GB2182036A - Enzymatic process for producing L-amino acids - Google Patents

Abstract

A process for producing an L-amino acid from the corresponding DL- and/or L-amino acid amide represented by the general formula: <IMAGE> wherein R is substituted or unsubstituted alkyl group having one to 4 carbon atoms, a substituted or unsubstituted phenyl group, or a substituted or unsubstituted aralkyl group, by action of an enzyme having hydrolytic activity to L-amino acid amides which is produced by novel Enterobacter cloacae or Pseudomonas sp.

Description

SPECIFICATION Process for producing L-amino acids BACKGROUND OF THE INVENTION FIELD OF THE INVENTION This invention relates to a process for producing an L-amino acid. More particularly, it relates to a process for producing an L-amino acid from DL- and/or L-amino acid amide by action of an enzyme prepared by a specific microorganism.

L-amino acids are important compounds as food additives, feed additives, and intermediates of medicines and various industrial chemicals.

DESCRIPTION OF THE PRIOR ART In general, L-amino acids are produced by fermentation or by optical resolution of DL-amino acids prepared in an organic synthetic-chemical method. Recently, there have been proposed and made practicable a large number of so-called chemico-enzymatic processes which comprise converting precursors inexpensively obtainable by chemical synthesis into L-amino acids by using enzymes.

Typical examples of the chemico-enzymatic processes for producing L-amino acids include a process comprising acting acylase produced by a micro-organism on an N-acyl derivative of DLamino acid (Japanese Patent Publication No. 22380/66), a process comprising acting hydantoinase produced by a microorganism on a hydantoin-substituted DL-amino acid derivative (Japanese Patent Publication No. 2274/79), a process comprising acting aspartase produced by a microorganism on fumaric acid (Japanese Patent Publication No. 18867/82 and Japanese Patent Application Kokai (Laid-Open) No. 14089/84), and a process comprising acting phenylaianine ammonia lyase produced by a microorganism on cinnamic acid (Appl. Environ, Microbiol. 42, 773 (1981)).

However, these processes involve problems, for example, complicated reaction systems, severe reaction conditions and expensive.starting materials, and can stand improvement as industrial production processes.

Recently, there have also been proposed processes comprising reactions for producing various L-amino acids from the corresponding DL- or L-amino acid amides by using enzymes produced by microorganisms, for example, a process using an enzyme L-amidase produced by microorganisms belonging to Bacillus, Bacteridium, Micrococcus and Brevibacterium (publicized in Japanese Official Patent Gazette No. 500319/81) and a process using L-amidase produced by various yeasts and bacteria (Japanese Patent Application Kokai (Laid-Open) Nos. 13000/82, 159789/84 and 36446/85).

However, all of these processes involve a problem of low L-amidase activity, and they are experiment examples in which production reactions of L-amino acid were carried out by using a large amount of cells, or nothing but a finding that well-known strains belonging to various genera hydrolyze various DL- or L-amino acid amides to give the corresponding L-amino acids.

Because of use of these microorganisms, they cannot possibly become economically advantageous production processes from the viewpoint of industrial processes for producing L-amino acids from DL- or L-amino acid amides by action of enzymes produced by microorganisms.

SUMMARY OF THE INVENTION Under these circumstances, in order to obtain a microorganism capable of producing an enzyme having high L-amidase activity which permits efficient production of L-amino acids particularly from the corresponding DL-amino acid amides which can be prepared by chemical synthesis easily and inexpensively, the present inventors have conducted screening of microorganisms from soils, sludges and the like in various places. Consequently, the present inventors have found that an enzyme produced by microorganisms, Enterobacter cloacae N-7901, Pseudomonas sp. N-7131 and Pseudomonas sp. N-2211 has a very high L-amidase activity and is very effective in achieving the object of this invention, whereby this invention has been accomplished.

That is to say, the gist of the invention is a process for producing an L-amino acid which comprises producing an L-amino acid from the corresponding DL- and/or L-amino acid amide represented by the general formula:

wherein R is a substituted or unsubstituted alkyl group having one to 4 carbon atoms, a substituted or unsubstituted phenyl group, or a substituted or unsubstituted aralkyl group, by action of an enzyme having hydrolytic activity to L-amino acid amides which is produced by a microorganism, Enterobacter cloacae N-7901 (FERM BP No. 873), Pseudomonas sp. N-7131 (FERM BP No. 874) or Pseudomonas sp. N-2211 (FERM BP No. 875).

DETAILED DESCRIPTION OF THE INVENTION The microorganisms used in this invention are those newly isolated and named by the present inventors as described above and were deposited in Fermentation Research Institute, Agency of Industrial Science and Technology (Bikoken), Ministry of International Trade and Industiry as FERM BP No. 873 (Enterobacter cloacae N-7901), FERM BP No. 874 (Pseudomonas sp.

N-7131) and FERM BP No. 875 (Pseudomonas sp. N-2211) according to Budapest Treaty, respectively.

Micological properties of these microorganisms are as follows: N-7901 strain (1) Morphological properties Bacillus 0.8--1.2x 1.0--1.5 ,um Flagellum mono or multitrichous Motility + Gram staining negative (2) Physiological properties Reduction of nitrate + Denitrification MR test VP test + Production of indole Production of hydrogen sulfide - Utilization of citrate + Production of pigment Urease Oxidase Catalase + Ranges for growth PH 4-10 Optimum temperature 35-37"C Behavior toward oxygen facultatively anaerabic O-F test F Production of acid and Production Production gas from sugars of acid of gas Adonitol Arabinose + + Xylose + + Glucose + + Mannose + + Fructose + + Galactose + + Maltose + + Sucrose + + Lactose + + Raffinose + Rhamnose + Glycerol + + Sorbitol + + Mannitol + + Inositol Dulcitol (3) Other properties Decomposition of starch Decomposition of gelatin Decomposition of urea slight Utilization of malonate + Decarboxylation of lysine Decarboxylation of ornithine + Deamination of phenylalanine Arginine dihydrolase + ss-Galactosidase + Resistance to potassium cyanide + N-7131 strain (1) Morphological properties Bacillus Flagellum polar mono- or multitrichous Motility + Endospore None Gram staining negative (2) Physiological properties Denitrification + Production of indole Production of hydrogen sulfide Utilization of citrate + Production of pigment water-soluble pigment fluorescent pigment Urease + Oxidase + Catalase + Growth at 40"C O-F test 0 Production of acid and Production Production and gas from sugars of acid of gas Xylose + Glucose + Mannose + Galactose + Lactose Mannitol + Utilization Glucose + Fructose + L-Arabinose + Sucrose Malonate Ethanol Trehalose meso-lnositol + fi-Alanine + DL-Arginine + (3) Other properties Decomposition of starch Decomposition of gelatin Decomposition of urea Accumulation of poly-fi- hydroxybutyrate + Anginine dihydrolase Aminopeptidase + Growth in the presence of 6.5% NaCI N-2211 strain (1) Morophological properties Bacillus Flagellum polar monotrichous Motility + Endospore None Gram staining negative (2) Physiological properties Denitrification slight Production of indole Production of hydrogen sulfide - Utilization of citrate + Production of pigment water-soluble pigment slight fluorescent pigment Urease + Oxidase + Catalase + Growth at 40"C O-F test 0 Production of acid and Production Production gas from sugars of acid of gas Xylose + Glucose + Mannose + Galactose + Lactose Mannitol slight Utilization Glucose + Fructose + L-Arabinose + Sucrose Malonate Ethanol Trehalose meso-lnositol slight ss-Alanine slight DL-Arginine (3) Other properties Decomposition of starch Decomposition of gelatin Decomposition of urea + Accumulation of poly-ss- hydroxybutyrate slight Arginine dihydrolase Aminopeptidase + Growth in the presence of 6.5% NaCI By reference to Bergy's Manual of Determinative Bacteriology, 8th ed. for the mycological properties described above, N-7901 strain was identified as Enterobacter cloacae and N-7131 and No2211 strains as bacterium belonging to Pseudomonas. N-7131 strain accumulated poly-fi- hydroxybutyrate in the cells, was negative in both growth at 40"C and arginine dihydrolase, and was positive in denitrification. Although this strain had untypical properties with repsect to utilization of sugars and the like, it seems to be Pseudomonas solanacearum or a strain nearly related thereto.

As is obvious to the artisan in the art, mutants of the above-mentioned strains can also be used. These mutants can easily be obtained by treatment with a mutagent such as nitrogen mustard and the like.

For culturing the above-mentioned microorganism to allow the same to produce the enzyme of this invention, it is sufficient that said microorganism is aerobically cultured by using a conventional medium containing a carbon source, a nitrogen source, inorganic salts and an organic nutrient.

As the carbon sources, there can be used sugars such as glucose, fructose, sucrose and maltose, organic acids such as acetic acid and citric acid, etc. The amount of carbon source used is usually 0.1 to 10% (by weight, as used hereinafter for all such amounts) based on the weight of the medium. As nitrogen sources, there can be used ordinary natural nitrogen sources, such as peptone, meat extract, yeast extract, corn steep liquor, protein hydrolyzates and amino acids, and ammonium salts of various organic or inorganic acids, etc. Inorganic salts which can be used include KH2PO4, K2HPO4, Na2HPO4 NaCI, CaCI2, MgSO4.7H2O and ions of heavy metals such as Fe, Mn, Zn, Co and the like.In this case, the addition of a small amount of an aliphatic amide having 2 to 5 carbon atoms (e.g., acetamide, propionamide, butylamide, or succinamide) is effective in inducing high enzymatic activity, and the amount thereof added is preferably 0.01% or more, more preferably about 0.1 to about 0.5%, based on the weight of the medium.

Culturing is aerobically carried out at pH 5 to 10 at a temperature of 20 to 40"C for one to 5 days.

This invention utilizes the action of an enzyme produced by a microorganism and the enzymatic action can be obtained by incubating a mixture of DL and/or L-amino acid amide and any culture broth of a microorganism obtained by culturing carried out in the manner described above, using separated viable cells, microorganism-derived fractions (e.g., sonicated cells and cell extracts), and immobilized cells or extracts thereof obtained by immobilizing cells or microorga nism-derived fractions on polyacrylamide, carragheen or the like by a conventional method. All of these forms for use can be applied to this invention.

Hydrolysis (incubation with the amide) is usually carried out under the following conditions: the concentration of DL- and/or L-amino acid amide of the above general formula is 0.5 to 50% (the substrate solution for reaction may be slurry); the amount of the micro-organism or the like is 0.01 to 10% (in terms of dried cells) based on the weight of the reaction solution; the reaction temperature is 20 to 60"C; the pH is 6 to 11; the reaction time is 5 min. to 100 hours.

The L-amino acid thus produced and accumulated in the reaction solution can be separated and purified by a combination of well-known methods such as ion exchange and the like.

The DL- and/or L-amino acid amide used in this invention is a compound represented by the above general formula in which R is a substituted or unsubstituted alkyl group having one to 4 carbon atoms, a substituted or unsubstituted phenyl group, or a substituted or unsubstituted aralkyl group. The substituents include the mercapto group, hydroxyl group, amino group, carboxyl group, phenyl group, indolyl group, pyridyl group and imidazolyl group.

Examples of L-amino acids which can be produced according to this invention are L-phenylalanine, L-tryptophan, L-leucine, L-methionine and L-serine.

This invention is explained further below referring to Examples, which are not by way of limitation but by way of illustration. In each Example, the identification and quantification of Lamino acid were conducted by thin-layer chromatography, followed by examination of positions of ninhydrin coloration, and high performance liquid chromatography.

Example 1 Shake culture of N-7901 strain was carried out at 300C for 48 hours by using a medium having the following composition: Sucrose 1 % Meat extract 0.5% MgSO4.7H20 0.01% FeSO4.7H20 0.001% Inorganic salts MnSO4.4H20 0.001% CaCI2.2H2O 0.001% ZnSO4.7H20 0.0001% Propionamide 0.5% pH 5 One hundred millimeters of the culture broth was centrifuged and the viable cells thus obtained were suspended in 50 ml of Tris-HCI buffer (pH9).

Subsequently, 1 ml each of the resulting suspensions was added to 4 ml of a 0.5% aqueous solution (prepared by using Tris-HCI buffer (pH9)) of each of the various amides shown in Table 1 and the resulting mixture solution were incubated, respectively at 30"C for 10-15 mininutes.

After the removal of fungous cells, the amounts of L-amino acid produced and the whole amino acid produced were measured to obtain the results shown in Table 1.

Table 1

Starting Amino acid Reac- Yield L-amino amino acid produced tion (based on acid/amino amide time DL-form acid (min) charged produced (%) (%) DL-Leucine L-Leucine 10 50 100 amide DL-Methionine L-Methionine 15 13 100 amide DL-Serine L-serine 15 3 100 amide DL-Phynyl- L-Phynyl- 15 50 100 alanine amide alanine DL-Tryptophan L-Tryptophan 15 34 100 amide DL-Phenyl- L-Phenyl- 15 48 100 glycine glycine amide Example 2 Shake culture of N-2211 strain was carried out at 30"C for 48 hours by using a medium having the following composition: Glycerol 1% Yeast extract 0.05% MgSO4.7H20 0.01% FeSO4.7H20 0.001% Inorganic salts MnSO4.4H20 0.001% CaCl2.2H20 0.001% ZnS04.7H20 0.0001% Isobutylamide 0.5% pH 7 Thereafter, the procedure in Example 1 was repeated, except that the reaction time was changed to 1 to 3 hours, to obtain the results shown in Table 2.

Table 2

Starting Amino acid Reac- Yield L-amino amino acid produced tion (based on acid/amino amide time DL-form acid (hr) charged produced (%) (t) DL-Leucine L-Leucine 1 50 100 amide DL-Methionine L-Methionine 3 19 100 amide DL-Serine L-Serine 3 6 100 amide DL-Phenyl- L-Phenyl- 3 24 100 alanine alanine amide DL-Tryptophan L-Tryptophan 3 50 100 amide DL-Phenyl- L-Phenyl- 3 20 100 glycine glycine amide Example 3 Shake culture of N-7131 strain was carried out at 30"C for 48 hours by using a medium having the following composition: Glycerol 1% Meat extract 0.5% MgSO4.7H20 0.01% FeSO4.7H20 0.001% Inorganic salts MnSO4.4H20 0.001% CaCl2.2H20 0.001% ZnSO4.7H20 0.0001% Isobutylamide 0.5% pH 7 Thereafter, the procedure in Example 2 was repeated to obtain the results shown in Table 3.

Table 3

Starting Amino acid Reac- Yield L-amino amino acid produced tion (based on acid/amino amide time DL- form acid (hr) charged produced (hr) charged produced D-Leucie L-Leucine 1 5G 100 amide DL-Methionine L-Methionine 3 31 100 amide DL-Serine L-Serine 3 0.3 100 amide DL-Phenyl- L-Phenyl- 3 50 100 alanine amide alanine DL-Tryptnphan L-Tryptophan 3 t8 amide DL-Phenyl- L-Phenyl- 3 48 100 glycine glycine amide Example 4 When 1 ml of a suspension of N-7901 strain obtained in the same manner as in Example 1 was added to 4 ml of a 1.25% L-phenylalanine amide solution and the resulting mixture was subjected to reaction at 40"C for 15 minutes, the yield of phenylalanine was 75% and the Lphenylalanine produced is the L-form alone.

Claims (10)

1. A process for producing an L-amino acid, in which the corresponding DL- and/or L-amino acid amide represented by the general formula

wherein R is a substituted or unsubstituted alkyl group having one to 4 carbon atoms, a substituted or unsubstituted phenyl group, or a substituted or unsubstituted aralkyl group, is subjected to the action of an enzyme having hydrolytic activity to L-amino acid amide and which is produced by a micro-organism selected from Enterobacter cloacae N-7901 (FERM BP No.

873), Pseudomonas sp. N-7131 (FERM BP No. 874), Pseudomonas sp. N-2211 (FERM BP No.

875), and mutant strains thereof.

2. A process according to claim 1, in which the microorganism is cultured in the presence of an aliphatic amide having 2 to 5 carbon atoms.

3. A process according to claim 1 or 2, in which the microorganism is obtained from a culture broth, by viable cell separation, by cell sonication or by means of cell extracts, immobilized cells or immobilized cell extracts.

4. A process according to claim 1, 2 or 3, in which cells of the microorganism are incubated with the amide, at a temperature of 20 to 60 C, for 5 min. to 100 hours and in a medium whose pH is 6 to 11.

5. A process for producing an L-amino acid, the process being substantially as hereinbefore described in any examples 1 to 4.

6. An L-amino acid obtained by a process according to any of claims 1 to 5.

7. Enterobacter cloacae N-7901 (FERM BP No. 873) or a mutant strain thereof.

8. Pseudomonas sp. N-7131 (FERM BP No. 874) or a mutant strain thereof.

9. Pseudomonas sp. N-2211 (FERM BP No. 875) or a mutant strain thereof.

10. A microorganism according to claim 7, 8 or 9, when isolated and substantially pure.