JPH0378992B2 - - Google Patents

Description

Detailed Description of the Invention (Industrial Technical Field) The present invention relates to Streptomyces twillus having the ability to produce D-aminoacylase.
tuirus). D-aminoacylase is an enzyme useful in the production of D-amino acids.

(Prior art) As a method for producing D-aminoacylase, a method using bacteria of the genus Pseudomonas (Nature, 181 , 1225) has been proposed.
(1952)), a method using methanol-assimilating bacteria (Japanese Unexamined Patent Publication No. 55-42534), and a method using Streptomyces genus actinomycetes by one of the inventors, Sugie et al.
-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 simultaneously produced, and the reaction is carried out using collected bacterial cells or crude enzyme waves extracted from the bacterial cells by ultrasonic waves or the like. 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 on improving D-aminoacylase-producing bacteria for the purpose of industrial production of D-aminoacylase, and as a result, D. - Among mutant strains of aminoacylase-producing bacteria, a strain was found that was substantially deficient in the ability to produce L-aminoacylase, an unnecessary enzyme, while maintaining the ability to produce D-aminoacylase, and the present invention was completed. 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.

(Microorganism of the present invention) The mutant strain of the present invention uses a strain belonging to the genus Streptomyces that has the ability to produce D-aminoacylase as a parent culture strain, and undergoes the mutation procedures described herein or other methods. You can get it by using: Specifically, ultraviolet rays, X-ray irradiation, or N
- It can be obtained by treatment with chemical agents such as nitrosoguanidine (abbreviated as NTG) and nitrous acid. Specifically exemplified in the present invention is Streptomyces twillus 0-33, which is a mutagenized strain of Streptomyces twillus IFO13418, which was submitted to the National Institute of Microbiology, Agency of Industrial Science and Technology.
It has been deposited as No. 8446. The morphology of Streptomyces twillus 0-33 is indistinguishable from the parent strain Streptomyces twillus IFO13418. However, Streptomyces twillus 0-33 is L-
It can be clearly distinguished from Streptomyces twillus IFO13418 in that it is substantially deficient in aminoacylase production ability.

(Method for culturing mutant strains) The mutant strains of the present invention are cultivated in the presence of conventional nutritional sources, ie, carbon sources, nitrogen sources, and inorganic salts. The enzyme production inducers include D- or DL-phenylglycine, D- or DL-valine, D-
or a D- or DL-amino acid, such as DL-leucine, or a derivative of a D- or DL-amino acid,
Examples include N-acetyl-D- or DL-phenylglycine. It is also necessary to add organic nitrogen as a nitrogen source, such as polypeptone,
Meat extract, yeast extract, corn steep liquor, etc. may be added as appropriate, and if necessary, about 100 μg of cobalt chloride may be added per 1 ml of the medium. Cultivation is possible in the PH5-8 range, but PH6
It is more desirable to carry out the cultivation within the range of 7 to 7 degrees Celsius, and the culturing temperature can be within the range of 20 to 37 degrees Celsius, but 30 degrees Celsius is more preferable. Culture requires oxygen supply and aeration and agitation.

(Enzyme recovery and reaction method) D-aminoacylase obtained from this strain is mainly produced within the bacterial cells, so the obtained bacterial cells may be washed with water and then recovered as an enzyme preparation, or by conventional methods. The enzyme may be extracted by crushing the bacterial cells or lysing them with lysozyme or the like, and then recovering this as an enzyme agent. To carry out continuous enzymatic reactions with enzyme agents, water-washed bacterial cells can be used as is, or they can be wrapped in polyacrylamide, etc., or enzymes extracted from bacterial cells can be immobilized using conventional methods. It is.

(Effects of the Invention) When producing useful optically active compounds such as D-amino acids using the bacterial strain obtained in the present invention, enzymes extracted from the bacterial cells or any method that uses the bacterial cells as they are for enzymatic reactions can be used. D-amino acids etc. with high optical purity can be obtained, and the industrial effect 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.

Example (a) Mutant strain Streptomyces twillus 0-33 from Streptomyces twillus IFO13418
Manufacturing of.

Streptomyces twillus IFO13418 was cultured on the agar slant medium shown in Table 1, and the grown bacterial cells were
Suspend in saline containing 0.01% Tween 80. Filter this through sterile gauze to obtain a spore suspension (10 ⁶
~ ¹⁰⁷ spores/ml). NTG for this
(concentration 29 μg/ml) and incubate at 30°C for 30 to 90 minutes. Subsequently, the spores were washed twice with physiological saline, then spread on the agar plate medium 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. The obtained bacterial cells were collected and washed with 0.1M phosphate buffer (PH7), and then N-acetyl-L-
React with amino acids at 30°C for 24 hours. The presence or absence of amino acids in the reaction solution from which the bacterial cells were removed was checked using a ninhydrin coloring method to select mutant strains with reduced or defective L-aminoacylase production ability. Furthermore, a secondary evaluation was performed using a reaction using N-acetyl-DL-amino acid as a substrate, and a mutant strain of Streptomyces that maintained a D-aminoacylase production ability equal to or higher than that of the parent strain, and had a significantly decreased L-aminoacylase production ability. - Obtained Twillus 0-33 (Feikoken Bibori No. 8446).

(b) Evaluation of mutant strains 2% soluble starch, 1% maltose, 1% glycerin, 0.5% polypeptone, 0.5 yeast extract
%, meat extract 1.5%, corn steep liquor 1%,
100 ml of nutrient medium (PH7.0) containing 10.5% NaC and 1.5% DL-valine was added to mutant strains 0-33
8446) and inoculated with spores at 30℃ and 110 revolutions per minute.
Cultured for 1 day. After collecting bacterial cells from the culture solution and thoroughly washing them with physiological saline, add them to 0.1M phosphate buffer (PH7.0).
and disrupt the cells using an ultrasonic disruptor (19.5KHz). Cell debris was removed by centrifugation and D-
A crude enzyme solution of aminoacylase was obtained. The enzymatic reaction
Using 0.05 M N-acetyl-DL-valine as a substrate and a crude enzyme solution equivalent to 1 ml of culture solution,
℃ for 24 hours. Analysis of reaction products is
This was done using HPLC. The analytical methods are a method using an optical resolution column WH manufactured by Daicel Chemical Industries, and a chiral mobile phase method (J.Am.Chem.Soc. 102 ,
5115 (1980)). As a result, after 24 hours of reaction, N-acetyl-D-valine
99.5% was converted to D-valine. On the other hand, N-acetyl-L-valine hardly reacted and only 0.4% was converted to L-valine. The analysis results using the chiral mobile phase method are shown in Figure 1. The D-aminoacylase 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.

On the other hand, spores of the parent strain Streptomyces twillus IFO13418 were inoculated into 100 ml of the same culture medium as above, and cultured for 4 days at 30°C and 110 revolutions per minute. A crude enzyme solution was obtained from this in the same manner as above. Using this crude enzyme solution corresponding to 1 ml of the culture solution, a reaction was carried out at 30° C. for 24 hours using 0.05 M N-acetyl-DL-valine as a substrate. Analysis of the reaction product revealed that 90% of N-acetyl-D-valine had been converted to D-valine after 24 hours of reaction. On the other hand, N-acetyl-L-valine is 48.6%
was converted to L-valine. The analysis results using the chiral mobile phase method are shown in Figure 2.

[Brief explanation of drawings]

FIG. 1 shows a chromatogram analyzed by HPLC of an enzyme reaction solution obtained using Streptomyces twirus 0-33. FIG. 2 shows a chromatogram analyzed by HPLC of an enzyme reaction solution obtained using Streptomyces twirus IFO13418. Table 1 Yeast extract 1g Meat extract 1g NZ amine type A 2g Maltose 10g Agar 20g Distilled water 1 PH=7.3 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 PH=7.0

Claims (1)

[Claims] 1 D-aminoacylase producing ability and L
- Streptomyces twillis characterized by being substantially deficient in aminoacylase producing ability.