JPS6224072B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing L-lysine by fermentation. L-lysine is useful as an essential amino acid, and an inexpensive industrial method for producing it is expected.

Amino acids are produced by fermentation 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 the large amount of impurities they contain. There are various problems such as the large amount of by-products produced and the coloring of the fermentation waste liquid, and fundamental improvements have been desired. As a countermeasure to these problems, production methods using fermentation raw materials such as hydrocarbons are being researched.
Hydrocarbons are difficult to use industrially because they are gaseous or insoluble in water, which limits the yield of products. Therefore, the present inventors believe that cheap and stable supply is possible,
As a result of intensive research focusing on ethanol, which does not have the above drawbacks, we found that among microorganisms that belong to the genus Acinetobacter and have the ability to assimilate ethanol, when cultured in a medium containing ethanol as the main carbon source, a significant amount of L.
- Discovered a microorganism that has the ability to produce and accumulate lysine, and completed the invention. That is, a microorganism belonging to the genus Acinetobacter, capable of assimilating ethanol, and capable of producing L-lysine is cultured in a medium containing ethanol as the main carbon source.
L-lysine can be efficiently produced by producing and accumulating lysine and collecting it from the culture.

The microorganisms used in the present invention belong to the genus Acinetobacter and have the ability to assimilate ethanol and produce and accumulate L-lysine, and may be isolated from nature, known strains, or artificially inherited from the future. It may be a mutant strain.

Acinetobacter calcoaceticum YK-1011 is shown as an example of a microorganism that produces and accumulates L-lysine from ethanol.

Acinetobacter Calcoaceticum YK-
1011 is the receipt number 4818 of the Institute of Microbiology, Agency of Industrial Science and Technology.
(February 9, 1978, Microtechnical Research Institute No. 4818).

This microorganism was isolated from Acinetobacter calcoaceticum ATCC19606 as a 5-methyl-DL-tryptophan-resistant strain, and other than its resistance to 5-methyl-DL-tryptophan, other characteristics are similar to that of Acinetobacter.
Identical to Calcoaceticum ATCC19606.

A concrete implementation method of the present invention is as follows.

Ethanol is used as the carbon source in the medium composition, and the initial concentration is selected from 1 to 5% v/v or less depending on the strain used.
Note that ethanol is added sequentially while paying attention to appropriate concentration conditions that do not inhibit the growth of the bacterial strain or the production of L-lysine 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, flavor liquid, yeast extract, inorganic salts, vitamins, etc. are added to the medium according to the required amount.

Regarding culture conditions, temperature: 20-37℃, pH: 4~
10. Preferably, the culture may be carried out at 25 to 35° C. and at a pH of 6 to 8, but these conditions are also selected as appropriate depending on the strain used. Culture is about 2~
It takes 7 days.

After completion of the culture, L-lysine produced 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-lysine produced was determined by subtracting the amount of L-lysine contained in the culture medium using a microbial quantitative method using Leuconostoc mesenteroides ATCC8042.

Example 1 Preculture medium (20 g of urea, 7.0 g of ammonium sulfate,
KH ₂ PO ₄ 0.5g, K ₂ HPO ₄ 0.5g, MgSO ₄・
7H ₂ O 0.5g, yeast extract 0.5g, casamino acid 0.5g,
FeSO ₄・7H ₂ O2mg, MnSO ₄・4～6H ₂ O2mg,
ZnSO ₄・7H ₂ O2mg, NaCl2mg, CaCl ₂・2H ₂ O2
mg, biotin 200μg, thiamine hydrochloride 100μg,
1) Dispense 10ml of tap water into a large test tube with a diameter of 24mm, sterilize it at 120℃ for 10 minutes, add 0.2ml of ethanol under aseptic conditions, inoculate it with Acinetobacter calcoaceticum YK-1011, and inoculate it at 30℃. Perform shaking culture for 2 days. Next, 10 ml of the same medium as the pre-culture medium.
Dispense the solution into a large test tube with a diameter of 24 mm, sterilize it at 120℃ for 10 minutes, add 0.2 g of calcium carbonate that has been dry heat sterilized in advance, add 0.2 ml of ethanol, and inoculate 0.2 ml of the preculture solution. Incubate the cells and culture 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, 6 g/accumulated L-lysine was collected.

Example 2 10 ml of the same preculture medium used in Example 1 was
The mixture was dispensed into large 24 mm test tubes and sterilized at 120°C for 10 minutes. Add 0.2ml of ethanol to this under sterile conditions.
In addition, Acinetobacter calcoaceticum YK-1011 (Feikoken Bibori No. 4818) was inoculated and cultured with shaking at 30°C for 2 days.

Next, 100 ml of the same medium as the preculture medium was dispensed into a 500 ml Erlenmeyer flask and sterilized at 120°C for 10 minutes.
To this was added 2 g of calcium carbonate that had been previously dried and sterilized, and then 2 ml of ethanol, followed by inoculation with 2 ml of the above preculture solution, and cultured with shaking at 30° C. for 7 days. At this time, ethanol is 3v/v as it is consumed.
It was added so as not to exceed %. On the 7th day of culture, a culture solution was obtained in which 6.5 g of L-lysine had been accumulated. This culture solution
The supernatant liquid obtained by centrifuging 100 ml was passed through a column of strongly acidic ion exchange resin [Amberlite IR-120, H type, manufactured by Rohm and Haas].
- Adsorbed lysine. The eluate was then eluted with 3% aqueous ammonia, the eluate was concentrated under reduced pressure, hydrochloric acid was added thereto, and the mixture was cooled, crystallized, and dried to obtain 560 ml of L-lysine hydrochloride.

Claims (1)

[Claims] 1. A microorganism that belongs to the genus Acinetobacter and has the ability to assimilate ethanol and produce L-lysine is cultured in a medium containing ethanol as the main carbon source to produce and accumulate L-lysine, and this is extracted from the culture. L by a fermentation method characterized by collecting
-Production method of lysine.