JPS6155957B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing L-valine by fermentation. L-valine is useful as an essential amino acid in medicine and as an additive to food for patients, and an inexpensive industrial production method is expected.

Amino acid production using the fermentation method is carried out using sugar (carbohydrate) substances as the main raw materials, but since the raw materials are agricultural products, there are problems with price and supply stability, and there are problems due to the large amount of impurities contained. There were various problems such as the large amount of by-products produced during fermentation and the coloring of the fermentation waste liquid, and fundamental improvements were desired. As a countermeasure to these problems, production methods using fermentation raw materials such as hydrocarbons are being researched, but hydrocarbons are difficult to use industrially because they are gaseous or insoluble in water. Therefore, there is a limit to the yield of the product. Therefore, the present inventors focused on ethanol, which is thought to be able to be supplied stably at low cost in the future, and which does not have the above drawbacks.
As a result of extensive research, we discovered a microorganism that belongs to the genus Acinetobacter and has the ability to produce and accumulate significant amounts of L-valine when cultured in a medium containing ethanol as the main carbon source. , hereby perfected the invention. Specifically, microorganisms belonging to the genus Acinetobacter and having the ability to assimilate ethanol and produce L-valine from ethanol are aerobically cultured in a medium containing ethanol as the main carbon source, and L-valine is added to the culture solution. - L-valine can be efficiently produced by producing and accumulating valine and collecting it.

The microorganism used in the present invention belongs to the genus Acinetobacter and has the ability to assimilate ethanol and produce and accumulate L-valine, and may be an isolated strain from the natural world, a known strain, or an artificial strain derived from these. It may also be a genetic mutant strain.

As a representative strain of the present invention, Acinetobacter calcoaceticum YK-1011 (National Institute of Fine Technology Deposit No.
4818). This strain is Acinetobacter
5-Methyl from Calcoaceticum ATCC19606
It was isolated as a DL-tryptophan-resistant strain, and other than the fact that it is 5-methyl-DL-tryptophan resistant, other characteristics are the same as Acinetobacter calcoaceticum ATCC19606. Acinetobacter Calcoaceticum
A method for inducing a 5-methyl-DL-tryptophan-resistant strain from ATCC19606 will be described. Acinetobacter calcoaceticum ATCC19606 uses 5-methyl-DL-tryptophan as a synthetic medium (urea 20
g, ammonium sulfate 7.0g, KH ₂ PO ₄ 0.5g, K ₂ HPO ₄ 0.5g,
MgSO ₄・7H ₂ O0.5g, FeSO ₄・7H ₂ O2mg,
MnSO ₄・4〜6H ₂ O2mg, NaC1 2mg, ZnSO ₄・
7H ₂ O2mg, water 1 ethanol 2% V/V) 2.00
Growth was almost completely inhibited in the plate medium supplemented with μ/ml. Mutant strains were induced by nitrosoguanidine treatment (nitrosoguanidine 200μg/
ml, 30℃, 15 minutes, PH7.0, Trismaleate buffer EAAdelberg et.al.Biochem.Biophys.Res.
Comm. 18 , 788 (1965)), and then cultured at 30°C for 3 to 5 days in a plate medium containing 200 μg/ml of 5-methyl-DL-tryptophan as described above.
Obtained by separating viable colonies. Naturally, the induction of mutant strains is not limited to the above-mentioned nitrosoguanine method, but also UV irradiation method,
In addition, various drugs can also be used.

A concrete implementation method of the present invention will be described.

Ethanol is used as the carbon source in the medium composition, but the initial concentration depends on the strain used.
Select appropriate conditions from within 1 to 5% V/V.
Incidentally, ethanol can also be added sequentially while paying attention to appropriate concentration conditions that do not inhibit the growth of the bacterial strain or the production of L-valine as it is consumed. The nitrogen source is selected from among ammonium sulfate, ammonium nitrate, ammonium phosphorus, urea, etc., depending on the utilization capacity of the bacterial strain. In addition, organic nutritional sources such as amino acids, cornstarch liquor, flavoring liquid, and yeast extract may be added as needed.
Add inorganic salts, vitamins, etc. and use it as a medium.

Regarding culture medium conditions, culture may be carried out at a temperature of 20 to 37°C, preferably 25 to 35°C, and a pH of 4 to 10, preferably PH6 to 8, but these conditions should also be selected as appropriate depending on the strain used. . Cultivation requires approximately 2 to 7 days. After the cultivation is completed, the produced L-valine can be recovered from the liquid by a known method such as an ion exchange resin method, an activated carbon method, or a concentration crystallization method. Examples are shown below.

The amount of L-valine produced was determined by subtracting the amount of L-valine contained in the culture medium using a microbial quantitative method using Leuconostocmesenteroides ATCC8042.

Example Preculture medium (urea 20g, ammonium sulfate 7.0g, KH ₂ PO ₄
0.5g, _{K2HPO4 0.5g} , _MgSO4・_7H2O0.5g , yeast extract 0.5g, casamino acid 0.5g, _FeSO4・
_7H2O2mg , NaCl2mg, _CaC12・_2H2O2mg , tap water 1) Dispense 10ml into a large test tube with a diameter of 24mm,
Sterilize at 120°C for 10 minutes, add 0.2 ml of ethanol under no conditions, inoculate with Acinetobacter calcoaceticum YK1011, and culture with shaking at 30°C for 2 days. Next, 10 ml of the same medium as the pre-culture medium
Calcium carbonate was dispensed into large test tubes with a diameter of 24 mm and sterilized at 120°C for 10 minutes, followed by dry heat sterilization.
After adding 0.2 g and then 0.2 ml of ethanol, 0.2 ml of the preculture solution was inoculated and cultured with shaking at 30°C for 7 days. Ethanol is added as it is consumed. (At this time, ethanol should not exceed 3% V/V.) On the 7th day of culture, 600 mg/L-valine was accumulated. Cells were removed from 100 ml of this culture solution by centrifugation, and the supernatant was collected using a strongly acidic cation exchange resin (Diaion SK-1B, H type).
After adsorbing L-valine, follow the usual method.
It was eluted with 0.5N ammonia water, the L-valine fraction was concentrated, and after decolorizing with activated carbon, cold ethanol was added to obtain 40 mg of crude crystals of L-valine. No production of L-valine was observed in Acinetobacter calcoaceticum ATCC19606, which was similarly cultured.

Claims (1)

[Claims] 1 A microorganism that belongs to the genus Acinetobacter and has the ability to assimilate ethanol and produce L-valine from ethanol is aerobically cultured in a medium containing ethanol as the main carbon source, and L-valine is added to the culture solution. A method for producing L-valine by a fermentation method, which is characterized by producing and accumulating valine and collecting it.