JPH0763367B2 - Novel alkaline protease - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of use] The present invention relates to a novel alkaline protease, and more particularly to an alkaline protease that can be widely used as a washing enzyme suitable for Japanese laundry conditions.

[Conventional technology]

Although the incorporation of protease into detergent has been performed for some time, the protease originally incorporated into detergent is one that has an optimum reaction pH near neutral with papain and pancreatic trypsin. Was used.

However, as is well known, the pH of the detergent is on the high alkali side, the enzyme for washing also has an optimum reaction pH on the alkali side, and it is desired and developed to be stable in the surfactant. As a result, the market for detergents containing alkaline protease has begun to grow sharply in Europe and America since 1967. Even in Japan, full-scale protease-containing detergents have been put on the market since 1979.

Currently, the typical alkaline proteases used as cleaning enzymes are alcalase, sabinase, esperase (manufactured by Novo Industry), maxatase (manufactured by Gist Procardis), etc. The optimum reaction temperature of these enzymes is around 60 ° C. It matches the washing style of Western countries.

However, Japanese washing methods generally wash around room temperature, so there is a need to provide proteases that are more active than conventional proteases in lower temperature regions. -21
(Showa Denko KK) has been developed.

[Problems to be solved by the invention]

It has been conventionally known that a protease capable of decomposing insoluble proteins such as keratin well contributes to washing (Motokawa Minamoto: Jinho 26 322 (1985)), and various properties required for the protease. That is, in addition to acting on the alkaline side and having high activity even at low temperatures,
Furthermore, if a protease having the property of having an excellent action on insoluble proteins such as keratin is added during washing, it is expected that a detergent composition having excellent detergency and ease of use can be obtained. Therefore, there has been a demand for the development of an alkaline protease having such properties.

[Means for solving problems]

Therefore, the present inventors have the ability to decompose keratin,
In addition, in order to obtain an alkaline protease having a low optimum reaction temperature (high activity even in a low temperature range), a thorough search was conducted for protease-producing bacteria.

As a result, they have found that a group of microorganisms obtained from the soil of Haga-gun, Tochigi Prefecture produce alkaline protease satisfying the above conditions, and completed the present invention.

The microorganisms producing the alkaline protease of the present invention are Bacillus sp. KSM-200, respectively.
1, Bacillus SP KSM-2002, Bacillus SP
They are named KSM-2003 and Bacillus sp. KSM-2005, and have the following mycological properties.

(A) Morphological properties (a) Cell shape and size Bacillus sp. KSM-2001: bacilli 0.5-1.0 x 1.0-3.5
μm Bacillus SP KSM-2002: Bacilli 0.5-0.8 × 1.5-3.0
μm Bacillus SP KSM-2003: Bacilli 0.5-0.8 × 0.8-2.5
μm Bacillus SP KSM-2005: Bacilli 0.5-0.8 × 1.5-5.0
μm (b) Polymorphism Neither strain is observed.

(C) Motility All strains have periflagellates and are motile.

(D) Spores (size, shape, position) Bacillus sp. KSM-2001: 0.4 to 0.6 x 0.4 to 0.6 μm,
Oval or circular, central quasi-end Bacillus sp. KSM-2002: 0.4-0.8 × 0.4-0.8 μm,
Round or oval, central quasi-end Bacillus sp. KSM-2003: 0.5-0.8 x 0.8-1.2 μm,
Oval or cylindrical, central quasi-end Bacillus sp. KSM-2005: 0.5-0.8 × 0.8-1.2 μm,
Oval, central quasi-end (e) Gram stain Positive for all strains (f) Negative for all acid strains (g) Growth pattern on broth agar plate Growing Bacillus sp. KSM-2001: pH 7.0 To form round, medium-convex, and smooth-wave colonies. The size of the colony is 1.0-4.0 mm in diameter, and the surface is smooth with a dewdrop-like pale yellow color. It is a translucent and oily colony.

Bacillus sp. KSM-2002: Forming round, convex, smooth wave colonies. The size is 1.0-5.0 mm in diameter, and the surface is smooth, dewdrop-like, and pale yellow to milky white. It is a translucent and oily colony.

Bacillus sp. KSM-2003: Grows at pH 7.0 and forms round, trapezoidal, and smooth-wave colonies. The surface of the colony is smooth, dewdrop-shaped and light yellow. It is a translucent and oily colony.

Bacillus sp. KSM-2005: Grows at pH 7.0 and forms round, medium-convex, and smooth-wave colonies. The surface of the colony is smooth, dewdrop-shaped and light yellow. It is a semi-transparent and oily colony.

(H) Growth on broth agar slope Bacillus sp. KSM-2001: Shows weak band-like growth at pH 7.0 and forms milky white, translucent colonies. It grows in a wide band at pH 10.0.

Bacillus sp. KSM-2002: Shows weak band-like growth at pH 7.0 and forms milky white, translucent colonies. It grows in a wide band at pH 10.0.

Bacillus sp. KSM-2003: Shows weak band-like growth at pH 7.0 and forms milky white, translucent colonies. It grows in a wide band at pH 10.0.

Bacillus sp. KSM-2005: It grows in a wide band at pH 7.0 and pH 10.0 and forms milky white, translucent colonies.

(I) Liquid culture of broth All bacteria grow slightly at pH 7.0 but do not form pellicle.

(J) Meat juice gelatin stab culture Bacillus sp. KSM-2001: No gelatin liquefaction at pH 7.0.

Bacillus SP KSM-2002, 2003, 2005: liquefy gelatin at pH 7.0.

(K) Litmus milk All strains grow slightly, but milk coagulation and litmus color change are not observed.

(B) Physiological properties (a) The results of the following physiological tests were negative for all strains.

Reduction of nitrate, denitrification reaction, MR test, VP test, formation of indole, formation of hydrogen sulfide, use of inorganic nitrogen source (sodium nitrate, ammonium sulfate), formation of pigment, use of citric acid (Christensen, Simons ), Urease,
OF test.

(B) The results of the following physiological tests were positive for all strains.

Oxidase, catalase.

(C) Hydrolysis of starch Bacillus sp. KSM-2001, 2003: Decomposes.

Bacillus sp. KSM-2002, 2005: No decomposition.

(D) Growth pH range Bacillus sp. KSM-2001: Good growth at pH 8 to 10.0. However, it can grow in the range of pH 7.0 to 11.0.

Bacillus sp. KSM-2002: Grows at pH 6.0 to 11.0.

Bacillus sp. KSM-2003: Grows at pH 7.0 to 11.0.

Bacillus sp. KSM-2005: Grows at pH 7.0 to 10.0.

(F) Growth temperature range Bacillus sp. KSM-2001: Good growth at 20 ° C to 37 ° C. However, it can grow within the range of 10 ℃ to 40 ℃.

Bacillus sp. KSM-2002,2003,2005: Good growth at 20 to 30 ° C. However, it can grow within the range of 10 ℃ -37 ℃.

(G) Attitude toward oxygen Any strain can grow under aerobic conditions.

(H) Production of Acid and Gas from Sugars None of the strains produced acid and gas from the following saccharides.

L-arabinose, D-xylose, D-glucose,
D-mannose, D-fructose, D-galactose, maltose, sucrose, lactose, trehalose, D-sorbitol, D-mannitol, inositol, glycerol, starch.

(I) Utilization of sugars (J) Resistance to sodium chloride Bacillus sp. KSM-2001, 2002, 2003: Grow in the presence of 10% sodium chloride.

Bacillus sp. KSM-2005: Grows in the presence of 7% sodium chloride.

The mycological properties of the above alkaline protease-producing bacteria,
"Barge Aise Manual of Date Terminator Bacteriology 8th Edition (1974)" and "The
Genus Bacillus, Agricultural Handbook
O.427 (1973) ”and the comparison with other strains is as follows.

It is clear that all the strains are Gram-positive spore-forming bacteria belonging to the genus Bacillus, but with some differences, they can assimilate glucose, mannitol, etc., but are acidogenic. Is not observed, it has no nitrate reducing ability, it does not utilize citric acid, and it cannot grow under anaerobic conditions.
It is considered to be closely related to Bacillus brevis. However, Bacillus brevis differs from the bacterium of the present invention in that it cannot grow in a medium containing 5% salt.
Further, although closely related to Bacillus pasteurii, the present invention is advantageous in that it grows under anaerobic conditions, does not require NH ₄ Cl in alkaline medium, and does not convert urea into ammonium carbonate. It is different from the fungus. Bacillus Freudenreich (Bacill) located between Bacillus Brevis and Bacillus pasteuri
us freudenreichii), but is different from the bacterium used in the present invention in that it converts urea and deaminates phenylalanine.

Further, among these strains used in the present invention, Bacillus sp.
Both KSM-2001 and -2003 have the ability to hydrolyze starch, and Bacillus alcalophilus (Bacillus a
Lactobacillus alcalophilus), but Bacillus alcalophilus cannot grow at pH7, whereas these strains differ at pH7 or higher.

From the above points, the present inventor has found that the alkaline protease producing bacterium used in the present invention, that is, Bacillus sp. KSM-2.
All of 001, KSM-2002, KSM-2003 and KSM-2005 were judged to be new Bacillus species different from the conventionally known Bacillus species. The strains were deposited at the Institute for Microbial Technology, National Institute of Industrial Science, as follows.

Bacillus sp. KSM-2001 (Microtechnical Lab. No. 9449) Bacillus sp. KSM-2002 (Microcosmic Lab. No. 9450) Bacillus sp. KSM-2003 (Microtechnical Lab. No. 9451) Bacillus SP KSM-2005 (Microtechnology Research Institute, No. 9453) To obtain the novel alkaline protease of the present invention using each of the above strains, inoculate the strain in an appropriate medium and culture according to a conventional method. Just do it. It is preferable to contain an appropriate amount of assimilable carbon source and nitrogen source in the medium.

The carbon source and the nitrogen source are not particularly limited, but examples thereof include, as the nitrogen source, corn gluten meal, soybean flour, corn steep liquor, casamino acid, yeast extract, pharmamedia, sardine meal, and meat extract. ,
Peptone, High Pro, Aji Power, Corn Meal, Soy Bean Meal, Coffee Meal, Cottonseed Oil Meal, Cultivator,
Amiflec and aciplon, zest, azix and the like can be mentioned. As the carbon source, assimilable carbon sources, for example, arabinose, xylose, glucose,
Examples thereof include mannose, fructose, galactose, sucrose, maltose, lactose, sorbitol, mannitol, inositol, glycerin, soluble starch, inexpensive molasses, invert sugar and the like, and organic acids such as acetic acid that can be assimilated. In addition, phosphoric acid, Mg ²⁺ , Ca ²⁺ , Mn ²⁺ , Zn ²⁺ , C
Inorganic salts such as o ²⁺ , Na ⁺ , and K ⁺ , and if necessary, inorganic and organic micronutrient sources can be appropriately added to the medium.

Collection and purification of the protease, which is a target substance, from the thus obtained culture can be carried out according to general means for collecting and purifying enzymes.

In other words, cells are separated by centrifuging or culturing the culture, and the cells are separated from the cells and the culture solution by a conventional method such as salting out method, isoelectric focusing method, solvent precipitation method. The protein of the present invention is obtained by precipitating the protein by a method (methanol, ethanol, isopropanol, acetone, etc.) or concentrating the protein by ultrafiltration (for example, Diaflow Membrane YC, manufactured by Amicon). In the salting out method, for example, ammonium sulfate (30 to 70% saturated fraction), in the solvent precipitation, for example, the enzyme is precipitated in 75% ethanol, and then freeze-dried by over- or centrifugation and desalting. It is also possible to use powder. As a desalting method, a general method such as dialysis or gel perfusion method using Sephadex G-25 or the like is used.

The enzyme solution thus obtained may be used as it is, or may be further purified and crystallized by a known method before use. To further purify the enzyme, for example, adsorption chromatography such as hydroxyapatite chromatography, ion exchange chromatography such as DEAE-Sephadex or DEAE-cellulose, and molecular sieve gel chromatography such as Sephadex or biogel are appropriately combined. It can be purified separately.

The alkaline protease of the present invention thus obtained has the following physicochemical properties. In the following, the alkaline proteases produced by Bacillus sp. KSM-2001 strain are referred to as alkaline protease K-2001 and KS.
The product of M-2002 strain is alkaline protease K-2.
002 and that produced by the KSM-2003 strain are referred to as alkaline protease K-2003, and those produced by the KSM-2005 strain are referred to as alkaline protease K-2005, respectively.

(A) Substrate specificity The activity of each enzyme was the same for urea-modified hemoglobin, and the activity for keratin, casein, myoglobin, and bovine serum albumin was measured. 100 mM borate buffer (p
H10.5) with 1% of each substrate (2.2% for urea-modified hemoglobin)
%), 1 × 10 ^-4 AU of each enzyme solution was added, and 10
Reaction was performed for 60 minutes (60 minutes when keratin was used as a substrate). Table 1 shows the relative activity of each enzyme when the activity of API-21 for each substrate is 1.

As is clear from this result, the alkaline protease of the present invention is an enzyme that is currently often used as a detergent enzyme.
Compared to API-21, Alcalase, Savinase, etc., it has excellent keratin degradability.

(B) Optimal pH of reaction Add urea-denatured hemoglobin at a final concentration of 2.2% to each buffer solution (100 mM), add 1.0 × 10 ⁻⁴ AU of each enzyme solution, and add 25
The reaction was carried out at 10 ° C for 10 minutes. The optimum pH value is 100%, and each pH is
The relative initial reaction rate was expressed. This result is
Shown in Figures A to 1-D. The buffer solutions used and their pH ranges are as follows.

Acetate buffer pH 4.0-6.0 Phosphate buffer pH 6.0-8.0 Borate buffer pH 8.0-10.0 Carbonate buffer pH 9-11.0 Sodium phosphate buffer pH 10.5-12.0 As is apparent from these figures, alkaline protease K
The optimum pH of -2001 and K-2002 was 9.5 to 10.5, the optimum pH of alkaline protease K-2003 was 8.5 to 11.5, and the optimum pH of alkaline protease K-2005 was 8.5 to 10.0.

(C) pH stability 2 in each same buffer (100 mM) used in (b) above.
An enzyme solution of × 10 ^-4 AU was added, and the mixture was left at 5 ° C for 24 hours. After diluting this treated solution 5 times with 0.5 M borate buffer, the reaction was carried out at pH 10.5 using urea-modified hemoglobin as a substrate, and the residual activity was measured. The enzyme activity before treatment was defined as 100%, and the activity of the sample treated in each pH range was relatively expressed. The results are shown in FIGS. 2-A to 2-D.

As is clear from these figures, alkaline protease K
-2001 and K-2002 are stable in the range of pH 4 to 12, and alkaline proteases K-2003 and K-2005 have a pH of 5
It was stable in the range of ~ 12.

(D) Optimum reaction temperature The results of carrying out the reaction at pH 10.5 using urea-modified hemoglobin as a substrate using each enzyme solution of 1 × 10 ⁻⁴ AU under each temperature are shown in FIGS. 3A to 3D. Shown in the figure.

As is clear from these figures, alkaline protease K
-2001 and K-2005 have an optimum temperature of 40 ° C, alkaline proteases K-2002 and K-2003 have an optimum temperature of 35 to 40 ° C, and 20 to 30 ° C for all enzymes.
It showed more than 50% of the maximum activity in the low temperature range.

(E) Effect of surfactants and builders About 2 × 10 ^-4 AU of each alkaline protease was added to a 50 mM borate buffer solution (pH 8.0) in which various kinds of surfactants or builders of predetermined concentrations were dissolved, and the mixture was added at 25 ° C. It was left for 1 hour. afterwards,
After diluting 5-fold with the same buffer, the residual activity was measured by the Anson-hemoglobin method. The enzyme activity treated in the same manner without the addition of a surfactant and a builder was defined as 100%, and the residual activity was expressed as a relative value. The results are shown in Table 2.

As is clear from this result, alkaline protease K
-2001 and K-2002 were stable to all surfactants and builders tested. Alkaline protease K-2003 was slightly unstable to LAS, SAS and EDTA, but very stable to other surfactants and builders. Furthermore, alkaline protease K-20
Although 05 is slightly inferior to the former alkaline protease, it can be said that it is relatively stable.

(F) Effects of enzyme inhibitors Regarding general enzyme inhibitors, that is, parachloromercury benzoate (PCMB), diisopropylfluoridate (DFP), antipain, leupeptin, pepstatin, and chymostatin, these are alkalis of the present invention. The effect on proteases was investigated. Each inhibitor is
100 mM borate buffer (pH 8.0) (DF
For P only, add to 100 mM Tris-HCl buffer (pH 7.0) 4
After the addition of × 10 ^-4 AU of alkaline protease, the mixture was treated at 25 ° C for 1 hour. Then, after diluting 10 times with the same buffer, the residual activity was measured by the Anson-hemoglobin method. The residual activity was expressed as a relative value to the enzyme activity treated in the same manner without the addition of an inhibitor. The results are shown in Table 3.

Since both enzymes are inhibited by DFP and chymostatin, it is suggested that they are serine proteases, but the inhibition rate is much smaller than that of conventional serine proteases, which is one of the features of the alkaline protease of the present invention. It has a strong resistance to these inhibitors.

(G) Effect of metal ions Table 4 shows the results of investigations on the effect of metal ions on the enzyme activity. Each metal salt was added to 0.1 M borate buffer (pH 8.0) so as to have a concentration of 5 mM, and each alkaline protease (4 × 10 ⁻⁴ AU) was added, followed by treatment at 25 ° C. for 10 minutes. Then, the enzyme activity was measured by the Anson-hemoglobin method after diluting 10 times with the same buffer solution. The system in which metal salt powder was added was used as a control, and the effect of each metal ion was expressed as a relative value when the activity was set to 100.

As a result, all of the proteases were strongly inhibited by Cu ²⁺ , but were very stable to other metal ions and had no effect.

(H) Molecular Weight The molecular weight of each alkaline protease was measured by gel perchromatography using Toyopearl HW-55.
In this gel filtration, as the eluent, 0.1 mM potassium chloride, 10 mM Tris-hydrochloric acid buffer solution (pH 7.0) containing 5 mM calcium chloride was used, and bovine serum albumin (MW: 68,000) and ovalbumin ( MW: 45,000), chymotrypsinogen A (MW: 25,000), and ribonuclease A (MW: 13,700). The results are shown in Table 5.

[Effects of the Invention] As described above, the alkaline protease of the present invention exhibits a high activity on the alkaline side, and this activity has 50% or more of the maximum activity even in a low temperature range. Further, this alkaline protease has excellent decomposing power for keratin and the like, and is present in various components such as a surfactant, a builder component, and a metal ion that are mixed in a detergent composition or mixed during washing. Almost no decrease in the action is observed even below. Therefore, the protease of the present invention is extremely excellent as a protease to be added to each detergent.

〔Example〕

 Next, the present invention will be described in more detail with reference to examples.

Reference Example 1 Separation and collection of protease-producing strain: (i) About 1 g of a soil sample collected in Haga-gun, Tochigi Prefecture was suspended in 10 ml of sterilized physiological saline, and heat-treated at 80 ° C for 20 minutes. The heat-treated supernatant (0.1 ml) was inoculated into a keratin halo agar medium, and statically cultured at 30 ° C. for 48 hours. The composition of the keratin halo agar medium used is as shown below.

Glucose 1% Yeast extract 0.2% Wool keratin 1% Carboxymethylcellulose 1% Potassium phosphate 0.1% Magnesium sulfate heptahydrate 0.02% Agar 1.5% (ii) 10% Carbonate obtained by separately sterilizing the above medium composition Add sodium 0.01%, 0.1%, 1%, final pH 7.0,
After adjusting to 9.0 and 10.5, a plate medium was prepared.

After culturing, those that produced halos around the grown colonies were selected and purified in the same medium 2 to 3 times to obtain uniform protease-producing bacteria. The number of strains obtained by plate culture at each pH from 300 soil samples was 120 strains.

(Iii) These strains obtained in (ii) above were inoculated into a keratin liquid medium shown below, and shake culture was aerobically performed at 30 ° C. for 48 hours.

Glucol 0.2% Keratin (wool) 0.5% Yeast extract 0.05% Potassium phosphate 0.1% Magnesium sulfate (7-hydrate) 0.02% Sodium carbonate (separate sterilization) 0.4% pH 9.0 Degradation of keratin during or after culturing A prominent strain was selected to obtain an alkaline protease-producing bacterium.

Each of the 4 strains thus obtained was subjected to Bacillus sp.
It was named KSM-2001, 2002, 2003 and 2005.

Example 1 Bacillus sp. KSM-2001 obtained in Reference Example 1
(FERM.P-9449), KSM-2002 (FERM.P-9450), KSM-
2003 (FERM.P-9451) and KSM-2005 (FERM.P-9453)
Inoculate each of the following liquid medium and aerobically at 30 ° C
Shaking culture was performed for 48 hours to produce alkaline protease.

Glucose 2% Fish meat extract 1% Soybean flour 1% Magnesium sulphate 0.02% Potassium potassium phosphate 0.1% pH 10.0 After completion of the culture, the culture obtained is centrifuged (3000 rpm; 10 minutes) to remove the bacteria and obtain The protease activity was measured using the obtained culture supernatant as an enzyme solution. The activity of alkaline protease was measured by the following two methods.

(A) Anson using urea-modified hemoglobin as a substrate
Hemoglobin method: According to Anson's method (Anson, ML: J.Ger.Physiol, 22 79
(1938)) Bovine serum hemoglobin was denatured with urea and adjusted to pH 10.5 with caustic soda. This substrate solution (2.2% as hemoglobin) was added to 0.5 ml with 0.1 ml of enzyme solution (1.0 x 10
^{-5 to} 1.0 × 10 ^-3 AU) was added and the reaction was carried out at 25 ° C. for 10 minutes, and 1.0 ml of 4.9% trichloroacetic acid was added to stop the reaction. After completion of the reaction, centrifugation (3000 rpm for 10 minutes) was performed, and the protein degradation product contained in the supernatant was quantified by the Foulin-Lowry method (OHLowry et al., J. Biol. Chem. 193 265 (1951)). .

1 A.U. is the amount of enzyme that liberates 1 mmole of tyrosine in 1 minute under the above reaction conditions.

(B) Method using keratin as a substrate: To 0.5 ml of a solution prepared by suspending 0.5% wool keratin in 20 mM carbonate buffer, 0.1 ml of enzyme solution was added and reacted at 25 ° C. for 2 hours to obtain 4.9% trichloroacetic acid. The reaction was stopped by adding 1.0 ml of an aqueous solution. The following operations were the same as those in (a), and the amount of proteolytic products was measured. 1 KU was the amount of enzyme that liberates 1 mmole of tyrosine in 1 minute under the above reaction conditions.

The protease activity of the enzyme solution obtained in this Example was 6th.
It is as shown in the table.

Example 2 Ammonium sulfate was added to each enzyme solution obtained in Example 1 so as to be 75% saturated. Centrifuge the resulting precipitate (8000 rpm
After 15 minutes) and dialysis against ion-exchanged water, the enzymatic properties (physical and chemical properties) were examined and the results were as described above.

Example 3 Cleaning test: A cleaning test was performed according to JIS K 3371. The cleaning system is
0.133% of commercial phosphorus-free heavy detergent (without addition of enzyme)
After being prepared with 4 ° DH of water, the alkaline protease K-2001, K-2002 and K-2003 protease solutions were added at 0.01 AU / each.

The test cloth should be the collar part of a shirt (wearing for 3 days) where dirt on human skin tends to adhere, so that paired comparisons can be made.
After cutting to about 11 × 13 cm, it was immersed in a cleaning system with or without addition of enzyme at 30 ° C. for 1 hour. After immersion, use a targotometer (Kamishima Seisakusho) at 20 ° C, 120 rpm
And washed for 10 minutes. After rinsing and drying, a pair of collar cloths (15
(3 sets), the degree of stain removal was visually judged by three skilled judges. The judgment method is 5 points when the stains are almost completely removed, and 1 point when the stains are almost completely removed. The total evaluation score of 15 pieces of the collar cloth is calculated, and the evaluation score by the cleaning system with no enzyme added. Was expressed as a detergency index. The results are shown in Table 7.

Example 4 Regarding the protease solutions of alkaline proteases K-2002 and K-2005, the enzyme concentration was 0.016 AU /, and 2 at 10 ° C.
A cleaning test at a low temperature was conducted in the same manner as in Example 4 except that the immersion was carried out for a period of time. The results are shown in Table 8.

From the above results, the alkaline protease of the present invention,
It is clear that the enzyme is an excellent detergent enzyme that can exert its action sufficiently at lower temperatures as well as the detergency at ordinary washing temperatures.

[Brief description of drawings]

FIGS. 1-A to 1-D are drawings showing the effect of pH on the activities of the alkaline proteases K-2001 to K-2005 of the present invention. FIGS. 2-A to 2-D are drawings showing the effect of pH on the stability of the alkaline proteases K-2001 to K-2005 of the present invention, respectively. FIGS. 3-A to 3-D are drawings showing the influence of temperature on the activity of the alkaline proteases K-2001 to K-2005 of the present invention.

Claims (1)

[Claims] 1. An alkaline protease selected from the group consisting of the following A, B, C and D. A. Alkaline protease having the following physicochemical properties (K
-2001). (1) Action It acts on urea-modified hemoglobin, keratin, casein, myoglobin, and bovine serum albumin. (2) Substrate specificity It has high activity against urea-modified hemoglobin, keratin, casein, myoglobin, and bovine serum albumin. Especially, it has high activity against keratin. (3) Effect of metal ion It is strongly inhibited by Cu ²⁺ . (4) Working pH and optimum pH The working pH range is 5.5 to 12.0. The optimum pH is 9.5 to 10.5, and even in the range of 7.5 to 11.5, the activity at the optimum pH is 5
It has a relative activity of 0% or more. (5) pH stability It is extremely stable at pH 4 to 11 and does not inactivate, and maintains an activity of 50% or more even at pH 4 to 12.5. (6) Optimum temperature The operating temperature is in a wide range from 10 to 70 ℃, and the optimum temperature is
40 ° C. Further, it has 50% or more of the activity at the optimum temperature even in the range of 18 to 48 ° C. (7) Molecular weight: about 17,000 (by gel filtration method) B. Alkaline protease with the following physicochemical properties (K
-2002). (1) Action It acts on urea-modified hemoglobin, keratin, casein, myoglobin, and bovine serum albumin. (2) Substrate specificity It has high activity against urea-modified hemoglobin, keratin, casein, myoglobin, and bovine serum albumin. Especially, it has high activity against keratin. (3) Effect of metal ion It is strongly inhibited by Cu ²⁺ . (4) Working pH and optimum pH The working pH range is 5.5 to 12.0. The optimum pH is 9.5 to 10.5, and even in the range of 7.0 to 11.5, the activity at the optimum pH is 5
It has a relative activity of 0% or more. (5) pH stability It is extremely stable at pH 7.0 to 10.5 and does not deactivate, and maintains activity of 80% or more even at pH 4 to 12. (6) Optimum temperature The operating temperature is in a wide range from 10 to 70 ℃, and the optimum temperature is
35-40 ° C. Further, it has 50% or more of the activity at the optimum temperature even in the range of 18 to 45 ° C. (7) Molecular weight approx. 67,000 (by gel filtration method) C. Alkaline protease having the following physicochemical properties (K
-2003). (1) Action It acts on urea-modified hemoglobin, keratin, casein, myoglobin, and bovine serum albumin. (2) Substrate specificity It has high activity against urea-modified hemoglobin, keratin, casein, myoglobin, and bovine serum albumin. Especially, it has high activity against keratin. (3) Effect of metal ion It is strongly inhibited by Cu ²⁺ . (4) Working pH and optimum pH The working pH range is 5 to 12 or more. The optimum pH is 8.5 to 11.5, and the activity at the optimum pH is 50 even in the range of 7 to 12.
% Or more relative activity. (5) pH stability It is extremely stable at pH 5 to 10.5 and does not inactivate, and maintains an activity of 50% or more even at pH 4 to 12.5. (6) Optimum temperature The operating temperature is in a wide range from 10 to 70 ℃, and the optimum temperature is
35 ° C. Further, it has 50% or more of the activity at the optimum temperature even in the range of 18 to 48 ° C. (7) Molecular weight of about 53,000 (by gel filtration method) D. Alkaline protease having the following physicochemical properties (K
-2005). (1) Action It acts on urea-modified hemoglobin, keratin, casein, myoglobin, and bovine serum albumin. (2) Substrate specificity It has high activity against urea-modified hemoglobin, keratin, casein, myoglobin, and bovine serum albumin. Especially, it has high activity against keratin. (3) Effect of metal ion It is strongly inhibited by Cu ²⁺ . (4) Working pH and optimum pH The working pH range is 5 to 12 or more. The optimum pH is 8.5 to 10.0, and even in the range of 7.5 to 12 or more, the relative activity is 50% or more of the activity at the optimum pH. (5) pH stability It is extremely stable at pH 5 to 12 and does not inactivate, and maintains an activity of 50% or more even at pH 4.5 to 12.5. (6) Optimum temperature The operating temperature is in a wide range from 10 to 70 ℃, and the optimum temperature is
40 ° C. Further, it has 50% or more of the activity at the optimum temperature even in the range of 22 to 55 ° C. (7) Molecular weight about 40,000 (by gel filtration method)