JPS62126976A - Production of d-aminoacylase - Google Patents

Abstract

PURPOSE:To obtain D-aminoacylase, by cultivating a variant strain, obtained by subjecting a microorganism of the genus Streptomyces having the ability to produce D-aminoacylase and deficient in the ability to produce L- aminoacylase to variation treatment in a nutrient culture medium. CONSTITUTION:A microorganism, belonging to the genus Streptromyces, substantially deficient in the ability to produce L-aminoacylase and having the ability to produce D-aminoacylase is cultivated in a nutrient culture medium to produce and accumulate D-aminoacylase, which is collected. The microorganism to be used in the above-mentioned cultivation may be any one obtained by subjecting a microorganism, belonging to the genus Streptomyces and having the ability to produce D-aminoacylase as a parent strain to ordinary variation inducing operation, e.g. irradiation with ultraviolet rays or X-rays or treatment with a chemical agent, e.g. N-nitrosoguanidine or nitrous acid, etc., and selecting a strain deficient in L-aminoacylase from the strains subjected to variation treatment.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a method for producing D-aminoacylase. More specifically, a mutant strain deficient in the ability to produce mono-aminoacylase obtained by mutating a microorganism of the genus Streptomyces that has the ability to produce D-aminoanlase is cultured in a nutrient medium to produce and accumulate D-aminoacylase. Concerning how to collect. This invention aims to provide a method for producing enzymes that is extremely effective for, for example, producing D-amino acids with high optical purity, which are required in recent years for applications such as antibiotic side chains and peptide medicines.

(Prior Art) As a method for producing D-aminoacylase, a method using bacteria of the genus Pseudomonas (Nature), 181.
1225 (1952)), the method using facultative methanol-assimilating bacteria (Japanese Patent Application Laid-Open No. 55-42534), and Streptomyces (S.
method using actinomycetes of the genus treptoBces (Special Ij
FJ 1977-59092) is known.

(Problems to be Solved by the Invention) However, all of these methods have problems with D-selectivity. That is, when D-aminoacylase is produced using these microorganisms, L-aminoacylase is produced at the same time, and a reaction using collected microorganisms or a crude enzyme solution extracted from the microorganisms by ultrasound, etc. The optical purity of the target product obtained is not fully satisfactory.

(Means for Solving the Problems) In view of the above circumstances, the present inventors conducted intensive studies to improve D-aminoacylase producing bacteria for the purpose of industrial production of D-aminoacylase, and as a result, Streptomyces spp. Among the mutant strains of D-aminoacylase-producing bacteria, we found a strain that substantially lacks the ability to produce mono-aminoacylase, which is an unnecessary enzyme, while maintaining the ability to produce D-aminoacylase, and completed the present invention. Until now, there have been no reports on the breeding of D-aminoacylase-producing bacteria, and this was obtained for the first time by the present inventors.

(Microorganisms used) The microorganisms used in the present invention are D-aminoacylase-producing bacteria belonging to the genus Streptomyces as a parent strain, and are subjected to conventional mutagenesis procedures such as ultraviolet rays, X-ray irradiation, or N
- Any strain obtained by treating with chemical agents such as nitrosoguanidine (abbreviated as NTG) or nitrous acid and selecting a strain lacking L-aminoacylase activity from the mutagenized cells can be used. It is possible, L-
It can be distinguished from the bacteria used in the prior art by lacking aminoacylase activity. A specific example of a suitable strain is Streptomyces twillis.
cs tuirus) I F Ol :3418 i
Examples include Streptomyces filus 0-33G & Oken Bacteria No. 8446), which was derived from Streptomyces filus 0-33G and monoaminoanolase-deficient mutants derived from p et al.

(Mutation induction method) Next, the method for selecting mutant strains used in the present invention will be shown in the following experimental examples.

Experimental example: Streptomyces twillis IFO13/118
Cultured on the agar slant medium shown in the table, the grown bacterial cells were 0.0
Suspend in physiological saline containing 1% Tween 80. Filter this through sterile gauze to obtain a spore suspension (I08-107/
ml of spores).

Add NTG to this (a degree 29 u g/ml),
Incubate for 30-90 minutes at 0°C. Subsequently, the spores were washed twice with physiological saline, then spread on the agar plates shown in Table 1, and cultured at 30°C for 6 to 12 days. The grown colonies are cultured in the liquid medium shown in Table 2 at 30°C for 2 to 4 days. Collect the obtained bacterial cells and place in O, 1M phosphate buffer 1
← (pH 7.0), and then reacted with N-acetyl-thi-amino acid at 30°C for 24 hours. Mutant strains with decreased or deficient L-aminoacylase production ability were selected by checking the presence or absence of amino acids in the reaction solution from which the bacterial cells were removed. Furthermore, a secondary evaluation was performed using a reaction using N-acedel-DL-amino acid as a substrate, and a mutant strain was obtained that maintained D-aminoacylase production ability equivalent to or higher than that of the parent strain, and had a significantly reduced L-aminoacylase production ability. . This strain was deposited as Streptomyces twirus 0-33 (Feikoken Bacteria No. 8446).

(Method for culturing mutant strains) The mutant strains of the present invention are cultivated in the presence of conventional nutrients, ie, carbon sources, nitrogen sources, and inorganic salts. This enzyme production inducer includes D- or DL-phenylglycine, D- or DL-valine, D- or DL-leucine, etc.
- or DL-amino acids, or derivatives of D- or DL-amino acids, such as N-acetyl-D- or DL-
Examples include phenylglycine. In addition, it is necessary to add organic nitrogen as a nitrogen source, such as polypeptone, meat extract, yeast extract, corn steep liquor, etc., and if necessary, add 100 μg per fml of culture medium.
A certain amount of cobalt chloride may be added. Culture is pI-1
The pH can range from 5 to 8, but it is more desirable to have a pH of 6 to 7, and the culture temperature can range from 20 to 37°C, but 30°C is more desirable. Culture requires oxygen supply and aeration and agitation.

(Enzyme recovery and reaction method) Since the D-aminoanlase obtained in the present invention is mainly produced within bacterial cells, the obtained bacterial cells may be washed with water and then recovered as an enzyme preparation, or The enzyme can be extracted by crushing the bacterial cells or lysing them with lysozyme, etc., and recovering this as an enzyme agent.

To perform continuous enzymatic reactions with enzyme agents, it is best to use water-washed bacterial cells as they are, or it is possible to enclose them in polyacrylamide, etc., or to use them after immobilizing enzymes extracted from the bacterial cells using conventional methods. be.

(Effect of the invention) ■] - For the production of useful optically active compounds of the amino acid list (r
The greatest drawback to the industrial production of mono-aminoacylase, the intracellular aminoacylase, was overcome by breeding the bacterial strain. By using the mutant strain of the present invention, it is possible to easily obtain highly purified D-aminoacylase activity by extracting the enzyme from the bacterial cells or using the bacterial cells as they are for enzymatic reactions, resulting in industrial effects. is extremely large.

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

(Margin below) Table - Table Yeast extract 1g Meat extract 1g NZ amine type A 2g Maltose 10g Agar 20g Distilled water B.

Table 2 Soluble starch 20g Maltose 10g Glycerin 10g Polypeptone 5g Yeast extract 5g Meat extract 15g Corn steep liquor 10g Salt 5g DL-valine 15g Distilled water 1/2 Example 1 Soluble starch 2%, maltose 1%, glycerin 1%
, polypeptone 0.5%, yeast extract 0.5%, meat extract 1.5%, corn steep liquor 1%, NaC10,
Nutrient medium containing 5% and 1.5% DL-valine (pi-1
Spores of mutant strain 0-33 (Feikoken Bacteria No. 8446) were inoculated into 7.0) lOOml and cultured at 30° C. and 110 revolutions per minute for 4 days. After collecting bacterial cells from the culture solution and thoroughly washing them with physiological saline, they were added to a 0.1M phosphate buffered solution (pl-17,
0) and disrupt the cells using an ultrasonic disruptor (19.5KH2). Cell debris was removed by centrifugation to obtain a crude enzyme solution of D-aminoacylase. Enzyme reaction is 0.05
Culture solution 1 using N-acetyl-DL-valine of M as a substrate.
mH,: Using the corresponding crude enzyme solution, the reaction was carried out at 30°C for 24 hours. Analysis of the reaction product was performed by HPLC. The analysis method is optical resolution column W manufactured by Daicel Chemical Industries.
The method using H and the chiral mobile phase method (
J, Am, Chew, Soc, I O2
Two methods were used: ゜5115 (l 980)).

Sono result, 24 o'clock ItJ W, after fd, N 7
99.5% of cetyl-D-valine was converted to D-valine. On the other hand, N-acetyl-valine hardly reacted, and only 0.4% was converted to valine.

The analysis results using the chiral mobile phase method are shown in Figure 1. The D-aminoanlase activity was 37 units when the enzyme activity in 1 ml of the culture solution to produce 1 μmol of D-valine per hour is defined as 1 unit.

Comparative Example 1 Spores of the parent strain Streptomyces twillus IPO13418 strain were inoculated into 100+ nl of the same nutrient medium as in Example I, and cultured at 30° C. for 4 days at 110 revolutions per minute. A crude enzyme solution was obtained in the same manner as in Example 1. 0.05
Using N-acetyl-DL-valine of M as a substrate, a reaction was carried out at 30° C. for 24 hours using a crude enzyme solution corresponding to 1+nl of the culture solution.

Analysis of the reaction products was carried out in the same manner as in Example I. 2
After 4 hours of reaction, 90% of N-acetyl-D-valine was D
- Converted to valine. On the other hand, 6% of N-acediyl-valine td 4 B was converted to L-valine. The analysis results using the chiral mobile phase method are shown in Figure 2.

Example 2 Mutant strains 0-33 were cultured in the same nutrient medium as in Example 1, and a crude enzyme solution was obtained in the same manner. 1 m of culture solution using 0.05M N-acetyl-DL-phenylglycine as a substrate.
A reaction was carried out at 30° C. for 24 hours using a crude enzyme solution corresponding to 1 ml. Analysis of the reaction product was carried out in the same manner as in Example 1. After 24 hours of reaction, 100% of N-acetyl-D-phenylglycine had been converted to D-phenylglycine.

On the other hand, N-acetyl-phenylglycine did not react at all.

Comparative Example 2 A crude enzyme solution was obtained in the same manner as in Comparative Example 1. 0.05M N
-Acetyl-DL-phenylglycine was used as a substrate and a crude enzyme solution corresponding to fml of the culture solution was used to react at 30°C for 24 hours. Analysis of the reaction product was carried out in the same manner as in Example 1. After 24 hours of reaction, 84.8% of N-acetyl-D-phenylglycine was converted to 1]-phenylglycine 1
Turn around.

On the other hand, N-acetyl-L-phenylglinone is 13.3
% was converted to 17-phenylglycine.

Example 3 Mutant strains 0-33 were cultured in the nutrient medium shown in Example 1, and bacterial cells were collected. Furthermore, after thoroughly washing the bacteria,
An enzymatic reaction was carried out at 30° C. using 0.05 M N-acetyl-DL-valine as a substrate and a bacterial cell suspension equivalent to 1 ml of the culture solution. After 10 hours of reaction, 86% of N-acetyl-D-valine was converted to D-valine. On the other hand, N-acetyl-L-valine did not react at all.

[Brief explanation of drawings]

Figure 1 shows a chromatogram of the enzyme reaction solution obtained in Example 1 analyzed by HPLC. Figure 2 shows the I-I P L of the enzyme reaction solution obtained in Comparative Example 1.
This figure shows a chromatogram analyzed by C.

Claims (1)

[Claims] A microorganism belonging to the genus Streptomyces that is substantially deficient in the ability to produce L-aminoacylase and has the ability to produce D-aminoacylase is cultured in a nutrient medium to produce and collect D-aminoacylase. A method for producing D-aminoacylase.