JPH0552195B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD The present invention relates to a method for producing L-amino acids. More specifically, the present invention relates to a method for producing L-amino acids from DL- or L-amino acid amides by the action of enzymes produced by specific microorganisms. L-amino acids are important compounds as food additives, feed additives, pharmaceuticals, and intermediates for various industrial chemicals. PRIOR TECHNOLOGY L-amino acids are generally produced by direct fermentation or optical resolution of DL-amino acids produced by organic synthesis, but recently, precursors that can be obtained inexpensively by chemical synthesis have been produced using enzymes. Many production methods using so-called chemico-enzymatic methods have been proposed and put into practical use. For example, a method in which an acylase produced by a microorganism is applied to an N-acyl derivative of a DL-amino acid (Japanese Patent Publication No. 41-22380), a method in which a hydantoinase produced by a microorganism is applied to a hydantoin derivative of a DL-amino acid (Japanese Patent Publication No. 41-22380), -2274 publication),
A method in which fumaric acid is reacted with aspartase produced by a microorganism (Japanese Patent Publication No. 18867/1986 and JP Patent Publication No. 59/14089) and a method in which cinnamic acid is reacted with phenylalanine ammonia lyase produced by a microorganism ( Appl.Environ, Microbiol, 42
773 (1981)) are representative examples of chemical enzymatic production methods for L-amino acids. However, these methods have problems such as complicated reaction systems, harsh reaction conditions, and expensive raw materials, and there is still room for improvement as an industrial production method. . In addition, recently, enzymes produced by microorganisms have been used to
DL- or L-amino acid amide to the corresponding L
- A method using L-amidase, an enzyme produced by microorganisms of the genus Bacillus, Bacteridium, Micrococcus and Brevibacterium, for reactions that produce amino acids (Published in 1983-500319)
method using L-amidase produced by various yeasts and bacteria (Japanese Unexamined Patent Publication No. 13000/1983),
59-159789 and 60-36446). However, all of these methods have weak L-amidase activity, and are examples of experiments in which L-amino acid production reactions were carried out using a large amount of bacterial cells, or simply the use of known bacterial strains of various species to produce various DLs. −
or the discovery that L-amino acid amide can be hydrolyzed to produce the corresponding L-amino acid, but it is merely a finding that L- from DL- or L-amino acid amide is produced by the action of enzymes produced by microorganisms. From the viewpoint of an industrial production method for amino acids, the use of these microorganisms cannot be an economically advantageous production method. SUMMARY OF THE INVENTION Under these circumstances, the present inventors developed an L-amidase with high L-amidase activity that can efficiently produce the corresponding L-amino acid from DL-amino acid amide, which can be easily and inexpensively produced through chemical synthesis, as a raw material. In order to obtain microorganisms that produce enzymes with
As a result of screening for bacteria in soil, sludge, etc. from various places, Enterobacter cloatusei N-
The present inventors have discovered that the enzyme produced by the microorganism No. 7901 has extremely high L-amidase activity and is extremely effective in achieving the objects of the present invention, and has completed the present invention. That is, the present invention provides the ability to convert the general formula [In the formula, R represents an alkyl group (having 1 to 4 carbon atoms), a phenyl group, an aralkyl group, or a substituent-containing group. ] The gist of the present invention is a method for producing an L-amino acid, which is characterized by producing a corresponding L-amino acid from a DL- or L-amino acid amide represented by the following. Detailed Description of the Invention The microorganism used in the present invention was newly isolated and discovered by the present inventors as described above, and was introduced in FIKEN Article No. 873 (Enterobacter cloatusei N- 7901) and has been deposited with the Institute of Microbiology (Feikoken), Agency of Industrial Science and Technology. The mycological properties of these microorganisms are as follows. N-7901 strain (1) Morphology Bacillus 0.8-1.2 x 1.0-1.5 μm Flagella 1 or more pericyla Motility + Gram staining negative (2) Physiological properties Nitrate reduction + denitrification reaction − MR test − VP test + Production of indole - Production of hydrogen sulfide - Utilization of citrate + Production of pigment - Urease - Oxidase - Catalase + Growth range PH4-10 Optimum temperature 35-37℃ Attitude towards oxygen Facultative Anaerobic O- F test F Production of acid and gas from sugars Production of acid Production of gas Adonitol - - Arabinose + + Xylose + + Glucose + + Mannose + + Fructose + + Galactose + + Maltose + + Seuucrose + + Lactose + + Raffinose + Rhamnose + Glycerin + + Sorbitol + + Mannitol + + Inositol - - Dulcitol - - (3) Others Decomposition of starch - Decomposition of gelatin - Decomposition of urea Weak Utilization of malonate + Decarboxylation of lysine - Decarboxylation of ornithine + Deamination reaction of phenylalanine - Arginine dihydrolase + β-galactosidase + Potassium cyanide resistance + The above mycological properties are summarized in Bergy's Manual of Determinative Bacteriology, 8th edition. of Determinative
Bacteriology 8th.ed.), N
-7901 strain was identified as a bacterium belonging to Enterobacter cloatusei. In order to produce the enzyme of the present invention by culturing the above-mentioned microorganism, the microorganism may be cultured aerobically using a conventional medium containing a carbon source, a nitrogen source, an inorganic salt, and an organic nutrient source. As a carbon source, sugars such as glucose, fructose, sucrose, and maltose, organic acids such as acetic acid and citric acid, and others can be used as appropriate, and the amount used is usually 0.1 to 10% (wt%, the same hereinafter) in the medium.
It is. As the nitrogen source, in addition to general natural nitrogen sources such as peptone, meat extract, yeast extract, cornstarch liquor, protein hydrolyzate, and amino acids, ammonium salts of various inorganic or organic acids can be used. Inorganic salts include KH ₂ PO ₄ , K ₂ HPO ₄ ,
Na ₂ HPO ₄ , NaCl, CaCl ₂ , MgSO ₄ .7H ₂ O, and heavy metal ions such as Fe, Mn, Zn, Co, etc. are used as appropriate. At this time, in order to induce high enzyme activity, fatty amides having 2 to 5 carbon atoms (e.g., acetamide, propionamide,
It is effective to add a small amount of (butyramide, succinoamide, etc.), and the amount added varies slightly depending on the microorganism used, but it is usually 0.01% or more in the medium, and preferably about 0.1 to 0.5%. Culture at pH 5-10 and temperature 20-40℃.
Perform aerobically for days. The present invention utilizes the action of enzymes produced by microorganisms, and the enzymatic action is achieved by using the microorganism culture solution, isolated live microorganisms, or processed microorganisms (e.g. microorganisms) obtained by culturing as described above. It can be obtained by using any of the following methods: crushed microbial cells, microbial cell extracts, etc.), and microbial cells obtained by immobilizing microbial cells or processed microbial cells with polyacrylamide, carrageenan, etc. according to conventional methods. , all of these usage forms can be applied to the present invention. The hydrolysis reaction is usually represented by the above general formula.
DL- or L-amino acid amide concentration 0.5-50%
(The reaction substrate solution may be in the form of a slurry.) The amount of microorganisms used is as dry bacterial cells per reaction solution.
The reaction temperature is 20 to 60°C, the pH is 6 to 11, and the reaction time is 5 minutes to 100 hours. The L-amino acid produced and accumulated in the reaction solution can be separated and purified using a combination of the ion exchange method and other known methods. The DL- or L-amino acid amide used in the present invention is a compound in which R is an alkyl group (having 1 to 4 carbon atoms), a phenyl group, an aralkyl group, or a substituent-containing group in the above general formula. Examples of substituents include a mercapto group, a hydroxyl group, an amino group, a carboxyl group, a phenyl group, an indolyl group, a pyridyl group, and an imidazoline group. Specific examples of the production of L-amino acids of the present invention include L-phenylalanine, L-tryptophan, L-leucine, L-methionine, L-
Serine etc. can be mentioned. EXAMPLES Hereinafter, the present invention will be specifically explained with reference to Examples, but the present invention is not limited only to these Examples. In each example, L-amino acid was confirmed and quantified using ninhydrin color development using thin layer chromatography and high performance liquid chromatography. Example 1 Using a medium with the following composition, N-
7901 bacteria was cultured by shaking. Seuclose 1% Meat extract 0.5% Inorganic salts MgSO ₄・7H ₂ O 0.01% FeSO ₄・7H ₂ O 0.001% MnSO ₄・4H ₂ O 0.001% CaCl ₂・2H ₂ O 0.001% ZnSO ₄・7H ₂ O 0.0001% Propion Amide 0.5% PH5 100 ml of this culture solution was centrifuged, and the resulting viable cells were suspended in 50 ml of Tris-HCl buffer (PH9). Next, 1 ml of this suspension was added to 4 ml of a 0.5% aqueous solution of the various amides shown in Table 1 [using Tris-HCl buffer (PH9)], and the mixture was reacted at 30°C for 10 to 15 minutes. Amount of amino acids produced in the reaction solution after sterilization, L-
Amino acids/produced amino acids were measured and the results shown in Table 1 were obtained.

[Table] Example 2 1 ml of the suspension of N-7901 bacteria obtained in the same manner as in Example 1 was added to L-phenylalaninamide 1.25% solution 4.
ml and reacted at 40°C for 15 minutes, the yield of phenylalanine was 75%, and all of the phenylalanine produced was only the L-form.

Claims (1)

[Scope of Claims] 1. By the action of an enzyme having L-amino acid amide hydrolysis activity produced by the microorganism Enterobacter Cloacae N-7901 [Feikoken Jokyo No. 873], the general formula [In the formula, R represents an alkyl group (having 1 to 4 carbon atoms), a phenyl group, an aralkyl group, or a substituent-containing group. ] A method for producing an L-amino acid, which comprises producing a corresponding L-amino acid from a DL- or L-amino acid amide represented by: 2. The manufacturing method according to claim 1, wherein the DL- or L-amino acid amide represented by the above general formula is phenylalanine amide or tryptophan amide.