JPH07236482A - Alkaline protease, its production and microorganism producing the protease - Google Patents

Abstract

PURPOSE:To obtain an alkaline protease having excellent stability to various surfactants compared with conventional alkaline proteases produced by microorganisms of the genus Bacillus and suitable e.g. as an assistant for detergent. CONSTITUTION:There are provided a new microbial strain belonging to the genus Bacillus, an alkaline protease having the following properties and produced by culturing the new strain, its mutant or its genetic manipulation product and a process for the production of the alkaline protease. (1) The optimum pH of the enzyme is about 11-11.5 by the reaction at 30 deg.C for 10min using casein as the substrate, (2) the optimum working temperature is about 55 deg.C by the reaction at pH 10, (3) the temperature to reduce the activity half is about 46-49 deg.C when treated at pH 10 for 10min, (4) the molecular weight is 29,100+ or -1,000 measured by SDS polyacrylamide electrophoresis and (5) the isoelectric point is 9.4-9.5 measured by a sucrose density gradient isoelectric electrophoresis.

Description

[Detailed description of the invention]

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to novel bacterial alkaline proteases, and more particularly to novel alkaline proteases, methods for producing the same, and microorganisms producing the proteases.

[0002]

2. Description of the Related Art In recent years, environmental pollution has become a problem and the use of phosphate has been regulated. Detergents containing enzymes to enhance their detergency have been developed. Detergents containing enzymes containing cellulase, lipase, etc. are on the market.

Among them, alkaline protease accounts for 10 to 40% of the organic dirt adhering to clothes, and is indispensable as a detergent ingredient in order to efficiently decompose protein stains that cannot be completely removed by detergent ingredients alone and increase detergency. It's becoming

[0004] As a detergent protease, many conventional microorganism-derived alkaline proteases are known which exhibit enzymatic activity in the alkaline pH range under which detergents (surfactants) are used. These enzymes generally have sufficient stability as enzymes for solid detergent formulations, but are not sufficiently stable in liquid detergents or in the presence of detergent components at high concentrations, and are deactivated in a short period of time.

Therefore, (1) development of new surfactants,
A number of techniques have been disclosed for improving the stability of enzymes in liquid detergents by (2) adding stabilizers and (3) microencapsulating enzymes (Japanese Patent Laid-Open No. 41398/1990, U.S. Pat. No. 42).
87082, British Patent No. 2021142, JP-A-62-596, JP-A-63-137996, JP-B-60-55118, JP-A-5-252949
number, etc.). However, along with the technology for stabilizing enzymes, there is a strong demand for enzymes that are more stable than conventional products in liquid detergents.

[0006] Accordingly, it is an object of the present invention to provide novel alkaline proteases having excellent stability in detergent ingredients and microorganisms producing them. A further object of the present invention is to provide an alkaline protease that is highly stable in a detergent solution containing a relatively low concentration of surfactant during washing, and a microorganism that produces it. A further object of the present invention is to provide an alkaline protease that can be supplied efficiently and inexpensively, and a microorganism that produces it.

[0007]

In order to achieve the above object, the present inventors conducted experiments with various surfactant-added systems and screened bacteria growing in the coexistence of detergents. As a result, among the multiple proteases with different isoelectric points produced by one strain of the genus Bacillus isolated from the soil in Miyagi Prefecture, the protease present on the side of the higher isoelectric point was found to satisfy the above conditions, and the present invention was completed. reached.

[0008]

DISCLOSURE OF THE INVENTION That is, the present invention provides the following novel alkaline protease, its production method and new strain. 1) Alkaline protease having the following properties. (1) Action: hydrolyzes proteins. (2) Optimum pH: 10 at 30°C with casein as substrate
When reacted for minutes, the optimum working pH is around 11-11.5. (3) Optimal temperature: When casein is used as a substrate and the reaction is carried out at pH 10, the optimum working temperature is around 55°C. (4) Heat resistance: When treated at pH 10 for 10 minutes, the temperature at which the activity is halved is about 46-49°C. (5) Molecular weight: The molecular weight determined by SDS polyacrylamide electrophoresis is 29,100±1,000. (6) Isoelectric point: The isoelectric point measured by sucrose gradient isoelectric focusing is 9.4-9.5. (7) Stability to surfactant 6 in 0.2% sodium n-dodecylbenzenesulfonate (LAS) aqueous solution (40°C) at a concentration of 200 nkat/ml
When incubated for 0 min, residual activity is approximately 60
%. 2) The alkaline protease according to 1 above, wherein 49 residues of the N-terminal amino acid sequence are represented by SEQ ID NO:1. 3) The microorganism belonging to the genus Bacillus and having the alkaline protease-producing ability described in 1 or 2 above, or a mutant or genetically engineered strain thereof, is cultured, and the desired alkaline protease is collected from the culture solution. 3. The method for producing an alkaline protease according to 1 or 2 above. 4) the microorganism having alkaline protease-producing ability or its mutant strain or genetically engineered strain is Bacillus s
p. TS50093 strain (Acceptance number FERM P-14104, National Institute of Biotechnology and Biotechnology, Agency of Industrial Science and Technology), or a mutant or genetically engineered strain thereof. 5) The microorganism belonging to the genus Bacillus or its mutant or genetically engineered strain having alkaline protease-producing ability according to 1 or 2 above. 6) The microorganism, mutant strain or genetically engineered strain thereof according to 5 above, wherein the microorganism is Bacillus sp.

[0009]

[Microorganisms and their culture] As described below, the strains producing the enzyme of the present invention were subjected to morphological observation, physiological property tests, and bacterial component analysis. JGH
olt : "Bergey's Manual of Systematic Bacteriology"
Vol.2 (1986) Williams &Wilkins;REGordon, WC
Haynes and C.H.Pang: "The Genus Bacillus" (1973),
USD Department of Agriculture; D.Fritze, J.Flossdo
rf and D. Claus: Int. J. Syst. Bacteriol., 40, 92
(1990) ) was used as a reference for identification. The strain of the present invention is a catalase-positive, Gram-positive bacillus, and was thought to belong to Bacillus from its colony shape and cell morphology.

[0010] However, when the bacterial cell components were analyzed, all analytical results were consistent with the characteristics of bacteria belonging to the genus Bacillus. From these results, the bacterium was considered to be a bacterium belonging to the genus Bacillus, which had lost the ability to form spores, and was clearly different from other known strains, and was identified as a novel bacterium. The present inventors named this strain Bacillus TS50093 strain. The strain of the present invention has been deposited with the Institute of Biotechnology, Agency of Industrial Science and Technology under the accession number FERM P-14104.

[0011]

[Mycological properties] The new strain TS50093 according to the present invention exhibits the following mycological properties.

A: Morphological properties (a) Cell shape: Bacillus. (b) Cell size: 0.8-1.1 × 2.4-4.5 μm (pH
0.8-1.0×4.4-8.8 μm (after 2 days of culture at 30° C. in SCD broth medium adjusted to pH 9). (c) Adherence state of flagella: periculum (after culturing for 1 day at 30°C in SCD agar slants adjusted to pH 9). (d) Gram staining: positive. (e) Formation of spores: Incubation was carried out for one month under the medium (pH 9) and temperature conditions shown in the table below, and observation was made, but formation of spores was not observed under any conditions.

[0013]

[Table 1] Medium Culture conditions Sporulation nutrient agar (purified water) 30°C - nutrient agar (purified water) 45°C - soil exudate agar 30°C - soil exudate agar 45°C - nutrient agar (soil contamination) Exudate) 30°C - nutrient agar (soil exudate) 45°C - nutrient agar (mineral water) 30°C - 1/10 nutrient agar (mineral water) 30°C - L medium1 ⁾ (purified water) 30 ℃ - L medium (mineral water) 30℃ - 1/10L medium (mineral water) 30℃ - TA agar medium2 ⁾ (purified water) 30℃ - TA agar medium2 ⁾ (purified water) ³⁾ 30℃ No growth AK agar ²⁾ (purified water) 30°C - AK agar ²⁾ (purified water) ³⁾ No growth at 30°C Tryptic soy agar (purified water) 30°C - 1) Bacto trypton 1%, yeast extract 0.5%, Na
Cl 0.5% _{, Na2CO3} 0.3%. 2) Kamei et al.: J. Antibact. Antifung. Agents., 19, 61
(1991). 3) After heat-treating the bacteria at 60°C for 10 minutes,
culture.

(f) Motility: Has motility. (g) Attitude to oxygen: aerobic. (h) Anaerobic growth: no growth.

B: Growth conditions in various media (pH 9) (i) gelatin liquefaction: positive; (j) Starch degradation: positive. (k) Use of citric acid: Do not use. (l) Use of Propionate: Do not use.

C: Physiological Properties (m) Catalase: positive. (n) VP test: negative. (o) Egg yolk reaction: negative. (p) nitrate reduction: negative. (q) acid and gas production from sugars

[0017]

[Table 2] ND: No data.

(r) pH range for growth: No growth at pH 5.7. Does not grow at pH 6.8 (Nutrient Broth). Weak growth at pH 7.5. Good growth at pH 8.0-9.5. (s) Tolerance to sodium chloride: 5% and 7% Na
It grows in the presence of Cl. (t) Temperature range for growth: No growth below 10°C and above 50°C. Good growth at 30-40°C.

D: Cell components and bacterial cell fatty acid composition Cell components and bacterial cell fatty acid compositions of the strain of the present invention and a strain of B. alcalophilus for comparison are shown in Tables 3 and 4, respectively. show.

[0020]

[Table 3] This strain B. alcalophilus TS50093 (JCM5262) cell wall diamino acid meso-DAP meso-DAP quinone MK-6, MK-7 MK-7, MK-6 DNA GC content (mol%) 41 40 meso-DAP : meso-diaminopimelic acid

[0021]

[Table 4] Fatty acid composition (%) of this strain B. alcalophilus TS50093 (JCM5262) iso-14:0 3.7 0.4 14:0 1.6 1.8 iso-15:0 16.7 33.9 anteiso-15:0 52.7 32.4 15:0 2.0 − iso-16:0 5.6 2.6 16:0 5.8 8.9 16:1 1.6 0.7 iso-17:0 0.9 4.1 anteiso-17:0 5.5 6.7 17:0 0.4 0.8 18:0 0.3 0.4 18:1(n-9) 0.4 1.2 18:1(n-11) − 0.3

[0022]

[Enzyme Acquisition Method] The TS50093 strain of the present invention produces two to three types of proteases with different isoelectric points (see Example 1 below). , which had the aforementioned features of the present invention.
In order to obtain the alkaline protease of the present invention using the TS50093 strain, the strain may be inoculated into a suitable medium and cultured according to a conventional method.

The microorganisms used for producing the alkaline protease of the present invention are not limited to the TS50093 strain,
Any strain may be used as long as it has the productivity of the alkaline protease of the present invention. Also, TS50093
Strains also include natural or artificial mutants and genetically engineered strains thereof.

[0024] As a method for preparing an artificial mutant strain, according to a conventional method, for example, the original strain is subjected to artificial mutation treatment with ultraviolet irradiation or a chemical such as nitrosoguanidine (NTG), and the mutant strain is obtained from skimmed milk. The resulting colonies are isolated by planting them on an agar medium containing , and cultured on a protease-producing medium to check the identity of the protease produced, and the strain with the highest productivity is selected. As a method for preparing the spontaneous mutant strain, according to a conventional method, for example, culturing in a normal nutrient medium is performed to obtain a culture containing a small amount of the spontaneous mutant strain,
From there, strains with excellent protease productivity may be selected by the same method as the method for preparing the artificial mutant strains described above.

[0025] As for the method for preparing the genetically engineered strain, it is carried out according to the same conventional method as described above. For example, the DNA base sequence is deduced from the amino acid sequence of the protease, a characteristic portion of the DNA base sequence is synthesized, and the phosphorus atom of the phosphate group is converted to the radioisotope ³² P.
label with On the other hand, the total chromosomal DNA was taken out from the original strain, cleaved with an appropriate restriction enzyme, and the above labeled synthetic D was obtained.
Chromosomal DNA fragments that hybridize with NA are selected by the Southern Hybridization method. The chromosomal DNA fragment thus selected is inserted into an appropriate vector, introduced into a non-protease-producing strain, and checked for protease production. A strain with improved productivity can be obtained by introducing a DNA fragment that has been confirmed to produce protease into the original strain or a strain with high enzyme productivity (high protein secretion ability) using an appropriate vector such as a plasmid.

Any medium may be used in the present invention as long as the Bacillus bacterium of the present invention can grow and produce alkaline protease. For example, carbon sources include glucose, maltose, sucrose, soluble starch, and the like, and nitrogen sources include organic nitrogen sources such as soybean meal, rice bran meal, bran meal, and cornsteve liquor. In addition, inorganic salts such as phosphates, sodium salts, potassium salts and magnesium salts are added.

In the present invention, the culture is carried out under aerobic conditions, for example, by the aeration agitation method or shaking culture method. The culture temperature may be in the range of 20 to 40°C.
0 to 40°C is desirable. The initial pH is preferably 8-11, and the pH during cultivation is preferably 8.5-10.5. Incubation time is 16
It is about 60 hours, and the culture should be terminated when the protease activity reaches the maximum.

Separation and purification of the target protease from the thus-obtained culture solution can be carried out according to a general enzyme separation and purification method. That is, a supernatant or a filtrate can be obtained by removing cells and medium solids by centrifugation, filtration, or the like. The protease of the present invention is obtained by adding a soluble salt or a hydrophilic organic solvent to these separated liquids to precipitate proteins by salting out, solvent precipitation, spray drying, freeze drying, or the like. Furthermore, it can be purified by combining purification methods such as ion exchange chromatography and gel filtration chromatography.

[0029]

[Method for measuring enzyme activity] In the present invention, enzyme activity was measured by the following fluorescence method or phenol method. note that,
In the examples below, the activity was measured by the phenol method unless otherwise specified.

1. Fluorescence method: 100 mM Atki at pH 10
10 to 1300 μl of ns-Pantin borate buffer (APBB)
mM succinyl-leucyl-leucyl-valyl-tyrosyl-4-methylcoumaryl-7-amide (synthetic substrate; Su
c-Leu-Leu-Val-Tyr-MCA) dimethyl sulfoxide (D
MSO) solution and 5 μl of the enzyme solution were added and heated to 30°C.
for 1 minute with a fluorometer (Hitachi Fluorresence spectrophoto
Meter F3000), fluorescence of 7-amino-4-methylcoumarin (AMC) produced by cleavage of the substrate bond by excitation light of 360 nm is measured at a fluorescence wavelength of 440 nm.

2. Phenol method: 50 mM at pH 10
Add 50 µl of the enzyme solution to 500 µl of APBB and heat at 30°C.
pre-incubate for 10 minutes at 500 μl of 50 mM APBB solution containing 2% casein was added to the solution,
After incubating for 10 minutes at 30° C., 2 ml of 0.3 M trichloroacetic acid (TCA) solution is added to stop the reaction. After incubating for 20 minutes at 30° C., filtering
2.5 ml of 0.4 M sodium carbonate to 0.5 ml of filtrate and 6
Add 0.5 ml of 2-fold diluted phenol reagent and incubate at 30°C for 3
After incubating for 0 minutes to develop color, absorbance at 660 nm is measured. In the above measurement, the amount of enzyme that produces an acid-soluble degradation product equivalent to 1 mol of tyrosine per second is defined as 1 qatar (kat).

[0032]

EXAMPLES The enzyme of the present invention will now be described in detail with reference to examples, but the present invention is not limited to these examples.

Example 1: Incubation of the enzyme of the present invention 1% polypeptone, 1% meat extract, 1% sodium carbonate and 0.1% casein in a 5-liter Sakaguchi flask.
was placed in 2 liters of liquid medium containing and sterilized in an autoclave at 121° C. for 15 minutes. This was inoculated with TS50093 strain, which had been plate-cultured and further cultured in test tubes, and incubated at 30°C for 12 hours.
Sakaguchi culture was performed at 5 rpm for 2 days. After collecting the culture medium,
Centrifugation (8000 rpm) was carried out at 0°C for 30 minutes and the supernatant was collected. A double volume of -20°C acetone was added to the supernatant, and the mixture was again centrifuged (8000 rpm) at 0°C for 30 minutes. The precipitate was collected and dried under reduced pressure to obtain crude enzyme powder.

About 10 mg of the resulting crude enzyme was added to 50 mM M
ES (2-(N-morpholino)ethanesulfonic acid) 30
It was dissolved in μl and 3 μl of glycerin, and subjected to agarose gel electrophoresis under the following conditions. Similarly subtilisin A, A
PI-21 (NKS-described in JP-B-60-55118)
Protease produced by 21 strains) and protease C
360 (protease produced by strain C360 described in US Pat. No. 3,723,250) was also subjected to agarose gel electrophoresis.

Conditions; Gel concentration: 1% agarose, Gel area: 15 x 15 cm, Buffer: 50 mM MES (pH 6.5), Voltage: 40V, Temperature: 4°C, Run time: 4-6 hours.

The results are shown in FIG. 1. TS50093 strain produces 2-3 proteases with different isoelectric points,
Among these, an enzyme with a higher isoelectric point, which behaves similarly to the alkaline protease API-21 produced by the NKS-21 strain, was purified.

The activity of the enzyme was evaluated by adding 7.5 ml and 1 ml of 0.5 M APBB (pH 10) to the bottom of the gel after electrophoresis.
A filter paper impregnated with 15 μl of 0 mM Suc-Leu-Leu-Val-Tyr-MCA was affixed, incubated at 37° C. for 30 minutes, and then the gel was irradiated with ultraviolet light to confirm the formation of luminescent spots. .

Example 2 Purification of Enzyme The crude enzyme obtained in Example 1 was dissolved in about 200 ml of 10 mM MES buffer containing 1 mM calcium chloride (pH 6.0).
5) and dialyzed twice against 5 liters of the same buffer. Then, DEAE-Cellulofine A-500 (manufactured by Seikagaku Corporation) column chromatography (gel volume 50 ml) (same buffer) was applied to collect the flow-through fraction, followed by CM-Cellulofine C-5.
00 (manufactured by Seikagaku Corporation) column chromatography (gel volume: 50 ml). The adsorbed active fraction was subjected to gradient elution with 0 to 1 M sodium chloride to recover the alkaline protease of the present invention having a specific activity increased about 12 times that before ion exchange chromatography. S
When subjected to DS slab electrophoresis and using Coomassie Brilliant Blue (CBB) as a staining solution, it was confirmed that the enzyme purity was uniform. The purified enzyme was freeze-dried and stored frozen.

Example 3: Analysis of N-Terminal Amino Acid Sequence Lyophilized products of the alkaline protease of the present invention and API-21 were dissolved in 50% acetonitrile and 0.1% trifluoroacetic acid, and the main peak was collected by HPLC. 49 residues of the N-terminal amino acid sequence were analyzed using a sequencer. Results are shown in SEQ ID NO:1 and SEQ ID NO:2.

The alkaline protease of the present invention is API-
Approximately 90% homology to 21 is observed.

Example 4 Measurement of Properties of Purified Enzyme (1) Optimum Temperature and Temperature Stability The optimum temperature and temperature stability of the alkaline protease of the present invention were measured as follows.

(1-1) Optimal temperature: The alkaline protease of the present invention was added to 50 mM APBB (pH 10) containing 1% casein as a substrate and 0.05 mM calcium chloride as a metal salt, and reacted at each temperature for 10 minutes. rice field. 30
FIG. 2 shows the results of determining the relative activity at each temperature, with the activity at °C as 100%. From FIG. 2, the optimum temperature at pH 10 for the alkaline protease of the present invention is around 55 to 57°C.

(1-2) Temperature stability and stabilizing effect by calcium ions: 50 mL containing 10 nkat/ml alkaline protease of the present invention and 1 mM calcium chloride.
200 μl of M APBB (pH 10) was incubated at each temperature for 10 minutes, ice-cooled, and activity was measured. In addition, measurements were also made in the same manner for those containing no calcium chloride. Also, the activity of API-21 as an enzyme was determined in the same manner. The results are also shown in FIG. From FIG. 3, all enzymes show similar temperature stability, and the presence of calcium ions increases the temperature stability. The temperature at which the activity is halved in heat treatment at pH 10 for 10 minutes is 51-55°C in the presence of calcium ions and 46-49°C in the absence of calcium ions. The activity at 50°C was 77% in the presence of calcium ions and 3 in the absence of calcium ions.
2%.

(2) Optimal pH and stable pH range Optimal pH and stable p of the alkaline protease of the present invention
The H region was measured as follows.

(2-1) Optimal pH: The enzyme obtained in Example 2 was added to 50 mM Britton-Robinson broad range buffer (pH 4-12) containing 1% casein as a substrate, and
℃ for 10 minutes at each pH. The activity at pH 11.5 was regarded as 100%, and the relative activity at each pH was determined. The results are shown in FIG. From FIG. 4, the optimum pH was around 11-11.5.

(2-2) Stable pH range: 50 mM Britto
15 μl of 20 mM calcium chloride and an enzyme solution (100 nkat/ml, 50 mL of n-Robinson broad range buffer)
Add 30 µl of M APBB, pH 10) and incubate at 30°C for 2
After incubating for 4 hours, it was ice-cooled and the activity was measured. As the enzyme, the enzyme obtained in Example 2 was used, and the activity before incubation was defined as 100%, and the results of determining the residual activity at each pH are shown in FIG. From FIG. 5, it can be seen that the stable pH range is around 4 to 8.5 at 30° C. (residual activity of 50% or more).

(3) Immunity characteristics When the immunological characteristics of the alkaline protease of the present invention against API-21 antiserum and protease C360 antiserum were examined according to the Ouchterlony method, the alkaline protease of the present invention was found to have A sedimentation line was formed, but the protease C360 antiserum did not form a sedimentation line. That is, it can be seen that the alkaline protease of the present invention has the same immune properties as API-21, but different immune properties from protease C360.

(4) Determination of isoelectric point Using 1 mg of the dried powder enzyme obtained in Example 2, sucrose density gradient isoelectric focusing (Takeichi Horio, Nipei Yamashita, Fundamental Experiments on Proteins and Enzymes) (1981)), the isoelectric point of the enzyme was measured. As a result, the isoelectric point of the alkaline protease of the present invention was 9.4-9.5.

(5) Measurement of molecular weight The molecular weight of the alkaline protease of the present invention was measured by 15% SDS-polyacrylamide gel electrophoresis using cytochrome as a molecular weight marker.
was 9,100±1,000.

(6) Substrate Specificity The activity of the alkaline protease of the present invention to decompose various MCA synthetic substrates and various proteins was examined as follows.

(6-1) Decomposing activity against MCA synthetic substrate: Alkaline protease and API-21 of the present invention
were used, and various MCA synthetic substrates shown in the table were used as substrates, and their degradation activities were compared at pH 10 (fluorescence method). In the table, Suc is a succinyl group, Boc is a t-butoxycarbonyl group, Bzl is a benzyl group, Bz is a benzoyl group, Pyr is a pyroglutamyl group, and z is a carbobenzoxy group. Moreover, the activity is shown as a relative activity when the degradation activity of Suc-AlaAlaProPhe-MCA is set to 100.

[0052]

[Table 5]

(6-2) Degradation activity for various proteins: 50 mM APB to which 1% of various proteins were added
After leaving 1 ml of B (pH 10) at 30°C for 10 minutes,
After adding 50 μl of a 50 mM APBB (pH 10) solution of the enzymes shown in Table 6 and reacting at 30° C. for 10 minutes, 0.3
The reaction was stopped with 2 ml of M trichloroacetic acid (TCA) solution and the activity was measured. The activity was measured by the phenol method.

[0054]

[Table 6] Substrate Present invention API-21 Protease C360 casein 100 100 100 Hemoglobin (native) 58 40 32 Hemoglobin (denatured) 165 130 60 BSA 21 15 15 Ovalbumin 20 26 20 Keratin ( denatured) 68 50 39

The alkaline protease of the present invention has protease C3 in terms of degrading activity for various proteins.
60 and shows a different pattern from API-21. In addition, Japanese Patent Application Laid-Open No. 5-252949, which is the prior application of the present applicant,
The alkaline protease described in JP-A-5-252949 is very similar in substrate specificity to protease C360. Therefore, the alkaline protease of the present invention is significantly different from the alkaline protease described in JP-A-5-252949 in terms of substrate specificity.

(7) Stability to Surfactants The stability of the alkaline protease of the present invention to various surfactants was examined as follows.

(7-1) Stability to sodium n-dodecylbenzenesulfonate (LAS) Of the anionic surfactants that are most closely related to the deactivation of enzymes contained in detergent ingredients, LAS damages enzymes particularly strongly. Stability in LAS solution was compared. enzyme (alkaline protease of the invention and API-21) and 1 mM
100 mM APBB (p
H10) 25 μl and 25 μl of 2% LAS aqueous solution
200 μl of mM APBB (pH 10) (final concentration of enzyme: 200 nkat/ml, final concentration of LAS: 0.2%) was incubated at 40° C. for 60 minutes.
50 mM sampled (50 μl) every 5 minutes
It was added to 500 µl of APBB (pH 10), and the residual activity was determined by setting the casein-degrading activity immediately after addition of the enzyme to 100. The results are shown in FIG. API-21 has a residual activity of about 40% after 60 minutes, whereas the alkaline protease of the present invention has a residual activity of about 60% after 60 minutes.
%, and it can be seen that the stability in LAS is remarkably superior to that of other enzymes.

(7-2) As a stability surfactant for other surfactants, sodium todecyl sulfate (SD
S), Brigi 35 and Triton X-1
00 (Triton X-100), the resistance of the alkaline protease of the present invention to 1% of each surfactant was examined as follows.

25 µl of 100 mM APBB (pH 10) containing 25 nkat enzyme (alkaline protease of the invention, API-21) and 10 mM calcium chloride.
1 and 25 μl of a 10% surfactant aqueous solution to 50 mM A
Dissolve in 200 µl of PBB (pH 10) at 40°C
for 60 minutes, and samples (50 μl) were added every 15 minutes to 50 mM APBB (pH 1
0) In addition to 500 µl, the residual activity was determined by setting the casein-degrading activity immediately after addition of the enzyme to 100. Results are shown in FIGS. The residual activity of the alkaline protease of the present invention after 60 minutes is about 40% in a 1% SDS solution.
%, about 75% in a 1% Bridge 35 solution and about 70% in a 1% Triton X-100 solution, known API-2
1 (SDS solution: 10%, Bridge 35 solution: 55%, Triton X-100 solution: 60%).

[0060]

INDUSTRIAL APPLICABILITY As described above, the alkaline protease of the present invention is a known alkaline protease API-
It has 90% homology with 21 in its N-terminal amino acid sequence and similar substrate specificity. The alkaline protease of the present invention is used for leather processing,
In addition to the food industry, the textile industry, and the pharmaceutical industry, it can be effectively used as an auxiliary additive for detergents, especially liquid detergents, taking advantage of its high stability against surfactants.

[0061]

[Sequence list]

SEQ ID NO: 1 Sequence length: 49 Sequence type: amino acid Topology: Linear Sequence type: protein arrangement Gln Thr Val Pro Trp Gly Ile Pro Tyr Ile Tyr Ser Asp Val Val Gln 1 5 10 15 His Gln Gly Tyr Phe Gly Asn Gly Val Lys Val Ala Val Leu Asp Thr 20 25 30 Gly Val Ala Pro Gln Pro Asp Leu Leu Ile His His Gly Val Ser Phe 35 40 45 Ile 49

SEQ ID NO:2 Sequence length: 49 Sequence type: amino acid Topology: Linear Sequence type: protein arrangement Gln Thr Val Pro Trp Gly Ile Pro Tyr Ile Tyr Ser Asp Val Val His 1 5 10 15 Arg Gln Gly Tyr Phe Gly Asn Gly Val Lys Val Ala Val Leu Asp Thr 20 25 30 Gly Val Ala Pro His Pro Asp Leu His Ile Arg Gly Gly Val Ser Phe 35 40 45 Ile 49

[Brief description of the drawing]

FIG. 1 is the result of agarose gel electrophoresis for enzymes produced by microorganisms.

[Fig. 2] A graph showing the optimum temperature range of the alkaline protease of the present invention.

Figure 3 is a graph showing the temperature stability of an alkaline protease of the invention and a known enzyme (API-21).

FIG. 4 is a graph showing the optimum pH range of the alkaline protease of the present invention;

FIG. 5 is a graph showing pH stability of the alkaline protease of the present invention;

Figure 6 is a graph showing the stability of alkaline proteases of the invention and a known enzyme (API-21) in LAS solutions.

Figure 7 is a graph showing the stability of the alkaline protease of the invention and a known enzyme (API-21) in SDS solution.

Figure 8 is a graph showing the stability of alkaline proteases of the invention and a known enzyme (API-21) in Bridge 35 solution.

Figure 9 is a graph showing the stability of alkaline proteases of the invention and a known enzyme (API-21) in Triton X-100 solution.

Continuation of the front page (51) Int.Cl. ⁶ identification code Internal reference number FI Technical label // C11D 3/386 C12N 15/09 15/01 (C12N 9/54 C12R 1:07) (C12N 1/20 A C12R 1:07) (C12N 1/21 C12R 1:07)

Claims (6)

[Claims] 1. An alkaline protease having the following properties. (1) Action: hydrolyzes proteins. (2) Optimum pH: 10 at 30°C with casein as substrate
When reacted for minutes, the optimum working pH is around 11-11.5. (3) Optimal temperature: When casein is used as a substrate and the reaction is carried out at pH 10, the optimum working temperature is around 55°C. (4) Heat resistance: When treated at pH 10 for 10 minutes, the temperature at which the activity is halved is about 46-49°C. (5) Molecular weight: The molecular weight determined by SDS polyacrylamide electrophoresis is 29,100±1,000. (6) Isoelectric point: The isoelectric point measured by sucrose gradient isoelectric focusing is 9.4-9.5. (7) Stability to surfactant 6 in 0.2% sodium n-dodecylbenzenesulfonate (LAS) aqueous solution (40°C) at a concentration of 200 nkat/ml
When incubated for 0 min, residual activity is approximately 60
%. 2. The alkaline protease according to claim 1, wherein 49 residues of the N-terminal amino acid sequence are shown in SEQ ID NO:1. 3. Cultivating the microorganism belonging to the genus Bacillus and having the alkaline protease-producing ability according to claim 1 or 2, or its mutant or genetically engineered strain, and collecting the alkaline protease from the culture solution. 3. The method for producing an alkaline protease according to claim 1 or 2. 4. The microorganism having alkaline protease-producing ability or its mutant strain or genetically engineered strain is Bacillus sp. Alternatively, the production method according to claim 3, which is a genetically engineered strain. 5. A microorganism belonging to the genus Bacillus having alkaline protease-producing ability according to claim 1 or 2, or a mutant or genetically engineered strain thereof. [Claim 6] The microorganism is Bacillus sp.
4) The microorganism according to claim 5 or its mutant or genetically engineered strain.