JP2674078B2 - Process for producing D-α-amino acid - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing D-α-amino acid. More specifically, it relates to a method for producing a D-α-amino acid by biochemically asymmetrically hydrolyzing a DL-α-amino acid amide to selectively produce a corresponding D-α-amino acid.

D-α-amino acids are important as raw materials for antibiotics, bactericides, and intermediates for various industrial chemicals.

[Prior Art] Conventionally, as a method for biochemically asymmetrically hydrolyzing a DL-α-amino acid amide to produce a corresponding D-α-amino acid, DL-α-amino acid amide is converted into L-α-amino acid. An enzyme (L-amidase) that selectively hydrolyzes amide is allowed to act to obtain L-α-amino acid, and then unreacted D-α-amino acid amide is separated, and then DL-amidase is acted on Is known (eg,
Special publication No. 56-500319).

However, this method has the disadvantage that almost the same amount of L-α-amino acid as D-α-amino acid is co-produced.

Also known is a method of enzymatically hydrolyzing a D-α-amino acid amide to obtain a corresponding D-α-amino acid (for example, JP-A-60-184392 and JP-A-61-9698).
No. 9). However, in these methods, D-α-amino acid amide obtained by previously dividing DL-α-amino acid amide has to be used as a starting material.

The present inventors use DL-α-amino acid amide as a raw material,
For the purpose of developing a method for industrially advantageous production of D-α-amino acid directly from this DL-α-amino acid amide, investigations have been carried out, and a microorganism belonging to the genus Rhodococcus was previously identified as DL-α.
In the hydrolysis of amino acid amide, it was found that it has an activity of selectively hydrolyzing only D-α-amino acid amide (Japanese Patent Application No. 61-244023).

However, this enzyme is practically used in the reaction solution,
The activity was not sufficiently high, the yield of D-α-amino acid was low, the stability was low, the activity was not retained for a long time, and it could not be industrially sufficiently satisfied.

[Means and Actions for Solving Problems] As a result of further research to eliminate these drawbacks, the present inventors have found that the presence of a compound having a mercapto group in the reaction solution stabilizes the enzyme. The present invention has been completed by finding that it is possible to significantly increase the property.

That is, the present invention has the general formula (Wherein R represents 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, an imidazolyl group or an indolyl group). , D-α
-Acting with an amino acid amide hydrolase, the DL-α-
A method for producing a D-α-amino acid, which comprises allowing a compound having a mercapto group to be present in a reaction solution when producing a D-α-amino acid corresponding to an amino acid amide.

The lower alkyl group of R of the DL-α-amino acid amide represented by the general formula of the present invention and the lower alkyl group of the substituted lower alkyl group are not particularly limited, and examples thereof include methyl, ethyl, propyl, isopropyl, butyl, isobutyl and A C ₁ -C ₄ linear or branched lower alkyl group such as sec-butyl is preferred. Further, the substituents contained in each of the substituted lower alkyl group and the substituted phenyl group include, for example, hydroxy, methoxy, mercapto, methylmercapto, amino, guanyl, carboxyl, carboxamide, halogen, phenyl, hydroxyphenyl, imidazolyl and indolyl. is there.

Typical examples of the DL-α-amino acid amide represented by the general formula of the present invention include alanine amide (“DL-” is omitted. The same applies hereinafter), valine amide, leucine amide, isoleucine amide, serine amide, threonine amide, cysteine amide, cystine. Amides, methionine amides, lysine amides, arginine amides, asparagine amides, glutamine amides, phenylglycine amides, phenylalanine amides, tyrosine amides, tryptophan amides and histidine amides.

The microorganism used in the present invention is not particularly limited as long as it has an activity of hydrolyzing a D-α-amino acid amide, and is not particularly limited, for example, Bacillus genus, Bacteridium genus, Micrococcus genus. (Micro
coccus), Brevibacterium,
Achromobacter, Alcaligenes, Kurthia, Rhodococcus, Pseudom
onas) and microorganisms belonging to the Serratia genus (Serratia) and the like.

Of these, Practically, Rhodococcus erythropoli (Rhodococcus erythropoli
s) is particularly preferred.

Representative examples belonging to Rhodococcus erythropolis include Rhodococcus erythropolis NR-23 (Microtechnological Research Institute No. 8937) and NR-28 (Microtechnical Research Institute No. 8938). These strains are new strains isolated and identified by the present inventors.

As a nutrient medium used in the culture for growing these microorganisms, it is necessary that these bacteria contain at least a carbon source that can be assimilated, and a medium containing an appropriate amount of nitrogen source and inorganic salts. It does not matter if it is a synthetic medium or a natural medium without any special medium.

The carbon source is not particularly limited as long as it can be assimilated by these bacteria, and examples include molasses, peptone,
Meat extract and corn steep. Natural products such as liquor, glucose, fructose, sucrose, sorbitol, glycerin and mannitol etc., alcohols such as methanol, ethanol and n-propanol, organic acids such as acetic acid, citric acid and succinic acid, etc. Can be used.

As the nitrogen source, inorganic nitrogen compounds such as ammonium salts and nitrates and / or organic nitrogen-containing substances such as urea, corn steep liquor, casein, peptone and yeast extract are used.

As the inorganic component, for example, calcium salt, magnesium salt, potassium salt, sodium salt, phosphate salt, manganese salt, zinc salt, iron salt, copper salt, molybdenum salt, cobalt salt, boron compound and iodine compound are used. .

It is also effective to add D-α-amino acid amide or DL-α-amino acid amide to the medium in order to obtain high enzyme activity. The α-amino acid amide added at this time may be any α-amino acid amide represented by the general formula of the present invention, but it is preferable to use the α-amino acid amide corresponding to the desired D-α-amino acid. Particularly preferred. The concentration of the added α-amino acid amide in the medium is usually 0.1 to 10% by weight, preferably 0.2 to 2% by weight.

The culture conditions differ depending on the strain used, and it is sufficient to select culture conditions suitable for growth, proliferation and production of a selective hydrolase of D-α-amino acid amide for each strain. For example, the culture temperature is usually 20 to 42 ° C, preferably 25 to 40 ° C, pH 5 to 9, and preferably 6 to 8.

The microorganisms grown and grown in this manner are treated with D-α.
In order to act on the DL-α-amino acid amide represented by the above general formula as an amino acid amide hydrolase (D-amidase), a culture solution obtained by culturing a microorganism in a liquid medium, from this culture solution The isolated bacterial cells, disrupted bacterial cells, or crude enzyme of enzyme (D-amidase) isolated from the culture solution or bacterial cells, purified enzyme, enzyme-containing extract or concentrate thereof, and immobilized by a conventional method The cells are allowed to act in the state of bacterial cells or enzymes (hereinafter, other than bacterial cells may be referred to as “treated bacterial cells”).

The compound having a mercapto group used in the present invention may be an organic compound or an inorganic compound as long as it is water-soluble and is not particularly limited, but preferably 2-mercaptoethanol, cysteine, glutathione and Such as dithiothreitol.

In the method of the present invention, the above-mentioned DL-α-amino acid amide, bacterial cells and / or treated bacterial cells are usually added to a reaction solution in which an aqueous medium such as water is added with a mercapto compound to give an asymmetric reaction. The hydrolysis reaction is allowed to proceed.
However, DL-α-amino acid amide can be added to the above-mentioned culture solution to prepare a reaction solution.

The conditions of the hydrolysis reaction vary depending on the type of microorganism, the strength of the hydrolysis activity of the enzyme, the type of DL-α-amino acid amide, the type of compound having a mercapto group, etc., and cannot be unconditionally specified. For example, DL-α- in the reaction solution
The concentration of the amino acid amide is 1 to 40% by weight, the concentration of the compound having a mercapto group is 10 ^-6 to 10 ^-2 Mol / L, and the amount of the microorganism used for DL-α-amino acid amide is 0,005 as a dry cell weight ratio. -10, reaction temperature 0-70 ° C, and pH 5-13.

The D-α-amino acid produced by the hydrolysis reaction is concentrated under reduced pressure after removing microorganisms from the reaction product solution by a conventional method such as centrifugation and further removing the enzyme by a method such as ultrafiltration, if necessary. After that, add ethanol to add D-α-
The amino acids can be easily separated by a method such as precipitation of the amino acids and filtration of the D-α-amino acids.

The L-α-amino acid amide remaining after separation of the D-α-amino acid can be hydrolyzed with a method known per se, for example, acid or alkali to obtain the corresponding L-α-amino acid. Further, after racemizing the L-α-amino acid amide, the L-α-amino acid amide is circulated to the reaction system together with unreacted DL-α-amino acid, if necessary, to obtain D-α-amino acid amide in a high yield. It is also possible to produce amino acids.

[Examples] Hereinafter, the present invention will be described with reference to Examples, but the present invention is not limited thereto.

Example 1 Glucose 10 g, polypeptone 10 g, and yeast extract 10 g were dissolved in pure water 1 to prepare 100 ml of a medium adjusted to pH 7.0.
Place in a Erlenmeyer flask and sterilize for 20 minutes at 1 kg / cm ² G, and inoculate 1 ml of the culture solution of Rhodococcus erythropolis NR-28 (Microtechnology Research Institute No. 8938) precultured in the same medium. Then, shake culture at 30 ℃ for 48 hours,
The cells were obtained by centrifugation at pm for 10 minutes.

10 ml of a suspension of 20 mg of the above-mentioned cells in 100 ml of pure water in terms of dry cell weight, 10% by weight adjusted to pH 7, 20 ml of DL-valineamide aqueous solution, and various mercapto group-containing compounds 4 × 10 ^-4 M / L 10 ml of the solution was added, and the reaction was carried out at 40 ° C. with shaking, and the change with time in the amount of D-valine produced in the reaction solution was analyzed by high performance liquid chromatography. Table 1 shows the results.

Example 2 The procedure of Example 1 was repeated, except that the medium had the following composition, cysteine was used as the compound having a mercapto group, and the concentration was changed variously.

Glucose 10 g Peptone 5 g Meat extract 1 g Yeast extract 5 g KH ₂ PO ₄ 1 g MgSO ₄ / 7H ₂ O 0.4 g FeSO ₄ / 7H ₂ O 0.01 g MnCl ₂ / 4H ₂ O 0.01 g DL-valine amide 5 g Water The results of 1 pH 7 are shown in Table 2.

Example 3 The procedure of Example 2 was repeated except that the concentration of added cysteine in the reaction solution was 1 × 10 ⁴ M / L, the reaction temperature was 20 ° C., and various strains were used.

 Table 3 shows the results.

Example 4 The procedure of Example 2 was repeated except that the concentration of added cysteine in the reaction solution was 1 × 10 ⁻⁴ M / L and various DL-α-amino acid amides were used as the reaction raw materials. Table 4 shows the results.

[Effects of the Invention] By increasing the activity and stability of D-α-amino acid amide hydrolase by the method of the present invention, many useful D-α from DL-α-amino acid amide can be obtained.
-It has become possible to produce amino acids easily and efficiently.

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Claims (1)

(57) [Claims] (1) The general formula is (Wherein R represents 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, an imidazolyl group or an indolyl group). , D-α
-Acting with an amino acid amide hydrolase, the DL-α-
A method for producing a D-α-amino acid, which comprises allowing a compound having a mercapto group to be present in the reaction solution when producing a D-α-amino acid corresponding to an amino acid amide.