JPS61285995A - Production of l-amino acid - Google Patents

Abstract

PURPOSE:To obtain an L-amino acid without producing an N-carbamylamino acid as a by-product, by carrying out a reaction at pH of >=neutraility, converting a 5-substituted hydantoin to the L-amino acid and the N- carbamylamino acid and continuing the reaction at pH of <=neutrality. CONSTITUTION:A reaction wherein a 5-substituted hydantoin is treated with an enzymatic system of a bacterium [Arthrobactor (FERM P-7472), Pseudomonas (FERM P-7121), etc.] capable of converting the 5-substituted hydantoin to an L-amino acid is carried out at pH of >=neutrality and the substituted hydrantoin is partially or wholly converted to the L-amino acid and an N-carbamylamino acid. Then, a reaction is continued at pH of >=neutrality to form rapidly the L-amino acid and the N-carbamylamino acid can be converted in high yield to the L-amino acid without accumulating finally the N-carbamylamino acid.

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 microbial enzyme systems.

[Prior art]

Conventionally, examples of using various microbial sources for the production of L-amino acids from 5-substituted hydantoin using microbial enzyme systems have been known (% Kokoku No. 13850/1983, Japanese Patent Publication No. 54/2274, Special Public Service No. 54-8749
Publication No.).

The present inventors have already proposed a method for producing the corresponding L-amino acid by reacting Alithropacter pg-200 strain (ipFXRMp-7472) with 5-substituted hydantoin (%G. ).

These methods are extremely effective in producing monoamino moromi, which is useful as a medicine, a raw material for the chemical industry, or a food additive, and are expected to have practical effects.

[Problem that the invention seeks to solve]

However, in these methods for producing sea amino acids, when the enzyme system of a microorganism that has the ability to convert 5-substituted hydantoins into L-amino acids is allowed to act on 5-substituted hydantoins with t1 neutrality or higher, a decomposition reaction of the hydantoins occurs. is fast, but due to the presence of D-hydantoinase activity, D-N
-There was a problem in that a large amount of carbamyl amino acids was produced and accumulated as a by-product.On the other hand, if the enzyme system of the microorganism is allowed to act on the microorganism without keeping the temperature below neutral, D-N-carbamyl amino acids become a by-product. Although the amount of bioactivity is small, there is a problem in that the reactivity and stability of the enzyme system of the bacterial cells are significantly reduced and the reaction is not completed.

[Means for solving problems]

The present inventors have made various studies to solve the above problems, and have found that when the enzyme system of the microorganism acts on D-N-carbamyl amino acid at − below neutrality, D-N-carbamyl It has been found that amino acids are resynthesized into 5-substituted hydantoins by D-hydantoinase activity, and the entire amount of 5-substituted hydantoins is finally converted to the corresponding L-amino acids.

Therefore, the present inventors further investigated this method, and found that the reaction was carried out in - above neutrality, and part or all of the 5-substituted hydantoin was converted into iL-phenylalanine and N-carbamylphenylalanine. , f: If the reaction is further continued at - below neutrality, L-phenylalanine is produced extremely rapidly (and L-amino acid is produced in high yield without the final accumulation of N-carbamyl amino acid). The present invention has been completed based on the discovery that it can be converted into

That is, the present invention provides a method for producing L-amino acids from 5-substituted hydantoins by the action of an enzyme system of a microorganism that has the ability to convert 5-substituted hydantoins into L-amino acids, starting from a reaction pH of neutral or higher. Alternatively, it is a method for producing a monoamino acid, which is characterized by continuously lowering the pH to below neutrality.

In the present invention, the enzyme system of the microorganism is allowed to act on the 5-substituted hydantoin from 4 to 10, preferably p) 15.
Good results are obtained in the range of 9 to 9. Outside this range, the enzyme system is not suitable for practical use in terms of reactivity and activity stability.

Good results are obtained when the reaction is carried out at a neutral temperature of 7 or higher, preferably 7.5 or higher. If it is less than 7, the decomposition rate of the 5-substituted hydantoin is low and is not practical.

For stepwise or continuous lowering of Kp) (t-, good results are obtained by setting it below 7, preferably below 6.8. If this is not done, the accumulation of N-carbamyl ε) acid will be large and the L- Aminomit cannot be obtained in good yield. To adjust the reaction, methods such as neutralization with a neutralizing agent and removal of ammonia and carbon dioxide produced in the reaction from the reaction mixture can be used. Specific examples of the neutralizing agent include acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, or acetic acid, alkalis such as ammonia and hydroxide, or salts thereof.

In addition, the reaction pHt may be changed by, for example, adjusting the reaction pHt to above neutrality at the beginning or during the reaction, and lowering FJ'('t- to below neutrality) stepwise or continuously. - at the start and end of the reaction can be arbitrarily selected as long as it is within the above-mentioned range.

Further, the reaction at neutrality or higher and the reaction at neutrality or lower may be alternately switched.

The enzyme system of the microorganism used in the present invention may be any one having the ability to convert 5-substituted hydantoin into the corresponding L-amino acid, for example, a microorganism that has the ability to convert 5-substituted hydantoin into the corresponding L-amino acid. All may be used alone, or in combination with a microorganism capable of converting 5-benzylhydantoin into the corresponding N-carbamyl amino acid and a microorganism capable of converting N-carbamyl amino acid into the corresponding sea amino acid. You can also use

Examples of microorganisms having these activities include Arylobacter (FIRM P-7472), Flavobacterium! -ami ie net, (pzRMp-3133
), '/Needomonas (FIIRM P-7121),
Examples include microorganisms belonging to the genus Pseudomonas (FEBM P-7475).

The enzyme system of these microorganisms may be in a form that exhibits enzymatic activity (such as a microorganism culture solution, live cells, frozen cells, dried cells, ground bacterial cells, or bacterial cell extracts). The processed bacterial cells or these may be immobilized by known means.

Specific examples of the 5-substituted hydantoin used in the present invention include 5-benzylhydantoin, 5-indolylmethylhydantoin, and 5-(4-hydroxybenzyl)hydantoin.

The desired L-amino acid can be obtained in high yield from any of these DXLf: or a mixture of D and L.

The concentration of 5-substituted hydantoin used is 0.1 to 40% by weight.
, preferably in the range of 1 to 10% by weight.

The reaction temperature is preferably in the range of 15 to 50°C, and a small amount of metal ions, such as MnO2, CC00J, etc., is added to the reaction system.
Addition of about 0.1 to 1 QmM gives good results for the reactivity and activity stability of the enzyme.

The L-amino acid can be isolated and purified from the reaction solution by conventional methods such as using an ion exchange resin or isoelectric precipitation.

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to examples, but the present invention is not limited to these examples.

Incidentally, unless otherwise specified, ``chi'' in each example is % by weight.

Example 1 Glucose 0.5%, Yeast Process Φ 0.2, Disodium Hydrogen Phosphate 0.1%, Potassium Dihydrogen Phosphate 0.05
4b, am Medium containing 0.05% magnesium (p) (
7,0) After adjusting the tA, 6 to 7 of the mixture was put into a 5 volume culture tank and sterilized at 120°C for 15 minutes. Separately, sterilized DL-5-indolylmethylhydantoin was added to a final concentration of 0.1%, and then the Arylobacter genus D K-'l Q Q (FERMP-7472
)"fi: Inoculated and maintained at pH 7.0 at 60°G.
The cells were cultured for 24 hours under aeration and stirring.

After the culture was completed, the cells were centrifuged from the culture solution and washed with 0.9 g of saline to obtain wet cells.

Next, this wet bacterial cell was dispersed in 1 volume of water containing 45 gr of 1DIi-5-benzylhydantoin and 0.5 x 10-3 mol of cobalt chloride, and placed in a 1 volume flask equipped with a stirrer.

The reaction flask was equipped with an auto-regulator and the reaction temperature was controlled by adding 2N hydrochloric acid.

The reaction was carried out at a temperature of 37° C. for 20 hours after initiation, at pH 7.5, for the next 20 hours at p) 17.0 and for the last 20 hours at p).
l (6.5) to complete the reaction.

After the reaction was completed, the reaction solution was analyzed and the yield of L-phenylalanine was 98! , N-carbamylphenylalanine,! -5-penzylhydantoin was not detected.

This reaction solution was sterilized by ultrafiltration with a molecular weight cutoff of 3 minutes,
After concentration, a small amount of methanol gold was added and the mixture was cooled to obtain crystals.

The dry weight of the crystals obtained was 33-3 gr, and TLO, I (PLO, NMR, elemental analysis, and optical rotation analysis) showed that the crystals were identical to the r, 1 to futechnyla 2 nin preparation. This was confirmed. The results of optical rotation analysis are shown in Table M1.

Table 1 Comparative Example 1 The reaction was carried out in the same manner as in Example 1 except that the reaction pH'e was kept at 7.5, and when anti-G* was analyzed, L-Fe = /I
The yields of /7-ynin and N-carbamylphenylalanine were 71% and 24%, respectively, and 5-benzylhydantoin was not detected.

Comparative Example 2 The reaction was carried out in the same manner as in Example 1 except that the pJ of the reaction was kept at 6.5. When the reaction solution was analyzed, the yields of L-phenylalanine and N-carbamylphenylalanine were each 61q/b. The residual rate of 5-benzylhydantoin was 66.

Example 2 The reaction was carried out in the same manner as in Example 1 except that the reaction value was continuously lowered from 7.5 to 6.5. When the reaction solution was analyzed, the yield of L-phenylalanine was 98%. , N-carbamylphenylalanine and 5-benzylhydantoin were not detected.

Example 6 Bacterial cells obtained in the same manner as in Example 1, DL-5-indolylmethylhydantoin 20 gr 1 cobalt chloride o,
When the reaction mixture was analyzed in the same manner as in Example 1,
The yield of L-)lyptophan was 98%, and N-carbamyltryptophan and 5-yne Vlylmethylhydantoin were not detected.

Water was added to this reaction mixture, partially precipitated tryptophan was dissolved by heating, and after sterilization by ultrafiltration with a molecular weight cut off of 60,000, the mixture was concentrated and cooled to obtain crystals.

The obtained crystals had a dry weight of 15-8 gr (yield 88
．． 7%), and the crystals were analyzed by TLO1 NMR, elemental analysis, and optical rotation analysis, and it was confirmed that the crystals matched the L-)liptophan standard. Table 2 shows the results of optical rotation analysis.

Table 2 Comparative Example 3 The reaction was carried out in the same manner as in Example 3 except that the pH of the reaction was maintained at 7.5. When analyzed, the yields of L-tryptophan and N-carbamyltryptophan were 75% and 12%, respectively, and 5-indolylmethylhydantoin was not detected.

Comparative Example 4 The reaction was carried out in the same manner as in Example 3 except that the pH of the reaction was maintained at 6.5, and the entire reaction mixture was analyzed, and the yields of L-tryptophan and N-carbamyltricytophan were 62% each. 1%, and the residual rate of 5-(4-hydroxy)hydantoin was 62%.

Example 4 The bacterial cells obtained in the same manner as in Example 1 were dispersed in 1 volume of water containing 10 g of DL-5-(4-hydroxy)-benzylhydantoin and 0.5 × 10 moles of cobalt chloride,
When the reaction was carried out in the same manner as in Example 1 and the reaction mixture was analyzed,
The yield of L-tyrosine was 98%, and N-carbamyltyrosine and 5-(4-hydroxy)benzylhydantoin were not detected.

Add 6N-salt rI! to this reaction mixture! After adding all of the tyrosine "fc" which was partially precipitated, it was sterilized by centrifugation and diatomaceous earth filtration. 6N caustic soda was added to the filtrate and p!-1
6, concentrated and cooled to obtain crystals.

The obtained crystals had a dry weight of 8.1 gr (yield 92
h) and its crystal is T1. +O, HPLG, NM
When R, elemental analysis and optical rotation analysis were carried out, it was confirmed that they all matched the L-tyrosine specimen. The results of optical rotation analysis are shown in Table 6.

Table 6 [Effects of the Invention] As explained above, according to the present invention, there is no by-product of N-carbamylic acid, the reactivity and stability of the bacterial enzyme system are excellent, and the L-amino acid is highly efficient. Can be manufactured.

Patent applicant Denki Kagaku Kogyo Co., Ltd. Hand##l'''=-7ifr correct: 7 pieces November 130, 1985

Claims (1)

[Claims] When producing L-amino acids from 5-substituted hydantoins by the action of the enzyme system of microorganisms that have the ability to convert 5-substituted hydantoins into L-amino acids, the reaction pH is gradually or continuously neutralized from a neutral or higher reaction pH. pH below
1. A method for producing L-amino acids, characterized by reducing the amount of L-amino acids.