JPH055478B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD The present invention relates to a method for producing L-phenylalanine using microorganisms. Conventional technology Conventionally, as a method for producing L-phenylalanine by an enzymatic method using phenylpyruvic acid as a substrate, phenylpyruvic acid and various amino acids as amino group donors are made to act on various microbial cells. (Amino Acids, Vol. 2, p. 18, 1960; Special Publication No. 37-10672; Amino Acids, Vol. 5,
p. 61, 1962; Japanese Patent Publication No. 45-20556), phenylpyruvic acid and nylon cyclic oligomer (Japanese Patent Publication No. 17991-1962), and nylon 6 hydrolyzate (Japanese Patent Publication No. 17991-1962). 44−17992
A method is known in which a lactam (Japanese Patent Publication No. 44-17990) is used. Problems to be Solved by the Invention In the conventional methods, the amino group donor raw material used is expensive, and the yield of L-phenylalanine is still unsatisfactory. There is always a need for an excellent method for producing L-phenylalanine. Means for solving the problem According to the present invention, a significant amount of L-phenylalanine can be obtained by using a microorganism capable of converting phenylpyruvate into L-phenylalanine in the presence of fumaric acid and ammonium ions or urea. Enilalanine can be obtained. The microorganisms used in the present invention include Aslobacter, Bacillus, Brevibacterium, and Corynebacterium, which have the ability to convert phenylpyruvate to L-phenylalanine in the presence of fumaric acid and ammonium ions or urea. If it is a microorganism belonging to the genus P. genus, Flavobacterium genus, Klebsiella genus, Kluybella genus, or Micrococcus genus, wild strains, mutant strains, recombinant strains induced by cell fusion method, gene manipulation method, or other genetic methods, etc. Also used is Arthrobacter globiformis (Arthrobacter globiformis).
globiformis) ATCC8010, Bacillus sphaericus ATCC10208, Brevibacterium lactofermentum
ATCC13655, Corynebacterium hydrocarboclusterum
hydrocarboclastum) ATCC15108, Flavobacterium suaveolens (Flavobacterium
suaneolens) ATCC958, Klebsiella oxytoca ATCC8724, Kluyvera cryorescens
Examples include ATCC14237 and Micrococcus luteus ATCC10240. The medium for culturing these microorganisms to obtain L-phenylalanine may be a natural medium, as long as it contains a carbon source, a nitrogen source, an inorganic salt, etc.
Any artificial culture medium may be used. Carbon sources include glucose, fructose,
Sugars such as sucrose, maltose, starch, and blackstrap molasses; Sugar alcohols such as glycerol, sorbitol, and mannitol; Organic acids such as acetic acid, formic acid, fumaric acid, malic acid, citric acid, and gluconic acid; Alcohols such as methanol, ethanol, and propanol. class is used. Nitrogen sources include ammonium compounds such as aqueous ammonia, ammonium chloride, ammonium sulfate, ammonium carbonate, ammonium acetate, and ammonium phosphate, nitrogen compounds such as urea, and amino acids such as glutamic acid, aspartic acid, methionine, glycine, lysine, arginine, and ornithine. ,
Natural nutrients such as peptone, yeast extract, casein hydrolyzate, defatted soybean or its digested product are used. As the inorganic substance, primary potassium phosphate, secondary potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium carbonate, etc. are used. If the microorganisms used in the present invention require specific nutrients for growth, appropriate amounts of those nutrients must be present in the culture medium, but these substances are not included in the natural products exemplified as nitrogen sources. Sometimes it is added. Phenylpyruvic acid, fumaric acid, and ammonium ion or urea may be added to the medium from the beginning or during the cultivation of the microorganism. Phenylpyruvic acid and fumaric acid are used as various salts such as ammonium salt, sodium salt, potassium salt, and calcium salt. Ammonium ions are supplied as salts such as ammonium sulfate, ammonium phosphate, ammonium nitrate, ammonium chloride, ammonium acetate, ammonium formate, ammonium fumarate, ammonium malate, or as ammonia water or ammonia gas. Cultivation is performed at a temperature of 20 to 40°C and a pH of 5 to 8 for 1 to 5 days. Thus, L-phenylalanine is produced in the culture solution. Next, as the medium composition and culture conditions when obtaining the cells of the microorganism, the medium composition and culture conditions described above are used. The microbial cells thus obtained can be used as they are for the reaction, or the processed products obtained by subjecting the microorganisms to various treatments may be used for the reaction. Examples of microbial cells include mechanically triturated cells, ultrasonication processed products, freeze-dried processed products, solvent-treated products, enzyme-treated products, dry-processed products, surfactant-treated products, and protein fractions of microbial cells. , immobilized products of bacterial cells and treated bacterial cells, etc. are used. The reaction is carried out in an aqueous solution by using the bacterial cells obtained above or a treated product thereof as phenylpyruvic acid, fumaric acid, and ammonium ions or urea. The phenylpyruvic acid, fumaric acid and ammonium ions used in the reaction are the same as those described above. The concentration of phenylpyruvic acid used in the reaction is 0.01 to 1 mol, and the concentration of fumaric acid is 0.01 to 1 mol.
The concentration ranges from 0.01 to 2 moles, and the concentration of ammonium ions or urea ranges from 0.01 to 10 moles. These raw materials are supplied in bulk or intermittently. Working conditions include a temperature of 20-60°C, preferably
25~45℃, PH6~10, preferably 7~9, 10~
Do it for 48 hours. Thus, L-phenylalanine is produced in the aqueous liquid. As a method for recovering L-phenylalanine from a culture solution or an aqueous solution, an ion exchange resin method, a precipitation method, etc. are used. Examples are shown below. Example 1 A test tube containing 10 ml of a medium (PH7.0) with a composition of 0.5% glucose, 0.3% yeast extract, and 1.5% powdered meat extract was inoculated with the microorganisms shown in Table 1 to 1ase,
Shake culture at 210 rpm and 28°C for 18 hours. After culturing, collect the bacteria by centrifugation and store the cells frozen (-20°C). After freezing for one day, 10ml of
Add 0.85% saline solution to the test tube and after thawing, centrifuge and wash. The obtained microorganisms were suspended in 1.5 ml of a reaction solution with the following composition and allowed to react for 18 hours under standing conditions at 40°C. As a result, each microorganism produced L-phenylalanine at the concentration shown in Table 1. was generated. The composition of the reaction solution is as follows: sodium β-phenylpyruvate 100mM, pyridoxal phosphate 200μM, Tris buffer 100mM (PH8.4), ammonium fumarate 200mM.

[Table] Example 2 The same procedure as in Example 1 was conducted except that Klebsiella oxytoca ATCC8724 was used and the composition of the reaction solution was changed as shown below. As a result, 3.5 mg/ml of L was added to the reaction solution.
- Phenylalanine was produced. Reaction solution composition: Sodium β-phenylpyruvate 100mM, pyridoxal phosphate 200μM, sodium fumarate 100mM, urea 100mM, Tris buffer 100mM (PH8.4) Effects of the invention Significant amounts of L-phenylalanine can be obtained by the method of the invention be able to.

Claims (1)

[Scope of Claims] 1 Belongs to the genus Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Flavobacterium, Klebsiella, Kluybella, or Micrococcus, and the presence of fumaric acid and ammonium ions or urea Cultivating a microorganism capable of converting phenylpyruvic acid into L-phenylalanine in a medium in the presence of (a) phenylpyruvate, (b) fumaric acid, and (c) ammonium ion or urea. B. Bacterial cells obtained by culturing in a medium or a treated product thereof and an aqueous solution containing the above (a), (b) and (c), and L-F L characterized by producing and collecting enilalanine
- A method for producing phenylalanine.