DE1954218B2 - A method for producing protease by aerobically growing a strain of Bacillus - Google Patents

Description

The invention relates to a method for producing protease by aerobically growing a strain of bacillus in a nutrient medium that is customary for this purpose with the temperature and pH value conditions customary for this purpose.

The already known methods for protease production according to the German interpretation 1226 975, French patent 1491 343, U.S. Patents 3,390,054, 3,097,145, and 3,331,751 differ from this Process in that either they have a very long cultivation time, one in terms of pH stability restricted protease and / or a relatively temperature sensitive protease is obtained.

The present invention is based on the fact that the new Bacillus strain, namely the Bacillus Sp. 0-20, on February 20, 1969 at the Fermentation Research Institute Agency of Industrial Science and Technology, Japan, and has the depository number FERM-P No. 270 received has produced protease with an extremely high level of enzymatic activity.

The method of the present invention for producing protease by aerobically growing a strain of Bacillus in a nutrient medium that is customary for this purpose with the temperature and pH value conditions customary for this purpose is characterized in that the Bac'llus Sp. FERM-P No. 270 is used. The effect the temperature or the pH value on the activity of the protease obtained by the present invention is in FIG. 1 and FIG. 2 shown. This strain possesses the following opposite properties that published in the Japanese Journal of Bacteriology, Vol. 23, No. 2, 1968, pp. 145-151 Bacillus strain, to be referred to here as strain B.

Properties strain A strain B

b-11

Generation of hydrogen sulfide + -

c-4

Degradation of mannose - +

c-8

Breakdown of sucrose - +

c-16

Breakdown of lactose - +

65

The properties of strain B result from the cited reference »Japanese Journal of Bacteriology ", Vol. 23, No. 2 1968, pp. 145-151.

This protease-producing strain B is a gram-positive and spore-forming bacillus and possesses many ciliated flagella. The cell size is around 1.5 - 6 μ.

Regarding physiological properties of the strain in the reference "Japanese Journal of Bacteriology", Volume 23, No. 2, 1968, pp. 145 to 151, it should be noted that, according to the information there breaks down any of the sugars fructose, mannose, lactose, glucose, galactose and sucrose and arabinose, Xylose, raffinose and dextrin not decomposed. It also coagulates cow's milk and liquefies gelatin, however, no generation of hydrogen sulfide by him was observed there. Acetylmethyl carbinol is generated and the catalase effect is positive. The microorganism grows with 55 ° C not at all, and growth is good at 30 ° C. These properties became the Microorganism of the reference "Japanese Journal of Bacteriology", Volume 23, No. 2 1968, p. 145 to 151, regarded as a certain species of a strain similar to Bacillus Cereus.

The Bacillus Sp. 0-20 used in accordance with the invention, which was obtained from Fermentation Research on February 20, 1969 Institute Agency of Industrial Science and Technology, Japan, and deposited there the deposit number FERM-P No. 270 is a new variant that consists of a certain strain very similar to Bacillus cereus, which is published in the "Japanese Journal of Bacteriology", Vol. 23, No. 2, 1968, pp. 145 to 151, prepared by irradiation with ultraviolet light became.

The bacillus strain to be used according to the invention can be clearly identified mycologically, as can be seen from the following mycological properties.

a) Growth conditions in the nutrient medium

1. size 1.0 to 1.2 μ5.0 to 5.2 μ,

2. Shape: oval, with an angular end,

3. Spore: oval shaped, 1.0 μ ■ 2.0 to 2.2 μ,

4. Mobility: yes, is peritric,

5. Degree of growth on each medium

(a) Liquid culture:

Broth: Slightly cloudy with deposits, forms a thin film ring that is cloudy not lightened,

Peptone water: Evenly cloudy, forms a weak ring,

(b) Stick culture:
Gelatin culture medium; liquefies quickly,

(c) Plate culture:

Gelatin medium: hydrolyzed in a wide range,

(d) Land culture:
Broth agar medium:

Grows quickly, shiny and grayish-white in color, spread out moist and somewhat swollen.
Glucose-bouillon-agar medium: Grows quickly, gray-white in color, moist, shiny, spread out and slightly denser than the bouillon-agar medium.

Potato slice: Grows quickly, moist and creamy white in color.

6. Surface condition of the colony:
Generally spherical, and homogeneous, coarse berries, gray-white in color.

7. Color of the breed: off-white.

8. (a) pathogenic properties: not established

represents,

b) physiological properties:

1. Growth conditions: pH value 6.8 to 7.2 (optimum), temperature 30 to 32 ° C (optimum), no growth at 50 ° C, aerobic.

2. Gram stain positive

3. Acid resistance -

4. Methyl Rottest +

5. Voges-Proskauer test +

6. Production of indole -f-

7. Production of ammonium ions -Ιδ. Reduction of separating water salt -

9. Production of catalase +

10. Liquefaction of gelatin and
Casein +

11. Production of hydrogen sulfide -f-

12. Hydrolysis of starch ±

13. Utilization of citric acid +

14. Curdling of cow's milk +

15. Reduction of methylene blue .. +

c) utilization of carbon raw material, where a "+" indicates the property of producing acid and a "-" the lack of it Property indicates.

1. Arabinose -

2. Xylose -

3. Glucose +

4. Mannose -

5. fructose +

6. Cerebrose +

7. Maltose +

8. Sucrose -

9. Trehalose +

10. Raffinose -

11. Sorbitol -

12.Glycerin -

13. Inositol

14. Salicine +

15. a-methyl glycoside -

16. Lactose -

17. Inulin -

18. Dextrin -

19. Strength -

20. Cellulose -

The taxonomic position of the strain to be used according to the invention was determined by the comparison the mycological properties with those of other commonly known bacteria according to the method which is described in the "Manual of Determinative Bacteriology" by Berge y (7th edition), with the following Result determined.

Class: Schizomycetes
Order: Eubacteriales
Family: Bacillaceae
Genus: Bacillus

As stated above, the cultivation of the strain to be used according to the invention liquefies in aer Gelatin rod culture very quickly, and by lightly coloring the protoplasm it looks hollow, shows however, the Voges-Proskauer reaction is positive and mobile. In this respect, the Strain of the Bacillus cereus var. Mycoides. Furthermore, the present strain does not generate acid Arabinose and xylose, and he is in the growth

condition and physiological properties very similar to Bacillus cereus.

However, the present strain is apparently a novel strain, which, although the Bacillus cereus very similar, but

is that it has slightly better growth conditions in the glucose-separating water salt culture medium shows and that it has no properties to reduce septic water salt and acid off To generate strength. Meanwhile, the Bacillus produces

cereus in general acids from glycerine and sucrose. In addition, is that used in the invention Strain has been determined to be different from breeding KP 931, which belongs to Bacillus cereus is very similar and in the "Journal of the Agricultural Chemical Society of Japan", Volume 40, No. 6, 1966, pp. 252-256, in the preparation of acids from arabinose, glycerin and sucrose. It has also been determined to be different from the breed that the Bacillus cereus is very similar, which is in the "Japanese Journal of Bacteriology", Volume 23, No. 2, 1968, pp 145 to 151, has been reported in the manufacture of hydrogen sulfide and acids from lactose, mannose and sucrose. From the above findings, the strain used in the invention was identified as identified a novel strain belonging to the genus Bacillus and one similar to Bacillus cereus Variant is.

The nutrient medium used in the invention is prepared in the conventional manner with glucose, Fructose, cerebrose, maltose and other sugars as a raw material for carbon and with soybean cake and the like as a raw material for nitrogen. An inorganic salt can be added such as potassium dihydrogen phosphate, potassium chloride and / or a vitamin as required. The pH of the nutrient medium is in the range from 5.0 to 9.0, preferably 6.7 to 7.0. The bacillus used in the method of the invention is inoculated into the nutrient medium and in at a temperature of 30 to 38 ° C for 12 to 72 hours with shaking or venting and stirring bred. The protease generated in the nutrient medium is precipitated and released by various known ones Purified processes, such as by precipitation with salts or by dialysis processes.

example 1

_{1.4 g of Na 2} HPO ₄ , 1.0 g of KH ₂ PO ₄ , 2.0 g of NaCl, 0.1 g of MgSO ₄ , 6.0 g of acidic sodium glutamate and 10.0 g of glucose were dissolved in 1000 ml of water. to produce a nutrient medium with a pH of 7.0, whereupon the nutrient medium was then sterilized. Bacillus Sp. 0-20 was inoculated into the culture medium obtained and cultured with shaking at 30.degree. C. for 3 days. After the use

When the nutrient medium had been centrifuged, the excess liquid became enzymatic activity measured.

The enzymatic activity was 1050 units per ml of fermentation medium.

For comparison, the one very similar to Bacillus cereus and published in the Japanese Journal of Bacteriology, Vol. 23, No. 2, 1968, pp. 145 to 151, published strain grown in the same manner as in Example 1. The enzymatic activity per ml of the excess liquid obtained was measured. she was 215 units.

Example 2

Bacillus Sp. 0-20 was grown in the same manner as in Example 1 except that the temperature of the nutrient medium was kept at 36 ° C. The enzymatic activity per ml of the excess liquid obtained was measured. It was 3000 units.

1 sheet of drawings

Claims (1)

Claim: Process for the production of protease by aerobically growing a strain of Bacillus in a nutrient medium customary for this purpose with the customary temperature and pH value conditions, characterized in that the Bacillus Sp. FERM-P No. 270 is used.