JPH0646954B2 - Method for producing nicotinamide adenine dinucleotide - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing nicotinamide adenine dinucleotide (hereinafter abbreviated as NAD) from a microorganism.

(Prior Art) NAD has long been known as a coenzyme for a wide variety of oxidoreductases in the body, as an energy metabolism in the body, and as a coenzyme for various substances. Also, in recent years, a wide range of applications have expanded to clinical chemistry and medical fields such as clinical reagents and biochemical reagents, and the demand for them has increased significantly.
D is an expensive substance.

Conventionally, NAD has been produced by culturing yeast, bacteria and the like, 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, NAD precursors or NAD precursors and surfactants are added to the culture process to add N
A method for accumulating AD (Japanese Patent Publication No. 47-19748, Japanese Patent Publication No. 47-19749, Japanese Patent Publication No. 45-29)
No. 436, JP-A-48-56891),
Alternatively, after the completion of the culture, the cells are once separated from the culture solution, treated with a surfactant, and then further cultured in a solution containing a NAD precursor (Japanese Patent Laid-Open Nos. 54-80493 and 54-5493). -80494).

However, all of the microorganisms used in the above conventional methods are limited to those that grow with carbohydrate as a carbon source and an energy source, and as a raw material for industrial scale production, a common raw material of the present fermentation industry is used. 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.
Has become a major factor in rising prices.

In order to solve this problem, the present inventors first set up the Kyandeida 2
We proposed a method for producing NAD by culturing 5-A in a medium containing methanol as a main carbon source and collecting NAD from the culture (Agricultural Chemistry Society of Japan, 1986 Annual Meeting Abstracts P
505).

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

(Means for Solving Problems) The inventors of the present invention have made earnest studies as a result of solving the problems as described above and providing a method capable of producing a large amount of NAD. It was found that the above-mentioned objects can be achieved by culturing using a specific strain that has been subjected to mutation treatment, and has completed the present invention.

That is, the present invention is a method for producing NAD, which comprises culturing a mutant strain AP-15 or PC-31 of Kyandeida voidiniii in a medium containing methanol as a main carbon source and collecting NAD from the culture. Is the gist.

The microorganism used in the present invention is, for example, the methanol-assimilating yeast Candida 25-A Microtechnological Research Institute Contribution No. 87, which has been deposited by the present inventors at Microindustry Research Institute.
No. 25 (FERM P-8725) was used as a parent strain, which was one of the analogs of nicotinic acid by the UV irradiation method.
Mutant strain A that has become resistant to acetylpyridine
The P-15 strain and Candida 25-
This is a mutant PC-31 strain, which has A as a parent strain and has become resistant to picolinic acid, which is one of the analogs of nicotinic acid, by the ultraviolet irradiation method.

Next, the AP-15 strain and PC-31 used in the present invention
The mycological properties of the strain 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 or picolinic acid resistance and NAD content.

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

Methanol 2% by volume, ammonium chloride 0.5%, potassium dihydrogenphosphate 0.1%, potassium monohydrogenphosphate 0.
1%, magnesium sulfate 0.05%, ferric chloride 30μ
A methanol medium (pH 6.0) consisting of g / 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.

Other studies include classification and identification of yeast [Hiroshi Iizuka, Shoji Goto, (1969)], revised classification and identification of microorganisms (above)
[Takeshi Hasegawa, (1984)], THE YEASTS A taxonomic stud
y) [Edited by Lodder, (1970)] and The Yeasts a taxo
nomic study) (edited by Rij, (1984)].

First, the bacteriological properties of the AP-15 strain are shown.

(a) Growth state in each medium MY liquid medium, cultured at 28 ° C for 3 to 5 days Cell size: 1.5 to 3.5 µ x 2.0 to 12.0 µ Cell morphology: oval or cylindrical, not alone 5-
Form agglomerates with 30 cells Proliferation type: Multipolar budding Medium turbidity due to growth: Slight turbidity Precipitate: Powdery precipitate Film formation: Wrinkle film on the surface MY agar medium, 28 ° C, Cultivation for 3 to 5 days Growth degree: good Peripheral shape: wavy Raised state: semi-lenticular surface shape: rough Surface gloss: dull Surface texture: buttery Surface color: milky white Valeilillo glucose agar Slide culture in culture medium Pseudohyphae: Formed spores, chlamydospores: Not formed (b) Ascospore formation: None (c) Ejected spore formation: None (d) Physiological properties Optimal growth conditions : PH 4.0 to 7.0, 27 to 30 ° C Growth range: pH 2.5 to 9.5, 18 to 33 ° C Nitrate assimilation: A Fat decomposition: None Urea decomposition: None Gelatin liquefaction: None Resistance Osmolarity: Caro that does not grow above 10% sodium chloride Production of tinoids: None Production of significant organic acids: None Production of starch-like substances: None Vitamin requirement: Growth is weak even in a medium containing no vitamins, but growth is enhanced by addition of biotin Characteristics of strain 3-Acetylpyridine resistance: NAD in methanol medium supplemented with 0.3% quinolinic acid
Content: 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 assimilating carbon source: D-ribose, D-xylose, D-glucose, D-
Mannose, D-fructose, ethanol, adonite, erythritol, D-mannitol, D-sorbit,
Glycerin Carbon source with weak assimilation: L-arabinose, D-galactose, maltose, D-gluconate, DL-lactate, succinate Carbon source without assimilation: D-arabinose, L-rhamnose, L- Sorbose,
Sucrose, lactose, melibiose, cellobiose, trehalose, raffinose, D-merezitose, α-methyl-
D-Glucoside, Arbutin, Dextrin, Soluble starch, Inulin, Inosit, Dusit, 2-Keto-D
-Gluconate, Citrate Next, the mycological properties of the PC-31 strain are shown.

(a) Growth state in each medium MY liquid medium, cultured at 28 ° C for 3 to 5 days Cell size: 1.5 to 3.5 μx 2.0 to 12.0 μ Cell morphology: oval or cylindrical, alone or in duplicate, Rarely forms pseudohyphae Growth type: Multipolar budding Medium turbidity due to growth: Slightly turbid Precipitate: Powdery precipitate Film formation: Wrinkled film on the surface MY agar medium, Cultivated at 28 ℃ for 3-5 days Degree of growth: Good Edge shape: Full edge Raised state: Semi-lenticular Surface shape: Rough Surface gloss: Dull surface texture: Buttery Surface color: Milky white valley・ Slide culture in glucose agar medium Pseudohyphae: forming, spores forming spores, chlamydospores: not formed (b) Ascospore formation: None (c) Ejection spore formation: None (d) Physiological properties Optimal growth conditions: pH 4.0-7.0, 27-3 ℃ Growth range: pH 2.5 to 9.5, 18 to 33 ℃ Nitrate assimilation: Adipose decomposition: None Urea decomposition: None Gelatin liquefaction: None Osmotic resistance: Growth over 10% sodium chloride Not produced Carotenoids: None Produced significant organic acids: None Formed starch-like substances: None Vitamin requirement: Grows weakly in medium containing no vitamin, but growth is increased by addition of biotin. Characteristic physiological properties Picolinic acid resistance: NAD in methanol medium supplemented with 0.3% quinolinic acid
Content: 26 mg / g dry cells (79 mg / medium) (e) Fermentability and assimilation Fermentability carbon source: D-glucose Non-fermentation carbon source: D-galactose, maltose, sucrose, lactose, Raffinose assimilating carbon source: D-ribose, D-xylose, D-glucose, D-
Mannose, D-fructose, ethanol, adonite, erythritol, D-mannitol, D-sorbit,
Glycerin Carbon source with weak assimilation: D-galactose, maltose, D-gluconate, DL-
Lactate, succinate Carbon source without assimilation: D-arabinose, L-arabinose, L-rhamnose, L-sorbose, sucrose, lactose, melibiose, cellobiose, trehalose, raffinose, D-merezitose, α-methyl- D-Glucoside, Arbutin, Dextrin, Soluble Starch, Inulin, Inosit, Drusit, 2-Keto-D-Gluconate, Citrate (THE YEASTS
A taxonomic study) '' (1970) and Rij's `` The East Taxonomy Study (Th
e Yeasta a taxonomic study) ”(1984), both strains were found to be
Although the results were different for a few sugars that were judged to belong to the genus dida) and were not so important in terms of carbon source assimilation, they were in agreement in other properties. Ca
ndida boidinii) was judged to be appropriate.

The parent strain Kyandeida 25-A is not resistant to 3-acetylpyridine, whereas the AP-15 strain is now resistant to 3-acetylpyridine, showing a significant difference between the two. Was given. Moreover, the NAD content is about 2. compared to the parent strain Kyandeida 25-A.
It increased by 0 times.

In this way, the AP-15 strain is clearly different in bacteriological properties from the existing strains, so it was judged as a new strain and named as Candida boidinii AP-15, September 1986. It was deposited on the 25th with the Ministry of International Trade and Industry, Institute of Industrial Science, Institute of Microbial Technology (Microtechnology Research Institute of Microbiology No. 8974 (FERMP-8974)).

Moreover, while the parent strain, Kyandeida 25-A, is not resistant to picolinic acid, the PC-31 strain is now resistant to picolinic acid, which shows a significant difference between the two. Moreover, the NAD content was about 2.4 times higher than that of the parent strain, Kyandeida 25-A.

In this way, the PC-31 strain is clearly different in bacteriological properties from the existing strains, so it was judged as a new strain and named as Candida boidinii PC-31, September 1986. On the 25th, it was deposited with the Institute of Microbial Engineering, Ministry of International Trade and Industry (Ministry of Industrial Science and Technology, Microbial Research No. 8975 (FERMP-8975)).

In the present invention, the yeast is cultured in a medium containing methanol as a main carbon source. In culturing, methanol was added in an amount of about 0.5 to 3% by volume, and quinolinic acid was added in an amount of 0.1 to 0.
It is preferable to culture in a medium containing about 5%, and the other medium composition may be a culture composition usually used in yeast belonging to the genus Canydadea. For example, peptone, urea, ammonium sulfate, ammonium chloride, ammonium nitrate, sodium nitrate, etc. are used as the nitrogen source, and sodium dihydrogen phosphate is used as the phosphoric acid source.
Potassium dihydrogen phosphate, potassium monohydrogen phosphate and the like are used. Furthermore, as growth promoting factors, piotin, thiamine, yeast extract, iron, calcium, manganese,
A trace amount of a metal salt such as zinc can be added.

In the present invention, the culture is carried out at a pH of 4-7 and a temperature of 25-3.
It is preferable to carry out the culture with aeration and stirring at 3 ° 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.

Next, in order to isolate and purify NAD from the obtained culture, for example, cells are first collected from the culture by centrifugation, filtration, etc., and then the collected cells are subjected to a conventional method, namely, 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 crushing the cells and then centrifuging, or adsorbing on a carrier for purification.

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

In the Reference Examples and Examples,% means% by weight unless otherwise specified.

Reference Example 1 First, in order to examine the sensitivity of 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,
Potassium dihydrogen phosphate 0.1%, potassium monohydrogen phosphate 0.1%, magnesium sulfate 0.05%, ferric chloride 3
0 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 / m
1. A plate was prepared using a medium consisting of 1, agar 1.5% and pH 6.0 (hereinafter abbreviated as methanol medium),
The bacterial suspensions of various concentrations were sprinkled thereon, and ultraviolet rays were radiated on the bacterial suspension at an illuminance of 400 μw / cm ² , and the irradiation time for giving a survival rate of 0.1% was determined to be 34 seconds.

Next, the medium to which various concentrations of 3-acetylpyridine were added to methanol medium was spread with Kyandeida 25-A (Microtechnical Laboratory No. 8725), and the sensitivity to 3-acetylpyridine was examined. 20 mM
It was found that the contained medium could not grow at all.
Therefore, 20 ml of 3-acetylpyridine was added to methanol medium.
Make a plate with M added, and add Kyandeida 25 to it.
-A (Microtech Lab. No. 8725) was sprinkled, and ultraviolet rays were radiated from above on the illuminance of 400 μw / cm ² for 34 seconds.
After culturing at 28 ° C for 1 week, a large number of grown colonies were picked up. These strains were cultivated with shaking in a liquid medium prepared by adding 0.3% quinolinic acid to a methanol medium.
When the NAD content in the bacterial cells was measured after a day, the NAD content in these strains was about 2.0 times higher than that of the parent strain. Candida boidinii
AP-15 [Microtechnology Research Institute Microbiology No. 8974 (FERMP-8
974)] was obtained.

The NAD was quantified by collecting the cells with hot water at 85 ° C. after collection, and then performing an enzymatic method using alcohol dehydrogenase [Methods of Enzymatic Analysis 3rd Edition (198).
4) Volume 7, page 253].

Reference Example 2 In the same manner as in Reference Example 1, a picolinic acid-resistant mutant strain was obtained.

That is, Kyandeida 25-A
No. 5) was examined for sensitivity to pyriconic acid, and it was found that it could not grow at all in a medium containing picolinic acid 10 mM. Therefore, after irradiating a strain that grows even at this concentration under the same ultraviolet treatment conditions as in Reference Example 1, When the NAD content was measured in the same manner as in Reference Example 1, among them, the NAD content was about 2.4 times higher than that of the parent strain, Candida boidinii PC-31.
[Microtech Lab, No. 8975 (FERM P-897
5)] was obtained.

Example 1 Glucose 1%, ammonium chloride 0.4%, phosphoric acid 2
Potassium hydrogen 0.1%, magnesium sulfate 0.05%,
Candida boidin (Candida boidin) obtained in Reference Example 1 was added to a medium having a composition of 0.2% yeast extract.
ii) AP-15 [Microtechnology Research Institute, Microbiology No. 8974 (FERM
P-8974)] at pH 6.0 and 2 at 28 ° C.
The seed culture was prepared by shaking culture for 4 hours.

Next, methanol 2% by volume, ammonium chloride 0.5
%, Potassium dihydrogen phosphate 0.1%, potassium monohydrogen phosphate 0.1%, magnesium sulfate 0.05%, second chloride
Iron 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 /
100 ml of a medium having a composition (pH 6.0) consisting of 0.3 ml of quinolinic acid was placed in a 500 ml shake flask, and 1% of the above inoculum solution was added thereto, followed by shaking culture at 28 ° C.

100 hours after starting the culture, 22 mg in the yeast cells
/ G of dry cell NAD was accumulated.

Example 2 Cyandeida Boydini (Cand obtained in Reference Example 2)
ia boidinii) PC-31 [Microtechnology Research Institute of Microbiology No. 8975 (FERM P-8975)] was used and cultured in exactly the same manner as in Example 1 to obtain 26 mg / g dry matter in the yeast cells. NAD of the bacterial cells had accumulated.

Comparative Example 1 Yeast cells were obtained by culturing in exactly the same manner as in Example 1 except that the parent strain, Candida 25-A [Microtechnical Lab. No. 8725 (FERM P-8725)] was used. NAD of 11 mg / g dry cell was accumulated in the inside.

Example 3 A 2-volume mini jar fermenter was charged with 1.2 of the fermentation medium used in Example 1, inoculated in the same manner as in Example 1, and then cultured at a temperature of 28 ° C. and an aeration condition of 1 VVM and 400 rpm. It was About 100 hours after the start of culture, about 2
NAD of 2 mg / g dry cells and 53 mg of NAD accumulated in the culture medium 1.2.

Next, cells were obtained from this culture solution by centrifugation,
It was washed 3 times with 0.9% sodium chloride. 8 to this fungus
About 10 ml of hot water at 5 ° C. was added, NAD was extracted by stirring in a hot water bath at 85 ° C. for about 5 minutes, and after cooling, an extract was obtained by centrifugation. Dowex 1-X2 (200
~ 400 mesh, formic acid type) column (0.8 × 40 cm)
Gradient elution with formic acid 0-0.15 N (40
0 ml), and fractionated every 10 ml. When the absorbance 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. Collect this fraction,
After adjusting the pH to about 2, it was adsorbed on activated carbon. After thoroughly stirring this activated carbon, suction filter it and cool it to an appropriate amount with 0.0
Immediately after washing with 01N HCl, 50% ethanol:
It was added to a concentrated ammonia (200: 1) solution, and after stirring, suction filtration was performed to collect a filtrate containing NAD.

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

(Effects of the Invention) According to the present invention, productivity is improved and NAD can be efficiently obtained, as compared with the conventional method using a wild strain using methanol as a raw material for culture, whereby NAD becomes inexpensive. .

Claims (1)

[Claims] 1. A mutant strain AP of Kyandeida voidini
A method for producing nicotinamide adenine dinucleotide, which comprises culturing -15 strain or PC-31 strain in a medium containing methanol as a main carbon source, and collecting nicotinamide adenine dinucleotide from the culture.