JPH0315385A - Novel alkali protease apg 501-producing bacterium - Google Patents

Abstract

PURPOSE:To enable to produce an alkali protease APG 501 having physicochemical properties such as a protease activity especially high against sericin by culturing Bacillus S.P 9111-5B strain. CONSTITUTION:Bacillus S.P 9111-5B strain (FERM 8520) separated from soil is cultured in a liquid medium containing starch, etc., as a carbon source, soybean residues, etc., as a nitrogen source and inorganic salts at 20-40 deg.C under an aerobic condition. After the culture is finished, the cells and the residues of the medium are removed and the filtrate is concentrated by an ultrafiltration method at a fraction mol.wt. of 5000 to provide a liquid crude enzyme. The crude enzyme is purified by an ion exchange chromatography, etc., and lyophilized to provide pure alkali protease APG 501 having physicochemical properties: (a) the enzyme has the protease action in an alkaline region, (b) the enzyme has an especially high peculiar protease action on sericin, the optimal pH thereof being 6.5-9 and the optimal temperature thereof being 60-65 deg.C.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a novel alkaline protease APG501-producing bacterium belonging to the genus Bacillus.

Background of the Invention Various alkaline proteases have been known so far.
It is used in a wide range of fields including textiles, detergents, leather processing, and food.

In recent years, in the field of textiles, an enzymatic scouring method using this alkaline protease for scouring silk fabrics has been proposed, and the present inventors have conducted various studies on the alkaline protease used. In the meantime, certain new strains belonging to the genus Bacillus have developed a novel alkaline protease AP suitable for the enzyme scouring.
It was discovered that G501 could be produced, and the present invention was completed.

Disclosure of the invention Novel alkaline protease A produced by the producing bacteria of the present invention
PG501 has the following physical and chemical properties.

(a) Has protease action in the alkaline region; (b)
) It has a particularly high specific protease action against sericin, its optimum pH is 8.5-9, and its optimum temperature is 60-90.
65°C, (c) Stable at pH 5.5 to 11 at 4°C, (d) pH 7. 0, stable up to 50℃, 6
Inactivation begins at 0°C, complete inactivation at 70°C, (e) No metal ions are required for activity expression or stabilization, and activity is significantly inhibited by diisoprobyl fluorophosphate (DFP). ( r) Molecular weight by ultracentrifugation method: 16300, molecular weight by gel filtration method: 19000, (g) 7. In disk electrophoresis on a 5% polyacrylamide gel (pH 9.4), 0. When the migration of Ol% promophenol blue (BPB) is 1, the migration of enzyme protein is 0.14, (h) The isoelectric point by focal electrophoresis is 6.96, (i) Lysine 5, Histidine 3, Arginine 2. Aspartic acid! 4, threonine 13, serine 20, glutamic acid 6,
Proline l1 glycine 17, alanine 20, valine 15
, methionine 2, isoleucine 4, leucine 8, tyrosine 7, phenylalanine 2, tryptophan 0, and cysteine O.

Alkaline protease APG501 has particularly high specificity for sericin, has a relatively small molecular weight,
It is distinguished from known alkaline proteases by its low isoelectric point.

The activity of alkaline protease APG501 is measured as follows.

Silk thread waste from the silk spinning process was heated to 110°C with a bath ratio of 1:18.
Perform hot water elution for 0 minutes, and add this eluate to 275r+n+
A sericin eluate is prepared by quenching with water so that the absorbance at 4.0 is 4.0. This sericin eluate 2. 5
*Qs sample enzyme solution 0. 1 x (l and 0.3 M sodium carbonate-0.3 M sodium bicarbonate buffer (p
H 1 0.0) 0.5112 were mixed and heated to lO at 60°C.
Let it react for a minute. Then, 3.0% of a 10% trichloroacetic acid (TCA) solution was added to the reaction solution. 0 *Q was added to stop the reaction, left in ice water for 30 minutes, and then centrifuged (170
00G)I, Measure the absorbance of the supernatant at 275n. An absorbance of 1 at 275 r+m corresponds to 4.5 micromoles of tyrosine. Under these conditions, the enzyme activity that produces a soluble nitrogen compound corresponding to 1 micromole of tyrosine per minute is defined as 1 unit.

The novel alkaline protease APG501 is an enzyme produced by the novel microorganism belonging to the genus Bacillus of the present invention. The microorganism was isolated by the present inventors from soil collected in Ayabe City, Kyoto Prefecture, and is a representative strain of Bacillus sp.9.
1 1 1-5 B strain is 8520
It has been deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry under the following title:

The mycological properties of Bacillus sp. strain 9111-5B (Feikoken Bacterium No. 8520) are shown below.

Morphological characteristics (1) Cell size, shape vegetative cell 0.3-0.5 μl x 0.7-1.5 μl 1 short rod.

(2) Polymorphism Cells are 0.5 μ friend x 3. The wound may extend.

(3) Motility Has mobility.

(4) Flagella Has circumferential hair.

(5) Spores Oval heat-resistant spores with a shoulder size of 0.4-0.5 μl and 0.7-1.2 μl are formed approximately in the center of the cell.

(6) Durham staining Durham is positive.

(7) Anti-acidity No acid resistance.

Growth status on various media (1) Cultured on broth agar plates Formed circular colonies, pseudoplate-like, with ridges at the edges and center (semi-lenticular). Colonies are smooth, creamy white, and glossy.

(2) Grain agar slant culture. Good growth, rhizoid-like, rough surface, white-brown color, and wrinkles in some areas.

(3) Meat juice liquid culture Forms a fungal membrane and produces turbidity just below the bacterial membrane.

(4) Good growth on the surface of meat juice agar high-layer culture, growth at the puncture site, whitish-brown color, wrinkles, and water droplets.

(5) Meat juice gelatin puncture culture It liquefies in layers in about a week and reaches about 1 cal in 10 days.

Bacterial cells grow on the surface.

(6) Litmus milk After about 30 days, the milk coagulated and there was no litmus discoloration.

physiological properties As shown in Table I.

No.! Table **: Can grow at 16%! No growth of 6.5% or more.

Glycolysis test Table 2 shows the results. Symbols in the table have the meanings indicated.

+: acid production, ±: partial acid production, no diacid production,
No gas generation was observed in either case.

*: Decomposes sugar anaerobically.

Table 2 The above properties of the 9111-5B strain are summarized in Bergey's Manual of Determinative Bacteriology, 8th edition.
Terminal Bacteriology.
Bacillus firmus (B.
acillus firmus).

As a result (1) Bacillus 9111-5B strain can grow on an agar medium even under anaerobic conditions, but Bacillus firmus cannot grow under anaerobic conditions. (2) Bacillus 9
111-5B strain can grow at 52°C, but Bacillus firmus cannot grow at temperatures higher than 45°C. (3) Bacillus 9111-5B strain can grow in a wide pH range of pH 5 to 12, but Bacillus firmus can grow at pH 6.
9111-5 in that it cannot grow unless it is 0 or more.
Strain B constitutes a distinct bacterial species that can be clearly distinguished from Patillus firmus, and was therefore determined to be a new bacterial species belonging to the genus Bacillus.

Thus, the novel alkaline protease APG501 is produced by Bacillus sp.
-5B strain was cultured in a nutrient medium, and APG50 was added to the culture.
APG501 can be obtained by accumulating APG501 and collecting APG501 from the culture. Like other bacteria, the 9111-5B strain can be mutated by artificial mutagenic means, such as by irradiation with ultraviolet light, Co@11, X-rays, etc., and by the use of various mutagenic agents; Stocks too, APG
As long as it has a production capacity of 501, it is within the scope of the present invention. Furthermore, any strain other than the 9111-5B strain and its mutant strains is within the scope of the present invention as long as it is a quiet strain that has APG501-producing ability.

Below, as a representative example, A
The production of PG501 will be explained.

Culture of strain 9111-5B A conventional aerobic culture method using a liquid medium can be adopted for culturing this strain. Various saccharides such as starch, dextrin, and glucose are used singly or in combination as carbon sources for the culture medium. As the nitrogen source, soybean meal, soybean flour, yeast extract, milk casein, corn steep liquor, etc. can be used alone or in combination. others,
Inorganic salts and the like can be added as appropriate. Cultivation is generally carried out under aerobic conditions at 20-40°C and pH 7.0-10.
．． 0 for about 24 to 144 hours.

This culture allows alkaline protease AP to be present in the culture.
G501 is accumulated.

Collection of APG 501 Alkaline protease APG 50 from the prepared culture! For collection and purification of microbial metabolites, methods commonly employed for isolating and purifying microbial metabolites from their cultures are used.

For example, after the completion of culture, bacterial cells and medium residue present in the culture are removed by filtration or centrifugation using a filter aid such as diatomaceous earth, and concentrated by ultrafiltration with a molecular weight of 50 QO to obtain a liquid crude product. Get the enzyme. This crude enzyme is further purified by ion exchange chromatography and gel filtration, if necessary, and lyophilized to obtain the desired purified alkaline protease APG501.

As mentioned above, the obtained alkaline protease APG501 has particularly high substrate specificity for sericin, and is suitably used in the textile industry for enzyme purification of silk fabrics, etc. However, APG501 also has casein It also exhibits protease action on various other proteins such as hemoglobin and hemoglobin under similar conditions, and can be used not only in the textile industry but also in fields similar to known alkaline proteases, such as detergents, leather processing, and foods. I can do it. In addition, sufficient alkaline protease action can be obtained even in the above-mentioned concentrated crude enzyme solution and in the form of crude enzyme, powder, or granular material obtained by known drying methods, and therefore, the crude enzyme is also included in the alkaline protease of the present invention. It is something.

Next, the present invention will be explained in more detail with reference to reference examples and test examples.

Reference example! Soybean casein 0.5%, glucose 0.5%, yeast extract 0.1%, K, HP 0.0.1% and MgSQ. −
Contains 0.02% 7H,0 and pHed with NatCOs
100jIi2 portions of the liquid medium adjusted to a concentration of 9.9 were dispensed into Sakaguchi flasks manufactured by No. 500112 and sterilized. 911 in this medium
1-5B strain was inoculated and cultured with shaking at 135 times/min for 5 days at 30°C.

The cultured limbs are centrifuged at 18,000 G, and the supernatant is concentrated by ultrafiltration with a molecular weight cut-off of sooo to obtain 15 ml of a crude enzyme solution with a molecular weight cut-off of 108 units/jl.

This crude enzyme solution was subjected to cation exchange ion chromatography, then gel filtration using polyvinyl gel,
By performing freeze-drying, the desired purified alkaline protease APG501 1.58-inhibitor in powder form having the above-mentioned physicochemical properties (a) to (i) is obtained.

The attached FIGS. 1 and 2 show the effects of pi-1 and temperature on the activity of the alkaline protease APG501 obtained. These are determined according to the activity measurement method described above.
It is a graph showing the relative activity when the activity was measured by changing p}{ and temperature, respectively, and the maximum activity was taken as 100%. As shown in Figures 1 and 2, alkaline protease APG50 1 has an optimal p level of action on sericin.
H is 8.5-9, and the optimum temperature is 60-65°C.

Furthermore, the attached FIGS. 3 and 4 show the pH stability and temperature stability of the enzymatic activity of the alkaline protease APG501 obtained. p}{stability at 5°C in the pH range of 4 to 11; residual activity after 16 hours of treatment;
The temperature stability was determined by measuring the residual activity after treatment at pH 7 for 10 minutes in the range of 30 to 80° C. according to the activity measurement method described above. As shown in Figures 3 and 4, alkaline protease APG501 has a pH of 5.5 to 11 at 4°C.
It is stable within the pH range of 7. 50℃ at 0
It is stable up to 70°C, starts to deactivate at 60°C, and completely deactivates at 70°C.

Reference Example 2 Defatted soybean meal 0.5%, dextrin 1.0%, corn steep liquor 0.5%, K, HP 0. Ol% and M
g S O a・7H, O O. Contains 02%,
After sterilizing 1.3Q liquid medium adjusted to pH 9 with NatCOa, it is placed in a mini jar and inoculated with strain 9111-5B. Add this to 300r. p. Culture at 30° C. for 3 days while stirring at 0.3 f2/min and aerating at 0.3 f2/min.

The culture solution was filtered using a filter press coated with Celite, and concentrated 18.1 times by ultrafiltration with a molecular weight cutoff of 5000 to obtain 72 iC of crude enzyme solution with 930 units/xl2 (enzyme yield approximately 70%). obtain.

The silk fabric scouring effect of the obtained enzyme was tested.

Test 1 3.0φX19.5C1! An enzyme scouring test was carried out by treating each unscoured silk fabric 29 in a large test tube at a liquid ratio of 1:30 as follows.

(1) Pretreated sodium silicate (18°Be) 49/Q and polyoxyethylene lauryl ether 0. 5 9/
+2 using a treatment solution (pHlO) of 60 μm, 98
Treated at °C for 40 minutes.

(2) Enzyme-treated anhydrous sodium carbonate 0. 5 9/(1, sodium bicarbonate 39/Q and alkaline protease APG
501 Processing solution containing 20,000 units/Q (pH
90) Process at 60°C for 120 minutes using 60ml2.

(3) Post-treatment Sodium bicarbonate 9/Q and boroxyethylene lauryl ether 0. Treatment was performed at 98° C. for 40 minutes using 60×f treatment solution (pH 8.0) containing 59/12.

As shown in the following formula, the desericination rate after each treatment was calculated by taking the reduction rate of the dough weight after each treatment to the initial unscoured dough weight as the desericination rate.
2.4%, 25.7% after enzyme treatment, and 27.0% after post-treatment.
1%, indicating a good scouring effect.

Test 2 Using a 40Q square treatment tank, unscoured raw silk 8609 was
An enzyme scouring test was conducted on each sample at a liquid ratio of 1:35 and the following treatments.

(1) Using the same treatment liquid 3012 as in pretreatment test 1, boiling treatment was performed for 40 minutes under natural convection stirring.

(2) Enzyme-treated anhydrous sodium carbonate 0. Treatment solution (pH 9.0) containing 59/(1, 3.59IQ of sodium bicarbonate and 22,500 units/12 of alkaline protease APG501) was used at 60°C under stirring by bomb circulation at 512/min. Process for 120 minutes.

(3) Boiling treatment for 60 minutes under natural convection stirring using 30ff of the same treatment liquid as in post-treatment test 1.

When the desericinization rate was calculated in the same manner as in Test 1, it was 13.1% after pretreatment, 24.3% after enzyme treatment, and 26.5% after posttreatment, indicating a good scouring effect.

[Brief explanation of drawings]

Figures 1 and 2 show alkaline protease A, respectively.
1 is a graph showing the influence of p}{ and temperature on the activity of PG501. Figures 3 and 4 are graphs showing the effect of pH and temperature, respectively, on the stability of alkaline protease APG501.

Claims (2)

[Claims] (1) A new alkaline protease APG501-producing bacterium belonging to the genus Bacillus and having the following physicochemical properties: (a) having a protease action in the alkaline region; (b) having a protease action specific to sericin; The optimum pH is 8.5-9, the optimum temperature is 60-65.
°C, (c) Stable at pH 5.5 to 11 at 4 °C, (d) Stable up to 50 °C at pH 7.0, inactivation starts at 60 °C, completely deactivated at 70 °C, (e) Does not require metal ions for activity expression or stabilization, and activity is significantly inhibited by diisopropylfluorophosphate (DFP). (f) Molecular weight: 16,300 by ultracentrifugation; molecular weight: 19,000 by gel filtration; (g) 7 In disk electrophoresis (pH 9.4) using .5% polyacrylamide gel, if the migration of 0.01% bromophenol blue (BPB) is 1, the migration of enzyme protein is 0.14. (h) Focal electrophoresis The isoelectric point is 6.96, (i) 5 lysine, 3 histidine, 2 arginine, 14 aspartic acid, 13 threonine, 20 serine, 6 glutamic acid,
1 proline, 17 glycine, 20 alanine, 15 valine
, methionine 2, isoleucine 4, leucine 8, tyrosine 7, phenylalanine 2, tryptophan 0, and cysteine 0. (2) Bacillus sp.
．． ) 9111-5B (Feikoken Bacteria No. 8520), the producing bacterium of item (1) above.