JPS60214889A - Production of l-aminoacid - Google Patents

Abstract

PURPOSE:The culture mixture of a specific strain in Arthrobacter is allowed to act on a 5-substituted hydantoin whereby the corresponding L-aminoacid is obtained in high efficiency by utilizing an enzyme system of a microorganism. CONSTITUTION:The culture mixture, cell bodies or processed cell bodies of DK- 200 strain in Arthrobacter capable of converting 5-substituted hydantoin into the corresponding L-aminoacid (FERM P-7472) is allowed to act on a 5-substituted hydantoin such as 5-benzylhydantoin, preferably at a pH of 6-9 and 15- 50 deg.C in the presence of 0.1-10mM of a metal ion such as MnCl2. Then, the L- aminoacid formed is collected.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for producing L-amino acids from 5-substituted hydantoins using a microbial r+V elementary system.

[Prior art]

Since ijE, 5-1 by microbial enzyme system? Preparation of L-amino acids from hydantoin
! The microorganisms of J.
No. 74 and No. 54-8749.

However, each of these methods cannot be said to be a sufficient method.

[Purpose of the invention]

An object of the present invention is to provide a method for efficiently producing gold from a 5-substituted hydantoin using a microbial enzyme.

[Structure of the invention]

The present invention is summarized as follows: The present invention relates to a method for producing L-amino acids, and includes 5-1. A culture solution, strain or bacterial cell treatment of Arylobacter Ji% DK-200 strain having the ability to convert into hydantoin iL-amino acid, 5-1i! 11' is characterized by acting on converted hydantoin and collecting the corresponding L-amino acid produced.

Arislobacter-13g DK used for 1 time in "This issue"
-200 strain is a new strain newly isolated by the present inventors, and its various properties are shown below.

1. Form (1) Thin 1j← shape and size = 03~0.5 X
0.8 to 5.0 μm (2) many) Hi I no + no I ■ Ku1
11: Lee Jf31;) Morning Ii sid'F's Lee Jsjl
+,: Meha) 9 (4) Whip Shino-tj Su 11 Stone, R
f Raw state None (5) #? j child (1) -IT pHH
＋(: Voice Iqru (7) Anti-acidity: Sushi 2, Growth status in each type (1) Meat juice agar plate Sapphire colony packaging: Pale white colony shape 2 Circular colony 1 Orientation: Convex Peripheral line of colony: Gold gauze (2) Meat juice 2 suten 8f.) side n4 curing f4 condition:, 1 Degree claw υ (2 Jig shape: Thread shape (3) Inner needle a body culture (171 to 1 1 presentation IJt 2 yen - sinking fence: 1 1 ts: 1: Especially nothing (・1) Meat juice gelatin puncture culture 11'8: 2'e, 6, Physiological properties (1) 1111 acid Age of salt: Yin (2) Desorption reaction: Yang (31 MR test: Limited (4) VP test 2 Yin (5) Formation of indole 2 Yin (6) Formation of hydrogen sulfide: 隘 (7) Power of starch Decomposition: Yin 0 (1 pigment life Iy:,: No Qll Urease: Ken0bu Oki ε/dase: 1 01 Catalase: Yang Q4 Attitude towards oxygen: Aerobic 0li Growth range temperature: 17.8-332℃ pH2 5-10 5-1O test oxidative O
η Presence or absence of acid and gas production from sugar Al'l
JL-arabinose −- ■ D-xylose + − ■ D-curcose ± − (/D D-mannose 10 − ■ D-7 lactose ± − ■ D-caractose ± − maltose − (a sucrose − - 〇 Lactose - (lΦ Trehalose ± - (1 Li D-Kurvit - (Parrot D-Mannit) - (!8mi) Inojito - 〇 Glycerin + - O-Chorus Dibasicity in the thin 11 wall Amino acid - lysine (contains no meso-diaminopimelic acid and arabinose) DNA degradability: positive casein degradability: positive salt tolerance: grows at 2% Naaz, weak FF at 5% Hydrocarbon assimilation ■ p-Hydroxybenzoic acid − ■ Curconic acid − ■ Lactic acid − ■ Acetic acid − 〇 Glucose + ■ Sucrose 10 ■ Xylose + ■ Lactose − ■ Mannitol + The above copperological properties
y's Manual of determination
ve bacterio-1-ogy (8th edition)]
Based on the search, the above DK-200 strain is (1)
Polymorphism: Yes, (2) graphite sexual particle k ff1ll 1
(3) Contains lysine ligation in the cell wall;
Arylobacter (Arylobacter) f+ VCJ! X, and already 9.1
Since the properties did not match any of the other bacteria, it was determined that it was a new fungus.

The deceased, DK-200-Co., Ltd., is listed as FIRM P-7472.

As a cultivation method for obtaining a large amount of this DK-200 strain, known methods such as ordinary extraction liquid culture are sufficient. is included.

Furthermore, the enzyme activity is enhanced by adding L-carbamyltryptophan or the like. For example, culture at pH 4-1
1. It is best to carry out the test at a temperature of 15 to 40°C for 5 to 120 hours.

The conversion reaction of 5-substituted hydantoin to L-amino acid is
The microorganism culture solution obtained in this way can be used as it is, but it is also possible to use a 64-cell culture solution such as live, frozen, freeze-dried microorganisms, microbial crush material, or microbial cell extract. Shi-C is also good. Furthermore, it goes without saying that performance-enhancing 1ζ mutant strains can also be used more effectively by mutation treatment.

Specific examples of the 5-substituted hydantoin used in the present invention include 5-benzylhydantoin, 5-indolylmethylhydantoin, and rj5-(4-hydroxybenzyl)
- Hydantoin is praised, these glue, Dmataha DL
The desired L-amino acid can be obtained at a low yield from any source.

Use of 5-substituted hydantoin #thigh 0.1-40% preferably 1-10%, pH 4-11 preferably pH 6-
Good results in the range of 9'5g:? (do.

Outside this range, it is not suitable for practical use in terms of enzyme activity expression and stability. Since the pH changes as the reaction progresses, it is desirable to maintain the desired pH using a suitable neutralizing agent. The reaction temperature is preferably in the range of 15 to 50°C, and a small amount of gold ion, such as Mn0t, OoOtg, is added to the reaction system.
Addition of 1 to 10 mM of isostimulatory α gives good results in reactivity.

To separate L-amino acids from the reaction solution, for example, [￥1
After removing the insoluble matter from the centrifuge using a centrifuge such as 1411, it was passed through a cationic resin of H) to 14 strength. The L-amino acid can be obtained in the form of crystals by cooling to +%.

〔Example〕

This will be explained in more detail in the following four examples.
The present invention is not limited to these. In addition, 11
) Unless otherwise specified, the percentages in each example are representative.

Example 1 Curcose 0.5%, yeast extract 0.2%, ') disodium hydrogen phosphate 0.1%, potassium dihydrogen phosphate [1L
0.05%, magnesium sulfate 0.05% medium (pH
7,5) was dispensed into test tubes and sterilized at 120°C for 15 minutes. In addition to this, DL-N-rubamyltributophane was also killed, and the final concentration was 0.05%.
After f L fc, Arilobacter-13% D K -
21101ti''4 and photographed at 50℃ for 24 hours.''

To this culture solution, 5-1i'. and hydantoin listed in Table 1 below were added separately for 7JIIL, and shaking was continued at 50° C. for 24 hours. After the reaction, bacteria were sterilized by centrifugation, and amino acids in the supernatant were removed. (cHPLO and bioassay (Lactobaburus arabinozas AT)
As a result of capacity analysis using the OO8014 meeting, the values matched, so it was confirmed that it was an L-amino acid.

Table 1 Example 2 Curcose u 5%, Yeast extract 0.2%, Disodium hydrogen phosphate u 1%, Potassium dihydrogen phosphate 9 μm α 05%, Magnesium sulfate L 105%, 1) L-Indolyl methyl hydantoy 701 Jar containing %, ground (p+(7,5
) f 100 mf in a 500 mF flask! Container 1
Sterilized at 20°C for 15 minutes. In addition to this, glucose 05%,
ni'mother's raw extract 0%, polybegtone 05%, salt 0
．． Medium containing 5% salt (pH 7, 5J) was cultured at 50°C for 20 hours.
11A'+ and cultured at 30°C for 24 hours.

After this fFt is completed, the culture solution is centrifuged once with physiological saline. 4) I did. This bacterial cell i, DL-5-
Benzylhydantoin 0.2 M and cobalt chloride (1,
5771M and 0.1M Tris-HCl buffer (pH
7,0) Disperse in 100 ml and incubate at 65°C at 15°C.
Allowed time to react. After reaction, centrifuge 1 minute! The supernatant was sterilized by Lit, the supernatant was reduced to 20 rne, and the crystals were washed out by 1 small cycle. This, I'i11 crystal game, TI, O, H
PLO2NMI (and when the optical rotation t1 is calculated by minute MF,
Table 2 below (From the table 1, L-phenylalanine standard and 1) Table 2 Example 3 M-body obtained in the same manner as in Example 1 1. Synthetic Kobal) 0
．． 5mM and 1mM manganese chloride and 0.1M
Tris-HCl buffer (pH 7,5) 2mj! dispersed inside. This dispersion was treated with ultrasonic waves at 20 kHz for 5 minutes 5 times under ice conditions to prepare a disrupted bacterial cell solution.

This solution was treated to form D-5-benzylhydantoin iuIM, and reacted at 35°C for 400 hours. After the reaction, all impurities were removed by centrifugation, and the supernatant obtained was subjected to HLPO and bioassay, and it was confirmed that the produced phenylalanine IL was the product. The yield of this L-phenylalanine was 82%.

〔Effect of the invention〕

As explained above, according to the present invention, d:, 5-1^1-substituted hydantoin to alithrono (lid-IfT4D K-
Using 200 bacteria, it was possible to efficiently produce the corresponding L-amino acid.

Special W ``Department S Person'' I, Representative of Kiikagaku Kogyo Co., Ltd. Hiroki Nakamoto Ito Kaime Yoshi Akatsura

Claims (1)

[Claims] 1, 5- IM' hydantoin iL-Amino acid to hammer 4;
-2o o IA', + strain culture solution, bacterial strain or m-body treated product, react with 5-IFV-converted hydantoin, and collect the corresponding L-amino acid produced! [ηtK<
A method for producing L-amic acid).