloride and 2% of phosphoric acid were used.
Potassium hydrogen 0.1%, potassium monohydrogen phosphate 0.1%, magnesium sulfate 0.05%, ferric chloride 30 μg / ml, calcium chloride 10 μg / ml, manganese sulfate 10 μg / ml, zinc sulfate 10 μg / m
l, thiamine hydrochloride 2 μg / ml, biotin 0.02 μg / ml, agar 1.
5%, the plate to create a using a medium consisting of pH 6.0 (hereinafter abbreviated as methanol medium.), It seeded the bacterial suspension at various concentrations, irradiated with ultraviolet rays from above at an intensity of 40μw / cm ² Then, when the irradiation time giving a survival rate of 0.1% was calculated,
It was 34 seconds.

Next, a medium containing various concentrations of 3-acetylpyridine added to a methanol medium was added to Kyandeida 25-A.
No. 8725) and the sensitivity to 3-acetylpyridine was investigated, and it was found that it could not grow at all in a medium containing 20 mM 3-acetylpyridine. Therefore, a plate was prepared by adding 20 mM of 3-acetylpyridine to a methanol medium, and added with Kyandeida 25-A.
No. 25), and ultraviolet rays from above on it at an illuminance of 400 μw / cm ² .
After irradiation for 34 seconds, the cells were cultured at 28 ° C for 1 week, and a large number of growing colonies were picked up. These strains were cultivated with shaking in a liquid medium containing 0.3% quinolinic acid in methanol medium, and the NAD content in the cells was measured 4 days later. The NAD content of these strains was about the same as that of the parent strain. 2.0 times higher than Candida boidin
ii) AP-15 [Microtechnology Research Institute, Microbiology No. 8974 (FERM P-8974)]
was gotten.

In addition, NAD was quantified by extracting the cells with hot water at 85 ° C after harvesting the cells.
It was carried out by an enzymatic method using alcohol dehydrogenase [Methods of Enzymatic Analysis, 3rd edition (1984) 7
Volume P253].

Next, the above strain was inoculated into a medium having a composition of 1% glucose, 0.4% ammonium chloride, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate and 0.2% yeast extract,
An inoculum solution was prepared by shaking culture at pH 6.0 at 28 ° C for 24 hours. Then, the methanol concentration was adjusted to 2% by volume, 100 ml of agar medium without addition of agar was placed in a 500 ml shake flask, and the seed culture solution was added to this to 1%, and the temperature was adjusted to 28 ° C.
The culture was performed with shaking. NAD of 22 mg / g dry cells was accumulated in the yeast cells 100 hours after the start of the culture.

For comparison, the parent stock Candidida
a) When culture was performed in the same manner as described above using 25-A Microtechnical Research Institute No. 8725 (FERM P-8725), 11 mg / g dry cell NAD was accumulated in the yeast cells. Was there.

Example 2 A 2-volume mini jar fermenter was charged with methanol medium 1.2 having a methanol concentration of 2% by volume and no agar added, and inoculated in the same manner as in Example 1, followed by temperature 28 ° C., aeration condition 1VVM, 400 rpm. Culture was carried out in. After 100 hours from the start of culture, NAD of about 22 mg / g dry cells was found in the yeast cells.
53 mg of NAD was accumulated in culture medium 1.2.

Next, the cells were obtained from this culture solution by centrifugation, and then a few times.
Wash with 0.9% sodium chloride. About 10 ml of hot water at 85 ° C was added to the cells, and NAD was extracted by stirring in a hot water bath at 85 ° C for about 5 minutes. After cooling, an extract was obtained by centrifugation.

This was applied to a Dowex1-X2 (200 to 400 mesh, formic acid type) column (0.8 x 40 cm), and a gradient elution of formic acid 0 to 0.15 N (400 ml) was performed, and fractionation was performed every 10 ml. When the light intensity at 260 nm and NAD were quantified for each fraction, the UV absorption peak eluting near the formic acid concentration of 0.08 N was the NAD peak. This fraction was collected, adjusted to pH about 2, and then adsorbed on activated carbon. After thoroughly stirring this activated carbon, suction-filtering, washing with an appropriate amount of cold 0.001N HCl, and immediately adding it to a 50% ethanol: concentrated ammonia (200: 1) solution, stirring and filtering with suction to obtain a filtrate containing NAD. Recovered.

The filtrate was concentrated under reduced pressure to about several ml, 10 times the amount of ethanol was added thereto, and the resulting precipitate was collected by centrifugation. When this precipitate was washed and dried, 42 mg of white powder was obtained. This white powder is NAD with a purity of 89%
Met.

(Effect of the invention) Since the strain of the present invention has a high NAD content, NAD can be produced at low cost by culturing the strain.

Claims (1)

[Claims] 1. A Kyandeida voidini AP-15 strain which is resistant to 3-acetylpyridine and has a high nicotinamide adenine dinucleotide content.