JPS62158483A - Novel alkaline protease apg 501 and production thereof - Google Patents

Abstract

PURPOSE:To produce a novel alkaline protease APG 501 suitable for enzymatic degumming of woven silk fabric, by culturing a microbial strain belonging to Bacillus genus. CONSTITUTION:A microbial strain belonging to Bacillus genus and capable of producing novel alkaline protease APG 501, e.g. Bacillus sp. 9111-5B (FERM P-8520) is cultured under aerobic condition at 20-40 deg.C and 7.0-10.0pH for about 24-144hr and the objective alkaline protease APG 501 accumulated in the culture product is separated therefrom.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a novel alkaline protease APG501 and a process for its production. More specifically, the present invention involves culturing a novel alkaline protease APG50I produced by a microorganism belonging to the genus Bacillus and a bacterium producing alkaline protease APG501 belonging to the genus Bacillus, and collecting the alkaline protease from the resulting culture. The present invention relates to a method for producing the alkaline protease.

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 The novel alkaline protease APG501 of the present invention has the following physicochemical properties.

(a) Has protease action in the alkaline region; (b)
) It has a particularly high specific protease action against sericin, and its optimum 1) H is 8.5 to 9, and the optimum temperature is 60.
~65°C, (c) At 4°C, stable in the range of pH 5°5 to If, (d) At pH 7.0, stable up to 50°C, 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) Molecule 16,300 by ultracentrifugation; molecular weight 19,000 by gel I filtration; ( g) In disk electrophoresis using 7.5% polyacrylamide gel (p) (9,4), if the migration of 0.01% bromophenol blue (BPB) is 1, the migration of enzyme protein is 0.14; (h) Isoelectric focusing by focal electrophoresis 696, (i) 5 lysines, 3 histidines, 2 arginines, 1 aspartic acid
4, threonine +3, serine 20, glutamic acid 6, proline 1, glycine 17, alanine 20, valine 15,
It has an amino acid composition of 2 methionine, 4 isoleucine, 8 leucine, 7 tyrosine, 2 phenylalanine, 01 tryptophan, and 0 cysteine.

The alkaline protease AI''G501 of the present invention is distinguished from known alkaline proteases by having 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 was heated to 110°C at a bath ratio of 1:18.
Perform hot water elution for 0 minutes, and add this eluate to 275r++n
Prepare a sericin eluate by adjusting the absorbance at 4.0 with water. This sericin eluate 2, 5
mQs sample enzyme solution 0. lxQ and 0°3M sodium carbonate-0.3M sodium bicarbonate buffer (p+-[1
0,0)0.5m+2 and react at 60°C for 10 minutes. Then, 10% trichloroacetic acid (TC
A) Solution 3: Stop the reaction by adding OmQ, and after standing in ice water for 30 minutes, centrifuge (170000 L) and measure the absorbance of the supernatant at 275 nm. 275n
The absorbance at m corresponds to 4.5 micromoles. Under these conditions, the enzyme activity that produces soluble nitrogen compounds equivalent to 1 micromole of tyrosine per minute is reduced to 1
Unit.

The novel alkaline protease APG501 of the present invention is an enzyme produced by a novel microorganism belonging to the genus Bacillus. The microorganism was isolated from soil collected in Ayabe City, Kyoto Prefecture by Inventor et al., and its representative strain, Bacillus sp.
Strain 11-53 has been deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry under Microbiological Research Institute No. 8520.

The mycological properties of Bacillus nis bii 9111-5B strain (Feikoken Bacteria Kisaku No. 8520) are shown below.

Morphological characteristics (1) Cell size, shape vegetative cell 0.3-0.5μ butt x 07-1.5μ shavings, short bacillus.

(2) Pleomorphic cells may extend to 0.5 μm x 3 or 0 μm.

(3) Motility Has mobility.

(4) Flagella Has circumferential hair.

(5) Spores Oval heat-resistant spores of 0.4 to 0.5 μz x 0.7 to 1.2 μm 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) Flesh agar plate culture Forms circular colonies, plate-like, with ridges at the edges and center (semi-lenticular). Colonies are smooth, creamy white, and glossy.

(2) Cultured on meat juice agar slant. Good growth, plate-like, rough surface, whitish brown, partially wrinkled.

(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 cx in 10 days.

Bacterial cells grow on the surface.

(6) Ridmus milk After about 30 days, there was no milk curdling or lid mass discoloration.

physiological properties As shown in Table 1.

Table 1 *: Decomposes sugar anaerobically.

**: Possible to grow at 16%, did not grow at 16.5% or more.

Glycolysis test Table 2 shows the results. The symbol in the table indicates the next residence.

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

Table 2 The above properties of the 9+1l-5B strain are summarized in Bergey's Manual of Deconomic Bacteriology, 8th edition.
terminative I3acteriology
, 8th edition), which is similar to Bacillus firmus (among the known species of the genus Bacillus)
The properties of Bacillus rirmus were compared. Results (1) Bacillus 9111-5Bl! can grow on agar medium under anaerobic conditions, but 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 911+-5B strain [(5-12 wide p
Bacillus firmus grows at pH 6, although it grows in the I-1 range.

9111-5 in that it cannot grow unless it is 0 or more.
The B strain was clearly distinguishable from Bacillus firmus and constituted a different bacterial species, and was therefore judged to be a new bacterial species belonging to the genus Bacillus. Thus, the novel alkaline protease APG50 of the present invention! cultured Bacillus nis bii strain 9111-5B in a nutrient medium, accumulated Arc;501 in the culture, and extracted APG501 from the culture.
It can be obtained by collecting. Like other bacteria, 91
The +1-5B strain can be mutated, for example, by artificial mutation means such as irradiation with ultraviolet rays, COoo, X-rays, etc., and the use of various mutagenic agents, and such mutant strains have no ability to produce AI'G501. It can be used in the present invention as long as it has the following. Furthermore, any bacterial strain other than the 911I-5B strain and its mutants can be used in the present invention as long as it has the ability to produce APG501.

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

9111-5Bl! Culture of this bacterial strain A conventional aerobic culture method using a liquid medium can be adopted. As a carbon source for the culture medium, various kinds such as starch, degistrin, and glucose can be used alone or in combination41. reactor. As the nitrogen source, soybean meal, soybean flour, yeast extract, milk casein, cornstarch liquor, etc. can be used alone or in combination. In addition, inorganic salts and the like may be added as appropriate. Cultivation is generally carried out under aerobic conditions at 20-40°C, pL
I 7, 0~! 0.0 for about 24 to 144 hours.

This culture allows alkaline protease AP to be present in the culture.
G50I is accumulated.

Collection and Purification of APG501 For collection and purification of alkaline protease APG501 from a culture, a method commonly employed for isolating and purifying microbial metabolites from a culture is used.

For example, after the cultivation is completed, 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 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 enzymatic scouring 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 Examples and Test Examples.

Example 1 Soybean casein 0.5%, glucose 0.5%, yeast extract 0.1%, K! HP0,0.1% and MgSO4・
I) containing 7HtOO, 02% and N at CO3
Dispense 100xQ of a liquid medium adjusted to 9111 into a 500 volume Sakaro flask and sterilize it.
5B strain was inoculated and cultured at 30° C. for 5 days with shaking at 135 times/min.

The culture solution is centrifuged at 18,000 G, and the supernatant is concentrated by ultrafiltration with a molecular fraction of 15,000 to obtain a crude enzyme solution 1511Q of 108 units/11Q.

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

The attached FIGS. 1 and 2 show the effects of leaf I and temperature on the activity of the alkaline protease APG501 obtained. These are graphs showing the relative activity when the activity was measured according to the above-mentioned activity measurement method while changing p) [ and temperature, and the maximum activity was taken as 100%. As shown in Figures 1 and 2, the optimal pH for alkaline protease APG501 to act on sericin is 8.
．． 5-9, the optimum temperature is 60-65°C.

In addition, the enzymatic activity pL (stability and temperature stability) of alkaline protease APG501 obtained is shown in the attached Figures 3 and 4. The residual activity after treatment with plI7 for 10 minutes at a temperature range of 30 to 80°C was measured 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 +1 at 4°C.
It is stable in the range of I) H7.0 up to 50°C.
It is stable at 60°C, starts to deactivate at 60°C, and completely deactivates at 70°C.

Example 2 Contains 0.5% defatted soybean meal, 1.0% dextrin, 0.5% cornstarch liquor, K, HPO, 0°1% and Mg5O, 7I-I, 02%, Nat
After sterilizing 1.3Q liquid medium adjusted to pi-19 with COa, it is placed in a mini jar and inoculated with strain 9111-5B.

This was stirred at 3 (J Or, p, m, 0°31
Culture at 30° C. for 3 days with aeration at 2/min.

The culture solution was filtered once through a Celite-coated filter press and concentrated 18.1 times by ultrafiltration with a molecular fraction of 15,000 to obtain a crude enzyme solution of 930 units/11IQ (72rnQC enzyme yield of approximately 70%). obtain.

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

Test 1 Using a large test tube of 3.0φ x 19.5 cm, an enzyme scouring test was conducted by treating each unscoured silk fabric 29 at a liquid ratio of 1:30 as follows.

(1) Pretreated sodium silicate (18°Be) 4 g/ρ and polyoxyethylene lauryl ether 0, 5910.
Using 60 tp (l) of a treatment solution (pHIO) containing
Treated at 98°C for 40 minutes.

(2) Enzyme-treated anhydrous sodium carbonate 0, 59IQ, sodium bicarbonate 3g/σ and alkaline protease APG501
Treatment liquid containing 20,000 units/Q (pH 9).

0) Processed at 60°C for 120 minutes using 60J112.

(3) Post-treatment treatment solution containing sodium bicarbonate Ig/ρ and polyoxyethylene lauryl ether 0,59IQ
t18.0) Processed at 98°C for 40 minutes using 60mQ.

As shown in the following formula, the deserination rate after each treatment was calculated by taking the reduction rate of the dough weight after each treatment as the deserination rate with respect to the initial unscoured dough weight.
2.4%, 25.7% after enzyme treatment, 27% after post-treatment.
1%, indicating a good scouring effect.

Test 2 Using a 40Q square treatment tank, 860 g of unscoured raw silk was
An enzyme scouring test was conducted on each sample at a liquid ratio of I:35 and the following treatment.

(1) Using the same treatment solution 30C as in the pretreatment test 1, the
Boil for 0 minutes.

(2) Enzyme-treated anhydrous sodium carbonate 0.59IQ, sodium bicarbonate 3.5g/(l and alkaline protease A
Processing liquid containing PG501 22,500 units/12 (
pH 9,0) 3012 at 60° C. for 120 minutes under stirring with pump circulation at 512/min.

(3) Using the same treatment solution 30C as in post-treatment test 1, the
Boil for 0 minutes.

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.
FIG. 2 is a graph showing the effects of p11 and temperature on the activity of PG501. Figures 3 and 4 show the effect of pI-1 on the stability of alkaline protease APG501, respectively.
and is a graph showing the influence of temperature. Patent Applicant: Gunze Mashishiki Company, 3 Attorneys, Patent Attorney: 2 Attorneys: 2*1 % 5 7 9 11 pH Figure 2 %

Claims (4)

[Claims] (1) Novel alkaline protease APG501 with the following physical and chemical properties: (a) Has protease action in the alkaline region, (b)
) Has a specific protease action against sericin,
The optimum pH is 8.5-9, the optimum temperature is 60-65℃, (c) stable at 4℃ in the pH range of 5.5-11, (d) stable up to 50℃ at pH 7.0, Deactivation begins at 60°C and complete deactivation at 70°C. (e) No metal ions are required for activity expression or stabilization, and activity is significantly inhibited by diisopropylfluorophosphate (DFP). (f) Molecular weight: 16,300 by ultracentrifugation, 19,000 by gel filtration method. (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,
1 proline, 17 glycine, 20 alanine, 15 valine
, methionine 2, isoleucine 4, leucine 8, tyrosine 7, phenylalanine 2, tryptophan 0, and cysteine 0. (2) A method for producing alkaline protease APG501, which comprises culturing a bacterium belonging to the genus Bacillus and having the ability to produce the novel alkaline protease APG501 in a nutrient medium, and collecting the alkaline protease from the culture. . (3) Novel alkaline protease A belonging to the genus Bacillus
PG501 producing bacteria. (4) Bacillus sp.
．． ) 9111-5B (Feikoken Bacteria No. 8520), the producing bacterium of item (3) above.