JP3289349B2 - Method for producing D-alanine by fermentation method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing D-alanine by a fermentation method.

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

[0003]

2. Description of the Related Art A method for producing D-alanine by a fermentation method using a microorganism belonging to the genus Brevibacterium having resistance to D-cycloserine has been known (Japanese Patent Application Laid-Open No. 1-187091). According to this method, it is possible to selectively generate and accumulate only D-alanine.

[0004]

SUMMARY OF THE INVENTION However, the amount of D-alanine obtained and the optical purity have not been sufficient.

[0005] As a result, the present inventors have found that microorganisms belonging to the genus Brevibacterium, which have special resistance, selectively produce and accumulate D-alanine with an improved accumulation amount and optical purity. Thus, the present invention has been completed.

That is, the present invention provides a method for culturing a microorganism belonging to the genus Brevibacterium having D-alanine producing ability and having resistance to β-chloro-D-alanine. -A method for producing D-alanine by a fermentation method, which comprises producing and accumulating alanine and collecting D-alanine from the culture solution.

Next, the present invention will be described in detail.

[0008] The microorganism used in the present invention is a microorganism belonging to the genus Brevibacterium which has D-alanine producing ability and is resistant to β-chloro-D-alanine. As long as it has such properties, those having other required and drug-resistant properties are also included in the scope of the present invention.

[0009] Representative mutant strains used in the present invention include, for example, Brevibacterium lactofermentum HR5-43 (Microtechnical Laboratory No. 13338).
Is mentioned. This mutant is a D-cycloserine resistant strain Brevibacterium lactofermentum DCSR17-2 (FERM BP-20) derived from Brevibacterium lactofermentum ATCC13869.
24) A mutant strain having resistance to β-chloro-D-alanine.

[0010] Induction of a mutant strain can be relatively easily carried out by a usual mutation treatment method. That is, in order to obtain a mutant strain having resistance to β-chloro-D-alanine, the parent strain is irradiated with ultraviolet light or a mutagen (eg, N-methyl-
N'-nitro-N-nitrosoguanidine, ethyl methanesulfonic acid, etc.), the growth of the parent strain was significantly greater than that of the parent strain in a medium containing an amount of β-chloro-D-alanine such that the parent strain could not grow sufficiently. What is necessary is just to obtain a viable strain.

The β-chloro-D-alanine resistant strain in the present invention is a strain having a higher resistance than its parent strain, and preferably has a relative growth of 40% after 64 hours of the parent strain.
The relative growth rate when cultured in a medium containing a β-chloro-D-alanine concentration as shown below is 50% or more. Here, the growth degree is measured by measuring the absorbance at 660 nm, and is expressed as a relative absorbance when the absorbance of the culture solution of each strain to which β-chloro-D-alanine is not added is expressed as 100%.

As shown in Examples, β-chloro-D-alanine resistance is a property independent of D-cycloserine resistance and is a novel trait to improve D-alanine productivity.

As the culture medium used in the method of the present invention, various nutrients commonly used for culturing microorganisms can be used. For example, as a carbon source, glucose, molasses, sugars such as starch hydrolysate, organic acids such as acetic acid, alcohols such as ethanol, organic compounds such as benzoic acid,
As the nitrogen source, ammonium sulfate, ammonium nitrate, ammonium salt, ammonium phosphate, urea, ammonia, and others can be used. Depending on the type of inorganic ammonium salt, an inorganic salt such as phosphate or calcium carbonate may be required. In order to improve the growth of microorganisms and to accumulate a considerable amount of D-alanine, it is desirable to add, for example, an organic nitrogen source, vitamins, trace amounts of metal ions, etc. to the above-mentioned medium. The purpose can be sufficiently achieved by adding liquor and the like.

The culture is performed under aerobic conditions. During the cultivation, the pH of the medium was adjusted from 5 to 9, the temperature was adjusted to 24-37 ° C,
Preferred results are obtained by shaking or aeration culture for 48-120 hours.

To collect D-alanine from the culture,
Normal methods can be used. For example, after the culture filtrate from which the cells have been removed is adjusted to pH 2 with hydrochloric acid, the solution is passed through a strongly acidic cation ion exchange resin, the adsorbed components are eluted with dilute aqueous ammonia, deammoniaized, and then concentrated. Alcohol was added thereto, and the crystals formed under cooling and preservation were collected.
Alanine can be obtained.

[0016]

The present invention will be described below in detail with reference to examples.

Example 1 (Isolation of β-chloro-D-alanine resistant strain) Brevibacterium lactofermentum DCSR1
After the cells of 7-2 (FERM BP-2024) were treated with N-methyl-N'-nitro-N-nitrosoguanidine (300 μg / ml, 10 minutes at 30 ° C.) by a conventional method, the cells were subjected to β-chloroform. -Agar medium supplemented with 0.8 g / l of D-alanine (D-ribose 1%, ammonium sulfate 2%, potassium monophosphate 0.2%, magnesium sulfate heptahydrate 0.1%).
04%, sodium chloride 0.1%, urea 0.5%, ferrous sulfate heptahydrate 20 mg / l, manganese sulfate tetrahydrate 20 mg / l, bition 120 μg / l, thiamine hydrochloride 60 μg / 1 complete synthetic medium). Then 3
The cells were cultured at 0 ° C. for 5 to 7 days, and the resulting large colonies were separated by filtration to obtain β-chloro-D-alanine resistant strains (Brevibacterium lactofermentum HR5-43).

Example 2 (degree of resistance of β-chloro-D-alanine resistant strain HR5-43) Each strain shown in Table 1 was shake-cultured at 30 ° C. for 16 hours using a liquid broth medium, and the cells grown were cultured. The cells were collected and washed well with physiological saline. This cell suspension is a minimal medium containing β-chloro-D-alanine at concentrations of 0, 150, and 300 mg / l, respectively (medium composition: D-ribose 1%, ammonium sulfate 2%, monobasic potassium phosphate 0.1%). 2%, magnesium sulfate heptahydrate 0.04%, sodium chloride 0.1%, urea 0.5%,
Inoculated in 5 ml of a complete synthetic medium containing ferrous sulfate heptahydrate 20 mg / l, manganese sulfate tetrahydrate 20 mg / l, bition 120 μg / l, and thiamine hydrochloride 60 μg / l) at 30 ° C. Cultured. Table 1 shows the results.

[0019]

[Table 1] (Note) The absorbance at 660 nm of the culture solution is shown in parentheses. Relative growth was calculated assuming that the absorbance of the culture solution of each strain to which β-chloro-D-alanine was not added was 100%.

The β-chloro-D-alanine resistant strain (HR5-43) used in the method of the present invention includes Brevibacterium lactofermentum ATCC 13869 and D-
Brevibacterium lactofermentum DCSR17-2 (FERM BP-2) which is a cycloserine resistant strain
024), it is clear that growth was not inhibited by β-chloro-D-alanine and that the strain had acquired resistance to β-chloro-D-alanine.

Example 3 Each strain was placed in a liquid broth medium (50 ml) at 30 ° C.
After shaking culture for 16 hours, containing 950 ml of a main fermentation medium having the following composition, which was previously steam-sterilized at 120 ° C. for 20 minutes.
30 ° C planted in a liter mini jar fermenter
Aeration and agitation culture was started at 500 rpm and an aeration rate of 0.5 vvm.

Main fermentation medium Glucose (separately sterilized) 5%, (NH 4) 2 SO 4 0.5
%, KH2 PO4 0.15%, NaCl 1.0%, Mg
0.035% SO4.7H2O, FeSO4.7H2
O 0.002%, MnSO4.4H2 O 0.002
%, CaSO4.2H2 O 0.02%, biotin 60
μg / l, Thiamine / HCl 40 μg / l

The pH was adjusted and the nitrogen source was supplied with 25% aqueous ammonia, and the pH was maintained at 6.0 to 8.0. While adding glucose as appropriate, were cultured for 72 hours, accumulation density in any case also D- alanine production yield is the parent strain D- cycloserine resistant a strain Brevibacterium lactofermentum DCSR17-
2 (FERM BP-2024).

The cells were removed from the fermented broth of Brevibacterium lactofermentum HR5-43 and its 500 ml
Is a strong cation exchange resin Diaion SK1-B (H type)
Through the column. After washing the column with water, the adsorbed components of the column were eluted with 2N aqueous ammonia, and after decolorization, concentrated under reduced pressure. Ethanol was added thereto, the mixture was cooled, and the generated crystals were collected and dried. As a result, 19.1 g of D-alanine crystals were obtained. The optical purity of the obtained D-alanine crystal was 99% or more.

[0027]

According to the present invention, it has become possible to produce and accumulate D-alanine with high accumulation concentration and high optical purity by fermentation. Therefore, D-alanine can be obtained at lower cost, and its industrial value is great.

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Claims (1)

(57) [Claims] 1. A microorganism belonging to the genus Brevibacterium having D-alanine producing ability and having resistance to β-chloro-D-alanine is cultured,
A method for producing D-alanine by a fermentation method, wherein D-alanine is produced and accumulated in a culture solution, and D-alanine is collected from the culture solution.