DE1954218A1 - Manufacturing process of protease - Google Patents

Description

DR.-ING. DIPL-ING. Q. RIEBLING PATENT ADVOCATE Your message from My · message from

Otsuka Kagaku Yakuhin Kabushiki Kaisha No. 1o, Bungomachi, Higashi-ku, Oe aka - shi / Japan I my mark
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899 Lindau (Bodensee) Rennerle 10 Pottfach 365

October 28, 1969

Manufacturing process of protease

This invention relates to a method of making protease and, more particularly, to a method of making of protease by cultivating a specific novel microorganism belonging to the genus Bacillus heard.

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Telegraph: 05 4374 patent d

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Office tent

by appointment

Bank account:

Bayer. Staatibank Lindau (B) No. 1562

Postal checking account: Munich 2 * 5 25

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1-95A218

A main object of the invention is to provide protease with a high degree of enzymatic activity and a Method of making the same to provide «The above and other objects of the invention, including some from US Pat The following description can be obtained by breeding that belonging to the genus Bacillus Bacillus Sp.o-2o in a breeding medium in order to protease generate and the generated protease from the nutrient medium collect.

The present invention is based on a new discovery, that the Bacillus Sp. o-2o has a useful property has by being able to protease with a to generate extremely high levels of enzymatic activity, for comparison with other generally known species that belong to the genus Bacillus.

The Bacillus Sp. O-2o used in the invention is a novel variant which is produced by irradiating a certain species of a strain very similar to Bacillus Cereus with ultraviolet light and has been published in "Japanese Journal of Bacteriology" Vol. 23 No. 2 (1968), page 145 to 151. This type of bacillus Sp. a-2o was deposited with the accession number FERIB-P no. 27o in "fermentation Research Institute of Agency of Industrial Science and Technology, Japan, on 2o. February 1969 .

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The mycological properties of this breed are like follows:,

a) Growth conditions in the nutrient medium 1) Size 1, o to 1, -2. . ^ χ 5, ρ to 5, .2A.

2.). Shape; oual with angular end 3.) Spore: shaped oual Ι, ο / ίχ 2, ο to 2.2 Aa, 4.) Mobility: yes, is peritrich 5.) Gred of growth on any nutrient medium

(a) Liquid culture

Broth: Slightly cloudy with. Deposit} forms a thin film ring,

the cloudiness is not lightened Peptonu / assBr: Evenly cloudy, forms a weak one

(b) Stick culture: -

Gelatin medium; v / superfluous quickly

(c) Wine cellar culture:

Gelatin medium: hydrolyzed to a large extent Area,

(d) Land culture:

Broth agar — medium: grows quickly, shiny and of grayish-white color, spread out damp and etuias schujollen.

Glucose Broth AgaT Medium; Grows quickly, from gray-iueisser Color, moist, shiny, spread out, wetted and somewhat denser than the Bauillon agar culture medium.

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Potato slice: Grows quickly, moist and creamy.

white is the color
6.) Surface condition of the colony:

Generally spherical and homogeneous, coarse berries, gray-u / ice-cream in color,
7.) Color of the breed: gray-u / ice-cream

8.) Pathogenic properties: not determined (b) physiological property

1.) Growth conditions: pH 6.8 to 7.2 is the optimum, temperature 3o to 32 ⁰ C is the optimum and no growth at 50 ° C and is aerobic. 2o) Gram stain positive

3.) Acid resistance -

4.) Methyl Rottest +

5o) Voges-Proskauer test + 6.) Production won indole + 7.) Production won ammonium + 8.) Reduction won vaginal

luassersalt -

9.) Production won catalase +

10) l / liquefaction of gelatin
and Kaseion +

11) Production of Hydrogen Sulphide +

12) hydrolysis of starch + _

1-3) Use of citric acid +

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ORIGINAL INSPECTED

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14) Curdling of cow's milk +

15) Reduction υοή methylene blue +

c) Use of carbon starting material, wherein a

"+" indicates the property of generating υοη acid and a

ii_ii indicates the lack of this property.

1o) arabinose -

2.) Xylose

3.) Glucose +

4.) Mannose -

5.) Fruktpse +

6.) Cerebrose +

7.) Maltose +

8.) sucrose -

9.) Trehalose +

10) RaffinosB -

11) sorbitol

12) glycerin -

13) inositol -

14) salicine +

15) © CiKiethylglykosid -

16) lactose ~

17) inulin -

18) dextrin -

19) Strength -

20) cellulose. -

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The taxonomic position of the present breed ujurde determined by comparing their mycological properties with those of other well-known people Bacteria according to the method described in Bergey's Manual of Determinative Bacteriology (7, edition) with the following result:

Class: Schizomycetes Order: Eubacterial Family: Bazillakae Genus: Bacillus

Ulis stated above, the culture of the invention liquefies very quickly and in gelatin rod culture by lightly coloring the protoplasm it looks hollow, but shows itself in the Voges Proskauer reaction positive and mobile. In this respect it differs from the Bacillus Cersus var, Hflycoidas. Also produced the present cultivation does not contain any acid from arabinose and xylose and it is in the UJachetum condition and the physiological properties of the Bacillus cereus very much similar. However, the present breed is obvious a novel breed which is very similar to, but different from, the Bacillus Cereus in terms of the facts, that they have somewhat better growth conditions in the glucose

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Separating water salt medium shows and that it has no properties of reducing separating water salt and producing acidity from starch, whereas the Bacillus cereus generally produces acids from glycerol and sucrose. Moreover, "the breed used in the invention is found to be different from the KP 931 breed, which is very similar to Bacillus Cereus, and in the" Journal of the Agricultural Chemical Society of Japan "Volume 40, No. 6 (1966) page 252 to 256 was published, in the production of acids from arabinose, glycerine and sucrose and is also determined differently from the breeding, which is very similar to the Bacillus Cereus, soft in "Japanese Journal of Bacteriology" Volume 23, Mr «. 2 (1968), pages 145 to 151, was reported in the production of hydrogen sulfide and acids from lactose, mannose and sucrose. From the above findings, the breed used in the invention was determined to be a novel breed leading to dam Genus Bacillus belongs and is an ari ante similar to Bacillus Cereus and was recently infected with Bacillus Sp o-2o.

The nutrient medium used in the invention is prepared in the conventional way with glucose, fructose, Cerebrose, maltose and other sugars as a starting material

ORIGlNAL BATHROOM

for carbon and with soybean cake and the like as a raw material for nitrogen. It can be a. inorganic salt can be added, u / ie for example Potassium dihydrogen phosphate, potassium chloride etc. and / or a vitamin as required. The pH of the soil is adjusted is in the range of 5, o to 9, o, preferably 6.8 to 7, o.

ψ According to the method of the invention, the Bacillus Sp o-2o is inoculated into the nutrient medium, and the culture is cultured at a temperature of 3o to 38 ° C for 12 to 72 hours by the shaking or aerating agitation culture method to thereby produce protease. The protease accommodated and generated in the nutrient is excreted and purified by various known methods such as excretion from salts or the dialysis method. If the medium used is thin, concentration is carried out with ion exchange resin or under reduced pressure =

For a better understanding of the invention, examples are given below in which the enzymatic activity

C as.FR.B.
(PU)

T. t y r.

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the protease obtained, which produces a non-protein substance of folin color corresponding to a Y won tyrosine in one minute, is represented as a unit of enzymatic activity when the milk-casein-based protease to be examined is treated at 30 C for 10 minutes and the absorbency measured ujird of soluble in trichloroacetic acid substance at a wavelength of 660 AL.

Example 1 :

In 1000..ml water 1.4 grams Na ₂ HPO *, 1, o grams KH ₂ PO ₄ were, 2, o grams NaCl, o, 1 gram IYl ₉ SO _4, 6.0 grams of sodium glutamate and acidic 1o.o Grams of glucose dissolved to create a medium with a pH uiart of 7, o, whereupon the medium was then sterilized. Bacillus Sp o-2o was inoculated into the resulting culture medium and cultured with shaking at 30 ° C. for three days. Folin color measurement method B. 'The enzymatic activity (PU) v ^ i, * per ml. Was 1o5o _e

For comparison, the one very similar to the Bacillus Cereus and in "Japanese Gournal of Bacteriology", Vol. 23, No. 2 (1968)

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Pages 145 to 151 published in the same manner grown as in Example 1. The enzymatic activity per ml of the excess liquid obtained was measured with the result (PU) y ^ f "* * of 215.

Example 2:

Bacillus Sp. O-2o ui was grown in the same manner as in Example 1 with the exception that the temperature of the nutrient medium was kept at 35 ^° C. The enzymatic activity per ml of the excess liquid obtained was measured with the result (PU) “, ^a ? * * * Of 3,000.

Example 3:

In this example, a nutrient medium with a pH of 7.0 was used which contained 150 grams of glucose, 500 grams of alkali extract, the 500 grams of defatted soy cake extract, 50 grams of KH ₂ POx and 20 grams of CaCO ₃ dissolved in

Contained 10 liters of water. After the nutrient medium is sterilized the Bacillus Sp o-2o was inoculated into the culture medium

and grown by the BBlüftungs-stirring method at 37 ⁰ C for 17 hours at a rate of ventilation of 1 liter per liter of the culture medium and a rotation speed of 3oo Upot "From the obtained cultured product

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Murde the soil centrifugally separated, and the enzymatic activity per ml. excess liquid was measured which PU p5 ^{s "rR" B} · ^ _0n 6000 sulfate at 5 ⁰ C showed the protease salt out. The salted-out product was dissolved in 0.02 moles of calcium. '

The excess liquid was saturated with ammonium sulfate at 5 C to salt out the protease. The salted-out product was dissolved in _{0.02 mol calcium acetate and then dialyzed overnight at 3 C with ο λ} ο2 mol calcium acetate. To the dialyzed liquid was further added acetone 0.6 times the volume of the liquid, and after removing the generated liquid precipitate, acetone was added in an amount 2.5 times the liquid volume. The precipitate thus obtained was frozen to dry it, thereby obtaining a crude enzyme product. From one liter of the excess liquid, 600 grams of protease powder was obtained. The enzymatic activity per mg of the obtained protease was measured with the result

(PU) Cas.FR-.B.
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Claims

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Claims (4)

Claims π · l / experienced in the production of protease, characterized by breeding of the Bacillus Sp. o-2o, which belongs to the genus Bezillus, in a culture medium to produce protease and collecting the protease from the culture medium. 2. The method according to claim 1, d a du r c h characterized in that the pH value of the nutrient floor is in the range from 5, ο to 9, ο. 3. The method according to claim 2, characterized characterized in that the pH Ulert des The nutrient medium is in the range of 6.8 to 7, ο. 4. The method according to claim 1, characterized g e k a η η ζ calibrates that the temperature of the Nutrient medium in the range of 3o to 38 ° C. 009820/1573