JPH0563156B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Field of Application> The present invention relates to an industrially advantageous method for producing D-alanine.

D-alanine is a non-natural amino acid,
It is a compound itself useful as a reagent or as a raw material for the synthesis of peptides and the like, and demand for it has increased in recent years.

<Prior art> Many methods have been known to produce DL-alanine by fermentation using microorganisms belonging to the genus Brevibacterium [Amino Acid Fermentation (Vol. 2), p. 119, Kyoritsu Shuppan (1971). Publication)].

Additionally, a method for preparing D-alanine by using DL-alanine as a raw material and assimilating L-alanine into yeast is also known (Amino Acid.
Nuclic Acid, 1967, No. 15, 89-94).

<Problems to be Solved by the Invention> However, when producing DL-alanine, some kind of optical resolution operation is unavoidable in order to obtain only D-alanine, and microorganisms are used to separate L-alanine from DL-alanine. In the method of obtaining D-alanine by assimilating only alanine, only about half of the raw material can be effectively utilized.

Therefore, the present inventors conducted extensive research with the aim of providing a cheaper and simpler method for producing D-alanine by selectively converting L-alanine to D-alanine using microorganisms.

<Means for Solving the Problems> As a result, the present inventors discovered that a microorganism belonging to the genus Brevibacterium surprisingly efficiently converts L-alanine into D-alanine, and completed the present invention. did. By the way, the method for producing D-alanine using any microorganism has not yet been known, and is a completely new technology.

That is, the present invention converts L-alanine to D-alanine using a microorganism that belongs to the genus Brevibacterium and has the ability to convert L-alanine to D-alanine, and collects the D-alanine that has been produced and accumulated. The present invention relates to a method for producing D-alanine, which is characterized by the following. Note that it is not necessary to use L-alanine with a purity of 100%, and one containing the D-isomer in any proportion can also be used.

Next, the present invention will be explained in detail.

The microorganism used in the present invention belongs to the genus Brevibacterium and has the ability to convert L-alanine to D-alanine. It is within the scope of the present invention.

Typical strains used in the present invention include, for example, Brevibacterium lactofamentum DCSR17-2 (Feikoken Jokyo No. 2024)
can be mentioned. This strain is Brevibacterium
Lactofermentum ATCC13869 (biotin-requiring) is a mutant strain that is resistant to D-cycloserine, and can ferment and produce D-alanine under appropriate conditions.

Mutant strains can be induced relatively easily by conventional mutation treatment methods. That is, to obtain a mutant strain resistant to D-cycloserine, the parent strain must be irradiated with ultraviolet rays or a mutagenic agent (for example, N-
Methyl-N'-nitro-N-nitrocyguanidine,
After treatment with ethyl methanesulfonic acid, etc.),
What is necessary is to obtain a strain that can grow significantly compared to the parent strain in a medium containing an amount of D-cycloserine that does not allow the parent strain to grow sufficiently.

As the medium for culturing the bacteria used in the method of the present invention, various nutrient sources commonly used for culturing microorganisms can be used. For example, carbon sources include sugars such as glucose, molasses, and starch hydrolyzate, organic acids such as acetic acid, alcohols such as ethanol,
Organic compounds such as benzoic acid and nitrogen sources include ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium phosphorus, urea, ammonia, and others. Depending on the type of inorganic ammonium salt, inorganic compounds such as phosphate and calcium carbonate can be used. Some may require salt. In addition, it is preferable to add organic nitrogen sources, vitamins, trace amounts of metal ions, etc. to the above medium in order to improve the growth of microorganisms, but usually the addition of inexpensive flavoring liquids, corn steep liquor, etc. is sufficient. can achieve the objectives of

In the present invention, the cultivation of the microorganism is preferably carried out under aerobic conditions such as shaking or aerating the medium. Culture temperature is usually 20-40
℃, especially preferably around 30℃. In addition, the pH of the medium is usually 5 to 9, preferably
It is desirable to maintain it near neutrality.

After culturing in this way for several days, L-alanine or D,L containing L-alanine in any proportion is added directly to the culture solution or to the reaction solution containing the isolated bacterial cells.
- D-alanine is produced and accumulated in the medium by adding alanine and culturing for several more days.

Furthermore, even if the culture medium is sterilized by adding L-alanine in advance, inoculated with microorganisms, and cultured for several days, D-alanine is produced and accumulated in the medium. In addition,
Similar results can be obtained using D,L-alanine containing L-alanine in any proportion. After completion of the culture, the produced D-alanine can be easily collected by using a combination of known isolation and purification operations such as, for example, ion exchange method, adsorption method, and precipitation method.

<Example> Hereinafter, the present invention will be specifically explained with reference to Examples.

Example 1 (Acquisition of bacterial strain) Brevibacterium lactofamentum
After treating ATCC13869 (biotin auxotrophic) cells with N-methyl-N'-nitro-N-nitrosoguanidine (300 μg/ml, 10 minutes at 30°C) using a conventional method, the cells were treated with D-cycloserine 50 mg/l. Agar medium supplemented with (2% glucose, 1% ammonium sulfate, 0.1% potassium phosphate, 0.04% magnesium sulfate heptahydrate, 0.05% sodium chloride, 0.25% urea,
Ferrous sulfate heptahydrate 10mg/l, manganese sulfate
Completely synthetic medium containing 10 mg/l of tetrahydrate and 50 μg/l of biotin) was coated. Next, culture at 30℃ for 5 to 7 days, isolate the resulting large colonies, and
- A cycloserine-resistant strain (Brevibacterium lactofamentum DCSR-26) was obtained.

The obtained Brevibacterium lactofamentum DCSR-26 was further subjected to the same treatment and cultured in the same medium to obtain a D-cycloserine highly resistant strain (Brevibacterium lactofamentum).
DCSR17-2) was obtained.

Example 2 (Isolated cell method) Glucose 10%, Ammonium sulfate 3%, Potassium phosphate 0.05%, Potassium phosphate 0.05%, Magnesium sulfate heptahydrate 0.025%, Flavor liquid 2%, Calcium carbonate Dispense 40 ml of a medium (PH7.25) containing 3% biotin and 30 μg/l of biotin into a 1 liter Erlenmeyer flask, and add Brevibacterium lactofamentum DCSR17-2 to the culture medium that has been autoclaved at 120°C for 10 minutes. One platinum loop was inoculated and cultured at 30°C for 5 days. Centrifuge the bacterial cells from the culture solution,
Glucose 4%, Potassium phosphate 0.05%, Potassium phosphate 0.05%, Magnesium sulfate 7
Hydrate 0.025%, taste acid 2%, calcium carbonate 3%,
Suspend in 40 ml of ammonia-free source reaction solution (PH7.25) containing 30 μg/l of biotin and 4% L-alanine,
As a result of reacting at ℃ for 45 hours, 38.6 g/l was found in the reaction solution.
of D-alanine was accumulated. On the other hand, when a reaction solution to which L-alanine was not added was similarly reacted, the accumulation of alanine was 0.5 g/l or less.

D- and L-form alanine can be analyzed using commercially available D-
It was measured by an enzymatic method using amino acid oxidase and by high performance liquid chromatography (HPLC) using a Sumitomo Chemical OA-1000 optical resolution column.

Example 3 (Isolated cell method) Brevibacterium lactofamentum DCSR17- isolated after culturing in the same manner as in Example 2
2 bacterial cells with 4% glucose and potassium phosphate
Ammonia-free reaction solution containing 0.05% dipotassium phosphate, 0.05% magnesium sulfate heptahydrate, 2% taste liquid, 3% calcium carbonate, 30 μg/l biotin, 4% D,L-alanine. (PH7.25)
As a result of suspending the suspension in 40 ml and reacting at 30°C for 45 hours, 39.7 g/l of D-alanine was accumulated in the reaction solution.

On the other hand, when a reaction solution without D,L-alanine was reacted in the same way, the accumulation of alanine was 0.5
g/l or less.

D- and L-form alanine analysis was performed in the same manner as in Example 2.

Example 4 Brevibacterium lactofamentum DCSR17-2 was inoculated into the same culture medium as in Example 2, and cultured at 30°C for 3 days to produce 20 g/l of DL-alanine.
was added. As a result of further culturing for 64 hours, 48.8 g/l of D-alanine was produced and accumulated in the medium as shown in FIG.

The bacterial cells were removed from 200 ml of this culture solution by centrifugation, and the resulting supernatant was treated with decolorizing charcoal. This decolorizing charcoal treatment liquid is used as a strong cation exchange resin Diaion SK.
^D-
Alanine was adsorbed, washed with water, eluted with 2N ammonia water, the D-alanine fraction was concentrated, ethanol was added to the resulting concentrated solution, and the precipitated crystals were collected. The crystals were recrystallized from ethanol to obtain 7.2 g of D-alanine crystals.

Optical purity 99.4% Specific rotation [α] ²⁵ _D = -14.2° (C = 6, 1N-HCl) Analyzes of D-form and L-form alanine were conducted in the same manner as in Example 2.

On the other hand, when cultured without adding D,L-alanine, 30.9 g/l of D-
Only alanine is produced and accumulated, D, L-alanine 20
g/l, this means that 17.9 g/l of D-alanine was produced and accumulated.

<Effects of the Invention> According to the present invention, it has become possible to exchange L-alanine with D-alanine using microorganisms.
Therefore, optical resolution of DL-alanine or L-
No need to assimilate alanine, making it cheaper and easier
It has become possible to obtain DL-alanine, and its industrial value is great.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the relationship between culture time and the amount of D-alanine accumulated in the medium in Example 4.

Claims (1)

[Claims] 1. Using a microorganism that belongs to the genus Brevibacterium and has the ability to convert L-alanine to D-alanine, L-alanine is converted to D-alanine.
- A method for producing D-alanine, which comprises converting it into alanine and collecting D-alanine that has been produced and accumulated.