JPH01215297A - Production of l-alpha-amino acid - Google Patents

Abstract

PURPOSE:To industrially and advantageously obtain the title compound for food additive, etc., by reacting a specific DL-alpha-amino acid amide with bacteria strains belonging to Mycobacterium.methanolica and having ability hydrolyzing only L-alpha-amino acid amide. CONSTITUTION:DL-alpha-amino acid amide (e.g., DL-valine amide) expressed by the formula [R is (substituted)lower alkyl, (substituted)phenyl, furyl, pyridyl, thiazolyl or imidazolyl] is reacted with bacterium cell of bacterium strain [e.g., Mycobacterium.methanolica BT-84 (FERM P-8823)] belonging to Mycobacterium.methanolica and having ability hydrolyzing only L-alpha-amino acid amide or treated product thereof to provide the corresponding and aimed L-alpha-amino acid.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for producing L-α-amino acids. More specifically, we used Mycobacterium luteum, a new bacterial species discovered by one of the inventors, to obtain DI, -α-
The present invention relates to a method for producing a corresponding L-α-amino acid from an amino acid amide.

L-α-amino acids are important as intermediates for pharmaceuticals, food additives, feed additives, and various industrial chemicals.

[Prior art, problems to be solved by the invention] Conventionally,
When α-amino acids are produced by organic synthetic methods, the resulting α-amino acids are in the DL-form, so it has been a major challenge how to optically resolve them in an industrially advantageous manner.

Methods for optically resolving DL-α-amino acids include physicochemical methods, biochemical methods, and the like, and among these methods, for example, the following method has been put into practical use regarding the latter.

1) A method of causing an acylase possessed by a microorganism to act on the N-acyl form of DL-α-amino acid.

N112 AC20NIIAC? Schiller I N
tlAc Nll□(I R=C11C0011→R-CIIC0010-÷R-
CIICOOII + R-CIICOOII (DI,
-) (DI, -) (D-)
(L-)2) A method of causing a hydantoinase possessed by a microorganism to act on a hydantoin derivative of DI, -α-amino acid.

Hydantoinase NlI
C0NI+2(D-) However, these methods require expensive raw materials and
Moreover, since the reaction system is complicated, it has the disadvantage that economic disadvantages are inevitable.

On the other hand, using DL-α-amino acid amide as a substrate, amidase produced by microorganisms is applied to the DL-α-amino acid amide, and the corresponding L-
As a method for producing α-amino acids, a method using enzyme amidase produced by microorganisms belonging to the genus Bacillus, Bacteridium, Micrococcus, and Previbacteridium (Publication No. 58-500319)
, a method using the enzyme amidase produced by bacteria such as various yeasts, such as microorganisms belonging to the genus Mycobacterium (Japanese Patent Application Laid-open Nos. 57-13000, 59-159789 and 60-38446), Enterobacter. Cloassay (N-7901) or Pseudomonas 5
A method using the enzyme amidase produced by P (N-7131 or N-2211) (Japanese Patent Application Laid-Open No. 62-55097) is known.

However, in all of these methods, the amidase activity of the microorganism used is weak, and DL-α-
Using these microorganisms to produce L-α-amino acids from amino acid amides cannot be an industrially advantageous production method.

Furthermore, many of the microorganisms belonging to the genus Mycobacterium are highly pathogenic, making them highly dangerous and unsuitable for practical use.

[Means and effects for solving the problem] The present inventors searched for microorganisms in nature that can efficiently convert DL-α-amino acid amide into monoα-amino acid using DL-α-amino acid amide as a substrate. We were able to obtain a strain suitable for the purpose, and completed the present invention using this strain.

This strain belongs to the genus Mycobacterium,
This is Mycobacterium methanolica, a new bacterial species discovered by one of the inventors.

It was discovered that this fungal species has strong amidase activity and is substantially free of pathogenicity, and based on this discovery, the present invention was achieved.

That is, in the present invention, the general formula of H2 is RCM CONH2 (wherein R is a lower alkyl group or a substituted lower alkyl group.

Phenyl group, substituted phenyl group, furyl group, pyridyl group,
DL-α-amino acid amide (representing a thiazolyl group and an imidazolyl group) is treated with the cells of a strain of Mycobacterium methanolica that has the ability to hydrolyze only L-α-amino acid amide or its processed product. This is a method for producing L-α-amino acids, which is characterized in that the corresponding L-α-amino acids are obtained.

The lower alkyl group of R in the DL-α-amino acid amide represented by the general formula of the present invention is not particularly limited, but C' t ” such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and 5ec-butyl, etc. ""'
C4 straight or branched lower alkyl groups are preferred, and the same applies to substituted lower alkyl groups. In addition, the substituents contained in each of the substituted lower alkyl group and the substituted phenyl group are, for example, hydroxy, methoxy,
These include recapto, methylmercapto, amino, guanyl, carboxyl, carbofusamide, halogen, phenyl, hydroxyphenyl, and imidazolyl.

The substrate of the present invention is a DL-α-amino acid amide represented by the general formula, and a representative example thereof is alanine amide (“
DL-" is omitted. The same applies hereinafter), valinamide, leucinamide, isoleucinamide, serine amide, threoninamide cysteinamide, cystinamide, methioninamide, lysinamide, argininamide, asparaginamide, glutamine amide, -phenylglycinamide, phenylalaninamide, These include tyrosinamide and histidineamide.

These substrates are asymmetrically hydrolyzed by bacterial cells or their processed products to obtain L-α-amino acids corresponding to the respective substrates.

The bacteria used in the present invention belong to Mycobacterium methanolica and may be any strain that has the ability to asymmetrically hydrolyze the DL-α-amino acid amide represented by the general formula to produce the corresponding L-α-amino acid. There are no particular restrictions.

Mycobacterium methanolica is a new bacterial species discovered by one of the inventors, and is a representative strain of bacteria belonging to this new bacterial species.

Mycobacterium methanolica BT-84 (Feweral Research Institute Bacteria No. 8823), Mycobacterium methanolica BT-143 (February Industrial Research Institute No. 8823)
No. 824), P-23 (Feikoken Bibori No. 8825)
, same P-26 (Feikoken Bibori No. 8826) and same
There is P-85 (Feikoken Bibori No. 8827).

The medium used for culturing this bacterium (the bacterium used in the present invention, hereinafter the same) must contain at least a carbon source that can be assimilated by this bacterium, and an appropriate amount of nitrogen source and inorganic salt. It may be a synthetic medium or a natural medium, and does not require any special medium.

The carbon source is not particularly limited as long as it can be assimilated by bacteria; for example, molasses.

Puffs, meat extracts, and natural products such as cornstarch liquor and sugars such as glucose, fructose, sucrose, sorbyl, glycerin, and mannitol, methanol, ethanol, and n-
Alcohols such as propatool can be used.

As nitrogen sources, inorganic nitrogen compounds such as ammonium salts, nitric acid salts, etc. and/or organic nitrogen-containing substances such as urea, cornstarch liquor, casein, peptone, yeast extract, etc. are used.

Inorganic components include, for example, calcium salts, magnesium salts, potassium salts, sodium salts, phosphates, manganese salts, zinc salts, iron salts, copper salts, molybdenum salts, cobalt salts,
Boron compounds and iodine compounds are used.

It is also effective to add DL-α-amino acid amide to the medium to obtain high enzyme activity.

At this time, the DI, -α-amino acid amide to be added may be any DL-α-amino acid amide represented by the general formula of the present invention; It is still more effective to use -α-amino acid amides.

The culture conditions are a temperature of 20 to 42°C, preferably 25 to 40°C.
°C, pif 5-9. Preferably it is 6-8.

Growth will be poor, but this does not preclude culturing outside these conditions.

The bacteria cultured in this manner are expressed by the general formula DL-α
- To act on amino acid amides, use a culture solution obtained by culturing microorganisms in a liquid medium, bacterial cells isolated from this culture solution, crushed bacterial cells, or an enzyme (amidase) isolated from the culture solution or bacterial cells. A crude enzyme, a purified enzyme, an enzyme-containing extract, or a concentrate thereof (hereinafter, substances other than bacterial cells may be referred to as "treated products") are used to act. In addition, each of the bacterial cells and enzymes can be immobilized on a carrier and used (this immobilized product will also be referred to as the processed material below).
).

Examples of carriers used for this immobilization include alginic acid, carrageenan, and collagen.

Cellulose, acetyl cellulose, agar, konnyaku,
Natural products such as cellophane, collodion or polyacrylamide, polystyrene.

polyethylene glycol, polypropylene glycol,
Commonly used carriers such as synthetic polymeric materials such as polyurethane, polybutadiene, etc. can be used. Immobilization is performed by conventional methods, but under conditions that do not impair amidase activity.

In the present invention, the reaction conditions for asymmetric hydrolysis of the DL-α-amino acid amide represented by the general formula by the action of Mycobacterium methanolica cells or a treated product thereof include a reaction temperature of 10 to 90°C; Preferably 20-70
℃, and the reaction pH is 5-13, preferably 6-12. The reaction time is usually about 0.5 to 36 hours, preferably about 1 to 24 hours. The reaction time can be shortened by raising the reaction temperature, increasing the amount of bacterial cells or their processed material, etc.

In the present invention, the amount of microorganisms used is the substrate DL-
Weight ratio of dry bacterial cells to α-amino acid amide is 0.
．． The range is from 0.001 to IO, preferably from 0.01 to l. When using a culture solution or a treated product, the amount to be used may be determined by converting it to the weight of dry bacterial cells.

The concentration of DL-α-amino acid amide used as a substrate is generally not limited as long as it is below the saturation concentration of DL-α-amino acid amide used as a raw material, but preferably 1 to 20
It is wt%.

In the present invention, when the hydrolysis reaction of L-α-amino acid amide is almost completed, the reaction is stopped as soon as possible, and then the target substance L-α-amino acid and unreacted D-α-amino acid amide and D-α-amino acid amide are each separated and recovered once.

This separation can be easily performed by fractional crystallization, solvent extraction, ion exchange, or other known methods.

In addition, in the above-mentioned asymmetric hydrolysis operation, D in the substrate
-α-amino acid amide is not susceptible to the action of bacterial cells or treated substances, but if the reaction time is too long, D-α-amino acids may be produced by the action of bacterial cells or treated substances. It is preferable to stop the reaction immediately upon completion of hydrolysis of the L-α-amino acid amide and to prevent the production of D-α-amino acids as much as possible.

To stop the reaction, for example, the bacterial cells and the treated product are removed from the reaction product solution, the D-α-amino acid amide is removed from the reaction product solution, the reaction product solution is changed, and/or the reaction product solution is Conventional methods such as changing the temperature can be used.

Further, unreacted D-α-amino acid amide can be hydrolyzed by a method known per se, for example, with an acid or an alkali, to obtain the corresponding D-α-amino acid. Alternatively, the entire amount of DL-α-amino acid amide can be converted to L-α-amino acid by racemizing the D-α-amino acid amide by a conventional method and then circulating it to the reaction system.

[Examples] The present invention will be explained in more detail with the following Examples, but the present invention is not limited thereto.
(Rhinoceros Golden Tongue) Example 1 Glucose Log, polypeptone Log, and yeast extract Log were dissolved in pure water IQ, and the pH was adjusted to 7.0.Pour medium 10071112 into an IQ Erlenmeyer flask, and mix at 1 kg/cJG. Into a medium that had been sterilized for 20 minutes, ldl of the culture solution of each strain of Mycobacterium methanolica precultured in the same medium was inoculated, cultured with shaking at 30°C for 5 hours, and the culture solution was incubated at 1800 rpm for 10 minutes. Centrifugation was performed to obtain bacterial cells.

0.5 g of the above-mentioned microbial cells (calculated as dry microbial weight) was added to 100% of pure water containing 5 g of DL-valinamide, the pH was adjusted to 9, and the reaction was carried out with shaking at 40° C. for 60 minutes. After the reaction is completed, the reaction product solution is heated at 1800 Orpm for 10 minutes.
Centrifugation was performed for a minute to obtain a supernatant. This supernatant liquid was analyzed by high performance liquid chromatography to determine the yield and optical purity of the produced L-valine.

In addition, when the specific optical rotation of the obtained L-valine was measured, all of them were [α] O'=+27.6 to 28.2. The results are shown in Table 1.

Example 2 The same procedure as in Example 1 was carried out except that the culture medium had the following composition.

glucose 10 g peptone
5g Meat extract 1g Yeast extract 5g K I'I 2 P O41g M g S O4・7H200-4g F e So<・'IHxOO,01gM n C1!
” 4 H200, 01gDL-Valinamide
5g water IQp)I
7 The results are shown in Table 2.

Example 3 Glycerol Log, meat extract 5g, MKSO4・
7l-I20 0.1 g, Fe So<・7tI
200,01 g, MnSO,・4HzOO,Ol
gCaC12・2H200,01g, ZnSO4・7
Hzo 0, 001 g, DL 2.5 g of L-phenylalaninamide was dissolved in pure water IQ, the pH was adjusted to 7.0, and a medium 100- was placed in an IQ Erlenmeyer flask, and the medium was sterilized at 1 kg/ajG for 20 minutes. , Mycobacterium methanolica BT precultured in the same medium
-84 culture solution was inoculated, cultured with shaking at 30℃ for 48 hours, and the culture solution was inoculated at 1800 rpm.
Centrifugation was performed for 0 minutes to obtain bacterial cells.

DL-Phenylalanine amide 0.5g in pure water for 100s
Reaction liquid A in which 0.03 g of the above bacterial cells (calculated as dry bacterial weight) was added to an aqueous solution dissolved in Jl, and the pH was adjusted to 9.0.
, B, were prepared nine times.

Using reaction solution A and reaction solution B, reactions were carried out with shaking at 40° C. and 30° C., respectively, and the yields of L-phenylalanine after 1 hour and 2 hours were determined. Also, 2
After a period of time, the L-form/D-form (molar ratio) of the reaction supernatant was measured. The results are shown in Table 3.

Table 3 Example 4 Each strain of Mycobacterium methanolica was reacted in the same manner as in Example 2 except that DL-leucinamide was used as the reaction raw material.

The results are shown in Table 4.

Example 5 The same procedure as in Example 2 was carried out, except that Mycobacterium methanolica BT-84 was used and various 1], 2, and -α-amino acid amides were used as reaction materials.

The results are shown in Table 5.

[Effects of the Invention] The method of the present invention has made it possible to easily and efficiently produce many useful L-α-amino acids from DL-α-amino acid amide.

Patent applicant Wangling Gas Chemical Co., Ltd. Representative: Nagano Washo Agent: Patent Attorney Sadafumi Kohori

Claims (1)

[Claims] The general formula may be a mathematical formula, a chemical formula, a table, etc. (wherein R is a lower alkyl group, a substituted lower alkyl group, a phenyl group, a substituted phenyl group, a furyl group, a pyridyl group, a thiazolyl group, or DL-α (representing an imidazolyl group)
- The corresponding L-α-amino acid is obtained by reacting the amino acid amide with the cells of a strain of Mycobacterium methanolica that has the ability to hydrolyze only L-α-amino acid amide or a processed product thereof. L-α-
Method for producing amino acids.