JPS6255097A - Production of l-amino acid - Google Patents

Abstract

PURPOSE:To produce the titled substance easily, by treating DL- or L-amino acid amide with an enzyme produced by Enterobacter or Pseudomonas genus microorganism and having L-amino acid amide hydrolyzing activity. CONSTITUTION:A microbial strain belonging to Enterobacter genus or Pseudomonas genus such as Enterobacter cloacae N-7901 (FERM BP-873) or Pseudomonas sp.N-2211 (FERM BP-875) is cultured aerobically in a conventional medium to obtain a culture liquid, separated bacterial cell or treated bacterial cell, etc., having L-amino acid amide hydrolyzing activity. The DL- or L-amino acid amide of formula is made to react with the above bacterial cell or culture liquid, etc., to obtain L-amino acid. The accumulated L-amino acid is separated and purified e.g. by ion exchange treatment.

Description

DETAILED DESCRIPTION OF THE INVENTION Production 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 diamino 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.

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 used. The so-called Chesico-Enzymatinoku (Chesico-Enzy+m) is converted by enzymes.
A number of manufacturing methods using atomic methods have also been proposed and put into practical use.

For example, a method in which acylase produced by microorganisms is allowed to act on N-acyl derivatives of DL-amino acids (Japanese Patent Publication No. 41-2
(Japanese Patent Publication No. 2380), a method in which hydantoin derivatives of DL-amino acids are reacted with hydantoinase produced by microorganisms (Japanese Patent Publication No. 2274/1983), and a method in which fumaric acid is reacted with aspartase produced by microorganisms (Japanese Patent Publication No. 57-1989). No. 18867 and JP-A No. 59-14089) and a method of causing phenylalanine ammonia lyase produced by microorganisms to act on cinnamic acid (^pp
1. EnvironSM Microbiol, 42773
(1981) ``I'' is a typical example of a chemical enzyme production method 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, regarding the reaction of producing the corresponding monoamino acid from various DL- or L-amino acid amides using enzymes produced by microorganisms, the enzyme L produced by microorganisms of the genus Bacillus, Batatteridium, Micrococcus and Previbacteridium has been reported. - A method using amidase (Publication No. 500319/1983), a method using Zurushi amidase produced by various yeasts and bacteria (Japanese Unexamined Patent Publication No. 57-198)
No. 3000, No. 59-159789 and No. 60-3
No. 6446) and the like have been proposed.

However, all of these methods are experimental examples in which L-amino acid production reactions were carried out using a large amount of bacterial cells with weak monoamidase activity, or simply the use of known bacterial strains of various species to produce various DL-amino acids. However, it is only a finding that the corresponding L-amino acid can be produced by hydrolyzing L-amino acid amide or L-amino acid amide. From the viewpoint of an industrial production method for amino acids, the use of these microorganisms cannot be an economically advantageous production method.

One support! Under such circumstances, the present inventors used DL-amino acid amide, which can be easily and inexpensively produced through chemical synthesis, as a raw material,
In order to obtain microorganisms that can produce enzymes with high amino acid activity that can efficiently produce amino acids, we screened soil, sludge, etc. from various locations, and found that Enterobacter. Cloanose 4N-7901, Pseudomonas SP, N-7131
and Pseudomonas sp., the enzyme produced by the microorganism N-2211 has extremely high amidase activity;
The present invention was found to be extremely effective in achieving the object of the present invention, and the present invention was completed.

That is, the present invention relates to Enterobacter cloacae N-
790I C'RI Koken Jyoki No. 873], Pseudomonas SP, N-7131 (
Microtechnical Research Institute No. 874] or Pseudomonas (Pse
The general formula is
R-CH-CONI+□N11□ [In the formula, R represents an alkyl group (having 1 to 4 carbon atoms), a phenyl group, an aralkyl group, or any of these groups having a substituent. ] A corresponding L-amino acid is produced from a DL- or L-amino acid amide represented by
The gist is a method for producing amino acids.

As mentioned above, the microorganisms used in the Japanese invention were newly isolated and discovered by the present inventors, and each microorganism was introduced in the Microorganism Research Institute No. 873 (Enterobacter Chloassay). N-7901), No. 874 (Pseudomonas SP, N-7131) and No. 875 (Pseudomonas SP, N-2211) have been deposited with the Institute of Microbiology, Agency of Industrial Science and Technology (Feikoken).

The mycological properties of these microorganisms are as follows.

N-7901 strain +11 rod shape bacteria 0.8-1.2 x 1.0-
1.5.17 m Flagella Motility of one or more pericyria Tengram staining Negative (2) Physiological properties Nitrate reduction Tendenitrification reaction - MR test - vP test + Indole production - Hydrogen sulfide production - Utilization of citrate Production of ten pigments - Urease - Oxidase - Catalase + Range of growth PI (4-10 Optimum temperature 35-37°C Attitude towards oxygen Facultative Anaerobic 0-F test F IJ! from acids and gases Production of acid Production of gas Adonitol −− Arabinose + + Xylose + + Glucose + + Mannose + +Fructose
+ + Galactose + + Maltose + + Shakrose + + Lactose + + Raffinose + Rhamnose + Glycerin + + Sorbitol + + Mannitol + + Inositol - - Dulcitol - Dulcitol - - (3) Decomposition of other starches - Decomposition of gelatin - Urea Decomposition Utilization of weak malonate Decarboxylation of decalysine − Decarboxylation of ornithine Deamination of decaphenylalanine − Arginine dihydrolase + β-galactosidase + Mu resistance TenN-7131 strain + 11 Shape Rod Fungal flagella Pole Hair 1 or more motile Ten spores Not observed Gram staining Negative (2) Physiological properties Denitrification reaction Generation of ten indole - Generation of hydrogen sulfide - Utilization of citrate Formation of ten pigments Water-soluble pigments - Fluorescent pigments − Urease + Oxidase + Catalase + Growth at 40°C − 0-F test 0 Xylose + Glucose + − Mannose + Galactose + Lactose − Mannitol + Assimilable glucose + Fructose + L-arabino-sucrose − Malonate − Ethanol - trehalose - meso-inositol + β-alanine + DL-arginine + (3) Decomposition of other starches - Decomposition of gelatin - Decomposition of urea - Accumulation of arginine hydrolase - Aminobebutidase + 1) Morphology Rod Bacterial flag Hair Polar motile Deca spores Not observed Gram staining Negative (2) Physiological properties Denitrification reaction Production of weak indole - Production of 0g hydrogen oxide - Utilization of citrate Deca pigments Formation Water-soluble dye Weakly fluorescent dye - Urease + Oxidase + Catalase + Growth at 40°C - 0-F test O Xylose + Glucose + - Mannose + Galactose + Lactose - Mannitol Weakly assimilable glucose + Fructose + L-arabino + - Susucrose - Malo name it - Ethanol - Trehalose - Meso-inositol Weak β-alanine Weak OL-arginine - (3) Decomposition of other starches - Decomposition of gelatin - Decomposition of urea Tenlase - Aminopeptidase + The above mycological Virginie's Manual of Nature
Bookumi Native Bacteriology 8th Edition (Berg
y's Manual or Determinati
ve[1acteriology 8th, ed,)
When searched according to
One strain was each identified as a bacterium belonging to the genus Shademonas. In addition, the N-7131 strain contains poly-β-
Accumulated hydroxybutyrate, growth at 40°C and arginine dihydrase were both negative, denitrification reaction was positive, and atypical characteristics such as sugar assimilation were observed.
This bacterium is thought to be Pseudomonas solanacearum or its related species.

In order to produce the enzyme of the present invention by culturing the above-mentioned microorganism, the cough 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 the 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 0.0% in the medium.
1 to 10% (rrL amount %, the same applies hereinafter). 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 KIIjPOa,
KzllPOn, Na211PO4, NaCl, C
aCI 1, Mg50m' IHzO and Fe,
Mn, ZnsC.

Heavy metal ions such as ions are used as necessary.

At this time, in order to induce high enzyme activity, the number of carbon atoms is 2.
It is effective to add a small amount of ~5 fatty amides (e.g., acetamide, propionamide, butyramide, succinoamide, etc.), and the amount added varies slightly depending on the microorganism used, but is usually 0.01% or more in the medium. In particular, it is preferably about 0.1 to 0.5χ.

Culture is P115-10 at a temperature range of 20-40℃.
Perform aerobically for 5 days.

The present invention utilizes the action of enzymes produced by microorganisms, and the enzymatic action is achieved by using the microorganism culture fluid, 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 fixing microbial cells or processed microbial cells with polyacrylamide, carrageenan, etc. according to conventional methods. All forms are applicable to the present invention.

In the hydrolysis reaction, the concentration of DL- or L-amino acid amide represented by the above general formula is usually 0.5 to 50% (the reaction substrate solution may be in the form of a slurry), and the amount of microorganisms used is 0.5 to 50%. As reaction liquid bubbles 0.01~10%, reaction temperature 20~60℃, PI+6~11, reaction time 5~10
Perform within the range of 0 hours.

In this way, the monoamino acid produced and accumulated in the reaction solution is
It can be separated and purified using a combination of ion exchange methods and other known methods.

In the above general formula, R is an alkyl group (carbon B1 to B4), a phenyl group, an aralkyl group, or a substituent-containing DL-amino acid amide used in the present invention. It is a compound, and examples of substituents include a mercapto group, a hydroxyl group, a 7-mino group, a carboxyl group, a phenyl group, an indolyl group, a pyridyl group, and an imidazolyl group.

Specific examples of the production of monoamino acids of the present invention include, for example, L-phenylalanine, L-1-lyptophan, L-
Examples include leucine, L-methionine, and L-serine.

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 by ninhydrin color development using WjWI chromatography and high performance liquid chromatography.

Example 1 N-79 was incubated at 30°C for 48 hours using a medium with the following composition.
A shaking culture of 01 bacteria was performed.

Sucrose 1% Meat extract 0.5% Propionamide 0.5% H5 100ml of this culture solution was centrifuged, and the resulting viable bacterial cells were collected at 50+w I! of Tris-HCl buffer (P119).

Next, 1 ml of this suspension was mixed with an O, S% aqueous solution of various amides shown in Table 1 (Tris-HCI buffer (PH9)).
The amount of amino acids produced and L-amino acids/amino acids produced were measured, and the results shown in Table 1 were obtained.

Table 1 Example 2 Using a medium with the following composition, N-22 was incubated at 30°C for 48 hours.
A shaking culture of 11 bacterial strains was performed.

Glycerol 1% Yeast extract 0.05χ Isobutyramide 0.5χ H7 Hereinafter, the same operations as in Example 1 were performed except that the reaction time was changed to 1 to 3 hours, and the results shown in Table 2 were obtained.

Table 2 Example 3 N-71 was incubated at 30°C for 48 hours using a medium with the following composition.
Glycerol 1% Meat extract 0.5% Isobutyramide 0.5% H7 The following operations were carried out in the same manner as in Example 2, and the results shown in Table 3 were obtained.

Table 3 Example 4 1 m of suspension of N-7901 bacteria obtained in the same manner as in Example 1
j! When added to L-phenylalanine amide and reacted at 40°C for 15 minutes, the yield of phenylalanine was 7.
At 5%, all the phenylalanine produced was only L.

Claims (2)

[Claims] (1) Enterobacter chromatography (Enterobacter chromatography)
-bacter Cloacae) N-7901 [Feikoken Jokyo No. 873] or Pseudomonas (Pseud
omonas) sp. N-7131 [Microtechnology Research Institute No. 87
No. 4] or Pseudomonas
) sp. Due to the action of an enzyme with L-amino acid amide hydrolysis activity produced by the microorganism of N-2211 [Feikoken Jokyo No. 875], the general formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [In the formula, R is It represents an alkyl group (having 1 to 4 carbon atoms), a phenyl group, an aralkyl group, or a substituent-containing group. ] An L-amino acid characterized by producing a corresponding L-amino acid from a DL- or L-amino acid amide represented by
Method for producing amino acids. (2) The production 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.