JPH0523741B2 - - Google Patents

Description

[Detailed description of the invention]

FIELD OF THE INVENTION The present invention relates to a novel alkaline protease APG501 producing strain belonging to the genus Bacillus. BACKGROUND OF THE INVENTION Various alkaline proteases have been known and are used in a wide range of fields such as textiles, detergents, leather processing, and foods. 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, we have discovered that a certain new strain belonging to the genus Bacillus produces a novel alkaline protease APG501 suitable for the enzyme refining, and have completed the present invention. Disclosure of the Invention The novel alkaline protease APG501 produced by the production strain of the present invention has the following physicochemical properties. (a) Has a protease action in an alkaline region; (b) Has a highly specific protease action against sericin; its optimum pH is 8.5-9; its optimum temperature is 60-65°C; (c) 4 ℃, stable in the pH range of 5.5 to 11 (d) At PH 7.0, stable up to 50℃, inactivation starts at 60℃, completely deactivated at 70℃ (e) Metals are used for activity development and stabilization. Does not require ions and activity is significantly inhibited by diisopropylfluorophosphate (DFP); (f) molecular weight 16,300 by ultracentrifugation; molecular weight 19,000 by gel filtration; (g) disc electrolysis with 7.5% polyacrylamide gel. In electrophoresis (PH9.4), if the migration of 0.01% bromophenol blue (BPB) is 1, the migration of enzyme protein is 0.14, (h) Isoelectric point by focal electrophoresis is 6.96, (i) Lysine 5, Histidine 3, arginine 2, aspartic acid 14, threonine 13, serine 20, glutamic acid 6, proline 1, glycine 17, alanine 20, valine 15, methionine 2, isoleucine 4, leucine 8, tyrosine 7, phenylalanine 2, tryptophan 0 , and has an amino acid composition of cysteine 0. Alkaline protease APG501 is distinguished from known alkaline proteases in that it has a particularly high specificity for sericin, a relatively small molecular weight, and a low isoelectric point. The activity of alkaline protease APG501 is measured as follows. Silk thread waste from the silk spinning process is heated at 110℃ at a bath ratio of 1:18.
Perform hot water elution for 60 minutes at
Prepare a sericin eluate by adjusting the absorbance at 4.0 with water. This sericin eluate
2.5 ml, sample enzyme solution 0.1 ml and 0.3 M sodium bicarbonate - 0.3 M sodium bicarbonate buffer (PH
10.0) Mix 0.5ml and react at 60℃ for 10 minutes.
Then, add 10% trichloroacetic acid (TCA) to the reaction solution.
Stop the reaction by adding 3.0 ml of the solution and place in ice water for 30 min.
After standing for a minute, centrifuge (17000G) and remove the supernatant.
Measure the absorbance at 275 nm. An absorbance of 1 at 275 nm 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 strain belonging to the genus Bacillus of the present invention. This strain was isolated by the present inventors from soil collected in Ayabe City, Kyoto Prefecture, and was named Bacillus sp. It has been deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry. The mycological properties of Bacillus sp. 9111-5B strain (Feikoken Bacterium No. 8520) are shown below. Morphological characteristics (1) Cell size and shape Vegetative cell 0.3-0.5 μm x 0.7-1.5 μm, short rod. (2) Pleomorphism Cells may grow to 0.5μm x 3.0μm. (3) Motility Motility exists. (4) Flagellum Has pericilla. (5) Spores Oval heat-resistant spores measuring 0.4-0.5 μm x 0.7-1.2 μm are formed approximately in the center of the cell. (6) Gram staining Gram positive. (7) Anti-acidity No anti-acidity. Growth status on various media (1) Broth agar plate culture Forms circular colonies, rhizoid-like, with ridges at the edges and center (semi-lenticular). Colonies are smooth, creamy white, and glossy. (2) Juicy agar slant culture: Good growth, rhizoid-like, rough surface, whitish-brown color, with wrinkles in some areas. (3) Meat juice liquid culture Forms a bacterial membrane and creates turbidity just below the bacterial membrane. (4) Meat juice agar high-rise culture Good growth on the surface, growth at the puncture site, whitish-brown color, wrinkles, and water droplets. (5) Meat juice gelatin puncture culture The gelatin liquefies in a layered form in about a week, and reaches a thickness of about 1 cm in 10 days.
Bacterial cells grow on the surface. (6) Litmus milk No milk coagulation or litmus discoloration after about 30 days. Physiological properties As shown in Table 1.

【table】

[Table] *: Decomposes sugar anaerobically.
**: Can grow at 16%, does not grow at 16.5% or higher.
Glycolysis test As shown in Table 2. The symbols in the table have the following meanings. +: Acid production, ±: Partial acid production, -: No acid production. In any case, no gas generation was observed.

[Table] The above properties of the 91110-5B strain were
Bergey's Manual of Determinative Bacteriology, 8th Edition
The properties of Bacillus firmus, which is a known species of the genus Bacillus described in Determinative Bacteriology, 8th edition, were compared. Results (1) Bacillus 9111−
Although the 5B strain can grow on agar medium under anaerobic conditions, Bacillus firmus cannot grow under anaerobic conditions. (2) Bacillus 9111-5B 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 5 to 12, but Bacillus firmus cannot grow unless the pH is above 6.0, so the 9111-5B strain can be clearly distinguished from Bacillus firmus. It constitutes a distinct bacterial species, and was therefore determined to be a new bacterial strain belonging to the genus Bacillus. Thus, the novel alkaline protease APG501
is a newly produced strain of the present invention, Bacillus S.
P.I. 9111-5B strain was cultured in a nutrient medium, APG501 was accumulated in the culture, and APG501 was extracted from the culture.
Obtained by collecting. The production of APG501 using the 9111-5B strain will be described below. Cultivation of strain 9111-5B For culturing this strain, a normal aerobic culture method using a liquid medium can be adopted. 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, cornstarch liquor, etc. can be used alone or in combination. Others, as appropriate.
Inorganic salts etc. can be added. Cultivation is generally carried out under aerobic conditions at 20-40°C and pH 7.0-10.0 for about 24-144 hours. This cultivation causes alkaline protease APG501 to accumulate in the culture. Collection and purification of APG501 Alkaline protease APG501 from 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 culturing, the 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 cutoff of 5000 to form a liquid crude product. Get the enzyme. If necessary, this crude enzyme is further purified by ion exchange chromatography and gel filtration, 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., but APG501 also has casein It also exhibits protease activity against 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. can. 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 1 Soybean casein 0.5%, glucose 0.5%, yeast extract 0.1%, K ₂ HPO ₄ 0.1% and MgSO ₄ 7H ₂
Dispense 100 ml of a liquid medium containing 0.02% O and adjusted to pH 9 with Na ₂ CO ₃ into a 500 ml Sakaguchi flask.
Sterilize. Inoculate the 9111-5B strain into this medium and
Culture with shaking at 135 times/min for 5 days at ℃. The culture solution is centrifuged at 18,000 G, and the supernatant is concentrated by ultrafiltration with a molecular weight cutoff of 5,000 to obtain 15 ml of a crude enzyme solution containing 108 units/ml. This crude enzyme solution was subjected to cation exchange ion chromatography, then gel filtration using a polyvinyl gel, and freeze-dried to obtain the above-mentioned results.
1.58 mg of the desired purified alkaline protease APG501 in powder form having the physicochemical properties shown in (a) to (i) is obtained. The attached FIGS. 1 and 2 show the influence of PH and temperature on the activity of the alkaline protease APG501 obtained. These are graphs showing the relative activity when the activity was measured by varying the pH and temperature according to the activity measurement method described above, and the maximum activity was taken as 100%. As shown in Figures 1 and 2, alkaline protease APG501 has an optimal pH of 8.5-9 and an optimal temperature for its action on sericin.
The temperature is 60-65℃. Furthermore, the attached FIGS. 3 and 4 show the PH stability and temperature stability of the enzymatic activity of the alkaline protease APG501 obtained. PH stability is PH4-11
The residual activity after treatment for 16 hours at 5℃ in the range of
In addition, the temperature stability is 10 at PH7 in the range of 30 to 80℃.
The residual activity after treatment for 1 minute was measured according to the activity measurement method described above. As shown in Figures 3 and 4, alkaline protease APG501 is stable at 4°C in the pH range of 5.5 to 11, and at pH 7.0.
It is stable up to 50°C, starts to deactivate at 60°C, and is completely inactivated at 70°C. Reference example 2 Defatted soybean meal 0.5%, dextrin 1.0%, corn staple liquor 0.5%, K ₂ HPO ₄ 0.1% and
Contains _MgSO4.7H2O 0.02 _% , _PH with _Na2CO3
After sterilizing the liquid medium 1.3 adjusted to 9111-5B strain, put it into a mini jar and inoculate it with the 9111-5B strain. this,
While stirring at 300rpm and venting at 0.3/min,
Culture at 30°C for 3 days. Transfer the culture solution to a filter coated with Celite.
Filtered with a press and ultrafiltrated with a molecular weight cutoff of 5000.
Concentrate 18.1 times to obtain 72 ml of crude enzyme solution with a concentration of 930 units/ml (enzyme yield: about 70%). The silk fabric scouring effect of the obtained enzyme was tested. Test 1 Using a large test tube of 3.0φ x 19.5cm, an enzyme scouring test was carried out by treating 2 g of unscoured silk fabric at a liquid ratio of 1:30 as follows. (1) Pretreatment Treated at 98°C for 40 minutes using 60ml of a treatment solution (PH10) containing 4g of sodium silicate (18°Be) and 0.5g of polyoxyethylene lauryl ether. (2) Enzyme treatment Anhydrous sodium carbonate 0.5g/, sodium hydrogen carbonate 3g/and alkaline protease
Processing solution containing 20,000 units of APG501 (PH
9.0) Process at 60℃ for 120 minutes using 60ml. (3) Post-treatment Treated at 98°C for 40 minutes using 60 ml of a treatment solution (PH8.0) containing 1 g of sodium hydrogen carbonate and 0.5 g of polyoxyethylene lauryl ether. As shown in the following formula, the desericination rate after each treatment was calculated by taking the reduction rate of dough weight after each treatment to the initial unscoured dough weight as the desericination rate, and found that it was 12.4% after pretreatment, and 12.4% after enzyme treatment. The rest is 25.7
%, and 27.1% after post-treatment, showing a good scouring effect. Weight loss rate (%) = Unrefined weight - Refined weight / Unrefined weight x
100 Test 2 860g of unscoured raw silk using a 40mm square treatment tank
were treated as follows at a liquid ratio of 1:35, respectively, and an enzyme scouring test was conducted. (1) Pretreatment Using the same treatment solution 30 as in Test 1, boiling treatment for 40 minutes under natural convection stirring. (2) Enzyme treatment Anhydrous sodium carbonate 0.5g/, sodium bicarbonate 3.5g/and alkaline protease
Processing solution (PH
9.0) Treated at 60°C for 120 minutes with stirring using a pump circulation of 5 minutes. (3) Post-treatment Using the same treatment solution as Test 1, boiling treatment was performed for 60 minutes under natural convection stirring. When the desericinization rate was calculated in the same manner as 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 are graphs showing the effects of PH and temperature, respectively, on the activity of alkaline protease APG501. Figures 3 and 4 are
2 is a graph showing the influence of PH and temperature on the stability of alkaline protease APG501, respectively.

Claims (1)

[Claims] 1. Bacillus sp. 9111-5B (Bacillus sp. 9111-5B), a novel alkaline protease APG501-producing strain having the following physical and chemical properties.
(a) Has a protease action in an alkaline region. (b) Has a protease action specific to sericin, with an optimal pH of 8.5 to 9 and an optimal temperature of 60.
~65℃, (c) At 4℃, stable in the PH5.5-11 range, (d) At PH7.0, stable up to 50℃, inactivation starts at 60℃, completely inactivated at 70℃, ( 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 ) In disk electrophoresis (PH9.4) using 7.5% polyacrylamide gel, if the migration of 0.01% bromophenol blue (BPB) is set to 1, the migration of enzyme protein is 0.14. (h) Isoelectric point by focal electrophoresis 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, 2 methionine, 4 isoleucine, 8 leucine, tyrosine 7, has an amino acid composition of 2 phenylalanine, 0 tryptophan, and 0 cysteine.