JPS6324895A - Production of l-amino acid or n-carbamyl-l-amino acid - Google Patents

Abstract

PURPOSE:To produce an L-amino acid, by bringing a 5-substituted hydantoin into contact with specific bacteria belonging to Bacillus genus to give the L- amino acid or the corresponding N-carbamyl-L-amino acid and further bringing the produced N-carbamyl-L-amino acid into contact with other specific bacteria. CONSTITUTION:Newly separated Bacillus sp. (FERM P-8837) is cultured in a nutritive medium containing a 5-substituted hydantoin or N-carbamylamino acid at 5-8pH and 25-40 deg.C for 1-10 days to give an L-amino acid. The L- amino acid or N-carbamyl-L-amino acid can be also produced by temporarily allowing to stand or stirring an aqueous solution at 10-70 deg.C, preferably 20-50 deg.C at 5-11pH, preferably 6.5-9pH, said aqueous solution being produced by dissolving or suspending the 5-substituted hydantoin or N-carbamylamino acid in the cells or treated cells of the above-mentioned microorganisms at a concentration of 0.1-30%, preferably 0.5-10%.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a process for producing rum-amino acids and a process for producing the corresponding monoamino acids from N-carbamyl amino acids.

[Conventional technology]

Conventionally, 5-substituted hydantoin was developed using microorganisms in 1980.
-214889) is known. Not like this.

A method for producing histidine (Japanese Unexamined Patent Publication No. 61-9292) and a method for producing histidine (Japanese Unexamined Patent Publication No. 61-9293) are known.

However, there is no known method for producing other L-amino acids from N-carbamyl amino acids.

[Problems to be solved by the present invention] To provide a method for producing cyanoethylglycine, N-carbamyl-L-/41J-n, N- from 5-substituted hydantoin.
To provide a method for producing carpami/I/ = L-leucine, N-carpami-L-methionine, N-carpami'〃-glycine, and to produce L-valine, L-leucine, L-from ICN-carbamyl amino acid. An object of the present invention is to provide a method for producing -isoleucine, L-7 enylalanine, L-tyrosine, L-alanine, and -L-Schiff/lualanine.

[Means for solving problems]

We have newly isolated a Bacillus strain that efficiently produces the corresponding L-amino acid from 5-substituted hydantoin. Microorganisms used in the present invention include the following.

Bacillus sp. A J-12299 (FEL'v
l l)-Tate 37) The above microorganism was found to belong to the genus Bacillus because it grows only under aerobic conditions and has spore-forming ability. In the production of L-amino acids from 5-substituted hydantoin or N-carbamyl amino acids, the method of allowing the present microorganism to act on the 5-substituted hydantoin or N-carbamyl amino acid is as follows: Alternatively, the microorganism may be cultured in a medium containing N-carbamyl amino acid, or the cells of the present microorganism or a treated product of the microorganism may be brought into contact with 5-substituted hydantoin or N-carbamyl amino acid in an aqueous solution.

Method of converting the present microorganism into 5-substituted hymenmino acid by culturing - e = h The microorganism of the present invention may be cultured in a medium containing 5-substituted hydantoin or N-carbamyl amino acid from the beginning of the culture. However, during the culture, 5
- Substituted hydantoins or N-carbamyl amino acids may be added to the medium.

The medium used for culturing this microorganism is a conventional medium containing 5-substituted hydantoin or N-carbamyl amino acid, as well as conventional carbon sources, nitrogen sources, inorganic ions, and organic nutrients if necessary. -0 As the carbon source, carbohydrates such as glucose, alcohols such as glycerol, organic acids, and others are used as appropriate.

As the nitrogen source, ammonia gas, aqueous ammonia, ammonium salt, and others are used. Inorganic ions include magnesium ions, phosphate ions, potassium ions,
Iron ions, manganese ions, and others are used as appropriate.

As the organic nutrient source, vitamins, amino acids, etc., yeast extracts, peptones, meat extracts, corn stay glycer, casein decomposition products, etc. containing these are used as appropriate.

Desired results can be obtained if the culturing is carried out under aerobic conditions with pH 5 to 8 and temperature controlled within an appropriate range of 25 to 40°C.

By culturing in this manner for up to 10 days, 5-substituted hydantoin or N-carbamyl amino acid is efficiently converted to only L-amino acid.

On the other hand, when the bacterial cells of the present microorganism or the treated product of the bacterial cells are brought into contact with 5-substituted hydantoin or N-carbamylamino acid in an aqueous solution, An aqueous solution prepared by dissolving or suspending the bacterial cells or the treated product of the bacterial cells is heated to a temperature of 10 to 70°C, preferably 20 to 50°C, and a pH of 5 to 11.
, preferably maintained at a temperature of 6.5 to 9, and allowed to stand for a while or be stirred. The concentration of 5-substituted hydantoin or N-carbamyl amino acid is between 0.1 and 30, preferably 0.
If necessary, 5-substituted hydantoin or N-carbamyl amino acid may be added during the reaction.

As the bacterial cells, a culture solution containing the bacterial cells may be used as is. Alternatively, this may be used after being separated from the culture solution and washed or without washing. The bacterial cell treatment products include mechanically ground bacterial cells, nine bacterial cells treated with ultrasonic waves, freeze-dried bacterial cells, acetone-dried bacterial cells, bacterial cells treated with enzymes such as lysozyme,
Bacterial cells treated with surfactants, toluene, etc., protein fractions of bacterial cells, and others may be used as appropriate.

As a method for obtaining such bacterial cells, the above-mentioned culture medium and culture method can be employed. If a small amount of 5-substituted hydantoin or N-carbamyl amino acid of the present invention is further added to the medium, bacterial cells with high activity of converting 5-substituted hydantoin or N-carbamyl amino acid into L-amino acid may be obtained. be. Furthermore, in this case, the culture may be completed in about 12 to 48 hours, as long as the microorganisms sufficiently proliferate.

The reaction yield may be improved if a surfactant, supplementary oxygen, hydroxylamine, coke ion, other metal ions, etc. are added to the aqueous solution as necessary.

Thus, after 5 to 100 hours have passed, a large amount of L-amino acids are produced and accumulated in the aqueous solution.

According to the method of the present invention, the L-amino acid obtained in this way is collected from a culture solution or an aqueous solution, since no D-amino acid is produced as a by-product. Ordinary methods such as precipitation can be used.

Quantification of L-amino acid was carried out by a microbial quantification method using a liquid chromatogelophyte leuconost, R. mesenteroides ATCC8042.

In addition, when the cells of this microorganism or the processed material of the microorganisms are brought into contact with 5-substituted hydantoin in an aqueous solution and allowed to act, the volume of the aqueous solution is maintained at 5.5 to 65, or the processed material of the microorganisms is fractionated. N-carbamyl-L-amino acids can be produced from cy5-substituted hydantoins by a method such as using a 5-substituted hydantoin. In order to collect the obtained N~carbamyl-L-amino acid from an aqueous solution, according to the method of the present invention, since N-carbamyl-D-amino acid is not produced as a by-product,
A method using an ion exchange resin, a method of precipitation at an isoelectric point, and a suitable method can be employed. N-carbamyl-L
-Amino acids were quantified using a colorimetric method by adding a concentrated hydrochloric acid solution of p-trimethylaminobenzaldehyde. kon spectrum,
All data such as X-ray diffraction spectra, liquid chromatography, bioassay quantitative values (for N-carbamyl amino acids, quantitative values after conversion to diamino acids by chemical methods), and specific optical rotation are L-amino acids. Alternatively, it was confirmed by matching with those of the standard N-carbamyl-L-amino acid.

Generally, 5-substituted hydantoin or N-carbamyl amino acid produced by organic synthesis method is racemic, but according to the method of the present invention, racemic 5-substituted hydantoin can be converted to the corresponding L-amino acid or N-carbamyl amino acid. -L-
It is capable of converting one or two semiamic N-carbamyl amino acids into the corresponding L-amino acids.

Example 1 Glycerol 2.01/French(NH4,)2So40.5
11/France KH2PO40, 11/dl, K2HPO4
0,31/dt, MgSO4-7H200,05JF
/dt1F' e S Oa ・7H201m9/d
11Mn SO4.4H201-w/a, yeast extract 1. oy7 Buddha Begton 1. Oji/dl, DL-
5-isopropylhydantoin 0.2 i/lu,
Charcoal a calcium 4. Pour 50ml of culture medium (voice 7°0) containing OJF/dt (separately sterilized) into a 500ml flask and incubate for 12 hours.
Sterilized at 0°C for 15 minutes.

This was inoculated with one platinum loop of AJ-12299, which had been cultured for 24 hours at 30°C on a bouillon agar medium, and cultured at 30°C for 16 hours. This culture solution and bacterial cells were collected by centrifugation, washed once with physiological saline in the same amount as the culture solution, and the bacterial cells were collected. This bacterial cell' L-, E)
-,1TaQ in 0.1M phosphate buffer (pH 7) containing 1117 dl of DL-isopropylhydantoin.
, 5) 5- to give a total weight of 5117 dt, and the mixture was reacted at 30° C. for 48 hours. After the reaction was completed, the bacterial cells were removed by centrifugation.

Generate 7? When L-valine was measured by bioassay method, it was 0.21117di and 0.2311/di from L-, D-, and Yupi DL-isopropylhydantoin, respectively.
dl.

0.22. ! L-valine of il/dt was produced.

The cells were cultured at 30° C. for 16 hours in the same manner as in Example 1. This culture solution and bacterial cells were centrifuged, washed once with physiological saline in the same amount as the culture solution, and further centrifuged to collect the bacterial cells. This bacterial cell was treated with 1 g/di of the hydantoin compound shown in Table 1.
Including 0. I M IJ acid buffer (pH 7,5) 5
- to give a concentration of 511/dt, and for 48 hours, 30
The reaction was maintained at ℃. After the reaction was completed, the bacterial cells were removed by centrifugation.

Table 1 shows the results of measuring each amino acid present in the supernatant using the method described above. Furthermore, after separating and purifying these amino acids and analyzing them using methods such as spectra, X-ray diffraction spectra, bioassays, liquid chromatography, and specific rotation measurements, we found that all of them matched the L-amino acid standard. Ta.

Table 1 The cells were then cultured at 30°C for 16 hours in the same manner as in Example 1.

Add 0 DL-5-isopropylhydantoin to this culture solution.
．． Add aseptically to give a concentration of 17 dt, and further heat at 30°C.
The cells were cultured for 24 hours. As a result, 0.11
L-valine of 1/dt was produced.

The bacterial cells were collected by centrifugation and diluted with 0.1M phosphate buffer containing 100 ppm of cobalt ions (the same amount as the culture solution).
Added to pH 7,5) 5- to give 511/di,
The reaction was maintained at 30°C for 24 hours. After the reaction was completed, the bacterial cells were removed by centrifugation. The generated L-valine is
As measured by Cey's method, each amount was 0.2117 dl from L-form ##PDL-form N-carbamylvaline.

0.20 i/lri of L-valine was produced.

Example 5 'tX' offspring Rus nisji-i-seikenba-hi-AJ-12299 was cultured in the same manner as in Example 1 at 30°C for 16 hours using the medium of Example 1. This culture solution and bacterial cells were centrifuged, washed once with physiological saline in the same amount as the culture solution, and further centrifuged to collect the bacterial cells. This face piece was added to a phosphate buffer (pH 7.5) containing 100 ppm cobalt ions to a concentration of 5 fi/di, and treated with 10 ka ultrasonic waves for 10 minutes. The treated product was centrifuged to obtain a supernatant. 5fnt of this supernatant liquid was added with N-carbamyl-DL-amino acid t-11 shown in Table 2.
Add it so that it becomes 1/#, correct the - to 7.5, and then add 3
The reaction was carried out at 0°C for 24 hours.

Table 2 shows the results of measuring various amino acids present in the reaction solution using the method described above. Furthermore, as a result of separating and purifying these amino acids and analyzing them by methods such as NMR spectroscopy, X-ray diffraction spectroscopy, PIO, SEI, liquid chromatography, and specific rotation measurement, it was found that they all matched the -amino acid sample.

Table 2 Example 6 The mixture was added aseptically and cultured at 30C for 24 hours. As a result, there was 0.061/di of L in the culture solution.
-Valine was produced.

The cells were then cultured at 30° C. for 16 hours in the same manner as in Example 1.

This culture solution and bacterial cells were collected by centrifugation, and cultured in 0.1M phosphate buffer (p
H6,0) 51 to 511Lt! /dt and kept at 30°C for 24 hours to react. After the reaction was completed, the bacterial cells were removed by centrifugation. Produced N-carbamyl-L
- When the amino acid was colorimetrically determined by adding a concentrated hydrochloric acid solution of p-dimethylaminobenzaldehyde, it was 98 ~/dt when the substrate was L form, and 96 m/di when the substrate was 35i1DL form. Example 8 Culture was carried out at 30° C. for 16 hours in the same manner as in Example 1.

This culture solution and bacterial cells were centrifuged, washed once with physiological saline in the same amount as the culture solution, and further centrifuged to collect the bacterial cells.

The bacterial cells were suspended in 0.05 M Tos 17 buffer (pH 7.0) and treated with 10 kc ultrasonic waves for 10 minutes. The treated product was centrifuged, the supernatant was dialyzed against the same buffer, and then fractionated by anion exchange chromatography. 5-
A fraction having the activity of converting substituted hydantoin into N-carpamy-L-amino acid was collected, and 0.1 M containing finally the hydantoin compound shown in Table 3 i p7ctt was collected.
Added to phosphate buffer (-6, O) and incubated at 30°C for 24 hours.
Retention reaction occurred.

Table 3 shows the results of measuring each N-carbamyl beak P amino acid present in the supernatant using the method described above.

Also, these N-forces Lupamil 壬; Air! As a result of separating and purifying the amino acid and analyzing it using methods such as bispectral, X-ray diffraction, liquid chromatography, and specific rotation, it was found that all of the amino acids matched the N-carbamyl-L-amino acid standard. .

Schedule 3 procedural amendment September 1986 date! , Indication of the case 1985 Patent Application No. 167535 2. Name of the invention Process for producing L-amino acids or N-carbamyl-amino acids 3. Person making the amendment Relationship to the case Patent applicant address Kyobashi, Chuo-ku, Tokyo -Chome 5-8 No. 6, Subject of amendment Detailed description of the invention column 7 of the specification, Contents of amendment, page 6, lines 6-9, "Bacillus sp.... has been discovered." Delete and insert the following description.

[The above microorganism AJ 12299 (FERM P-88
37) has the following mycological properties.

(凰) Morphology 1) Cell shape and size: Bacillus, 0.7-0.9 x 2-4 μm 2) Presence or absence of cell pleomorphism: None 3) Presence or absence of motility, epiphytic state of flagella: Yes ), periflagella 4)
Presence/absence of spores, shape, size, site: present, oval, 0.
3X O, 5X O, 4-0.6 Jim, neutral to quasi-terminal spore sacs expand.

5) Gram staining: Positive 6) Acid-fast bilayer (b) Growth status in each medium 1) Broth agar plate culture: Moderate growth, irregular, flat, wavy to lobed, puff-colored, semi- Clear, buttery, and does not produce water-soluble pigments.

2) Meat juice agar slant culture Medium growth, spread-like, platform-like, dull-glow, puff-colored.

3) Meat juice liquid culture: It becomes slightly cloudy. Uniform, no bacterial membrane formation. There is no precipitation.

4) Meat juice gelatin puncture culture: Liquefies in layers. The degree of liquefaction is weak.

5) Lidmus milk: Becomes alkaline. Peptonize. Redmass is not returned.

(c) Physiological properties 1) Nitrate reduction: positive 2) Denitrification reaction: negative 3) MR test: negative 4) VP test: negative 5) Indole production: negative 6) Generation of hydrogen sulfide: negative 7) Does not hydrolyze and bi-decompose starch 8) Use of citric acid: Not used in Kogsr medium.

Use in Christensen medium.

9) Utilization of inorganic nitrogen sources: Use nitrates Use ammonium salts.

10) Pigment production: Not produced 11) Urease: Very weakly produced 12) Oxidase: Positive 13) Catalase: Positive 14) Range of growth Grows at two temperatures: 40'' but not at 45°C.

pH 7-9 15) Attitude towards oxygen: QIqD facultative anaerobic, microaerobic, anaerobic 16) O-
F test (by Hugh & ElfaoH method) 20, F 17) Production of acids and gases from sugars
Gas production L-arabinose -- D-xylose -- D-glucose -- D-mannose -- D-fructose Ten - Acid production Gas production - galactose -- maltose -- sucrose -- lactose -- Trehalose -- D-Sorvit -- D-Mannit -- Inosit -- Glycerin -- Starch -- 18) Production of acid from sugars in Ayev's medium L-arabinose - D-xylose + D-glucose + D -Mannitol + trehalose + 19) Decomposition of hippuric acid dinegative 20) Utilization of f-ropionic acid: negative 21) Salt tolerance: Grows at 3% but not at 5%.

The above mycological properties are classified into Berger classification (Berg*f).
s Manual of Syatematla
Bacteriololog7+Volume 2 (
1986)], the above AJ-
The 12299 strain is Bacillus plebis (Baa i l
lugbrsvis Migula 1900). "that's all

Claims (1)

[Claims] The 1,5-substituted hydantoin is an L-amino acid or an N-
A culture solution of a microorganism belonging to the genus Bacillus that has the ability to convert into carbamyl-L-amino acid,
The bacterial cells or processed bacterial cells are expressed by the following general formula I: ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼... [I] (In the formula, R_1 is the formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼,
▲ There are mathematical formulas, chemical formulas, tables, etc. ▼, ▲ There are mathematical formulas, chemical formulas, tables, etc. or L-amino acid or N-carbamyl-L-amino acid, which is characterized by acting on the DL body to convert it into the corresponding L-amino acid or N-carbamyl-L-amino acid, and collecting the same.
Method for producing amino acids. 2. A culture solution, cells, or a processed product of a microorganism belonging to the genus Bacillus that has the ability to convert N-carbamyl-L-amino acids into L-amino acids according to the following general formula II: ▲Mathematical formula, chemical formula, table etc.▼...[II] (In the formula, R_2 is a formula▲There are mathematical formulas, chemical formulas, tables, etc.▼,
▲There are mathematical formulas, chemical formulas, tables, etc.▼, ▲Mathematical formulas, chemical formulas,
There are tables, etc. ▼, ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼
, ▲There are mathematical formulas, chemical formulas, tables, etc. ▼, CH_3-, N
It acts on the L-form or DL-form of N-carbamyl amino acid represented by CCH_2CH_2-, ▲Mathematical formula, chemical formula, table, etc. ▼ or ▲Mathematical formula, chemical formula, table, etc. ▼) A method for producing an L-amino acid, which comprises converting the L-amino acid into the corresponding L-amino acid and collecting the same.