JPH01101885A - Novel alkali protease and microorganism producing the same - Google Patents

Abstract

NEW MATERIAL:An enzyme of following physical, chemical properties: actions, it acts on urea-modified hemoglobin, keratin, casein, myoglobin, cattle serum albumen; substrate properties; it has high activity to afore-mentioned substrates, especially to keratin; effect of metallic ion, it is strongly inhibited by cupric ion; active and optimal pH, active in the pH range from 5.5 to 12.0, optimal pH from 9.5 to 10.5 and showing more than 50% activity of that in the optimal pH in the pH range from 7.5 to 11.5; pH stability, it is very stable, and not deactivated in the pH range from 4 to 11. USE:Alkali protease suitable for washing near room temperature. PREPARATION:A microorganism obtained from the soil at HAGA-GUN TOCHIGI prefecture, Japan, such as Bacillus sp. KSM-2001 (FERM 9449) is inoculated in a medium containing carbon and nitrogen sources and cultured according to the usual manner.

Description

[Detailed Description of the Invention] [Field of Industrial Application] The present invention relates to a novel alkaline protease and a microorganism that produces the same, and more specifically, to a novel alkaline protease that can be widely used as a cleaning enzyme suitable for Japanese laundry conditions. The present invention relates to an alkaline protease and an alkaline protease-producing bacterium belonging to the genus Bacillus.

[Conventional technology]

The combination of proteases into detergents has been practiced for some time, but the nine proteases originally added to detergents have an optimal reaction pH near neutrality, such as /Q-in and pancreatic trypsin. A friend who is being used.

However, as is well known, the pH of detergents is on the high alkaline side, and enzymes for detergents have an optimum reaction pH on the alkaline side, and it is desired that they be stable in surfactants and have been developed. As a result, the market for alkaline protease-containing detergents began to expand rapidly in Western countries from 1967 onwards. In Japan, full-fledged protease-containing detergents have been on the market since 1979.

Typical alkaline proteases currently used as detergent enzymes include alcalase, sabinase, esperase (manufactured by Novo Industries), and maxatase (manufactured by Gist Zolocardis), and the optimal reaction temperature for these enzymes is around 60°C. This is in line with the washing style in Western countries.

However, since Japanese laundry methods generally involve washing at around room temperature, there is a need to provide a protease that has stronger activity than conventional proteases in a lower temperature range. as API-2
1 (manufactured by Showa Denko) has been developed.

[Problem that the invention seeks to solve]

It has long been known that proteases, which have the ability to effectively degrade insoluble proteins such as keratin, contribute to cleaning (Minyouki: Senkaishi 26322 (1985)), and the various requirements of the proteases mentioned above have been known to contribute to cleaning. If a detergent contains a protease that has the properties of being active even in alkaline conditions and having high activity even at low temperatures, it also has excellent effects on insoluble proteins such as keratin, resulting in excellent cleaning. It is expected that detergent compositions with strength and ease of use will be obtained.

Therefore, there has been a demand for the development of an alkaline protease having such properties.

[Means to solve the problem]

Therefore, the present inventors have the ability to decompose keratin,
In order to obtain an alkaline protease with a low optimum reaction temperature (high activity even at low temperatures), we conducted an intensive search for zolotease producing bacteria.

As a result, they discovered that a group of microorganisms obtained from soil in Haga District, Tochigi Prefecture produce alkaline protease that satisfies the above conditions, and completed the present invention.

The microorganisms producing the alkaline protease of the present invention are Bacillus sp.
KSM-2001, Bacillus sp. KSM-200
2. They are named Bacillus O sp. 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: Bacillus 0.5-1
．． OX 1.0~35μm Pal≠Russp KSM-2002: Bacillus 0.5~0
．． 8 X 1.5-3.0μm Bacillus sp. KS
M-2003: Bacillus 0.5-0.8 X O, 8-23
μm Bacillus sp. KSM-2005: Bacillus 05=
OBX 1.5-5.0pm (b) No polymorphic strains are observed.

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

(d) Spores (size, shape, location) Bacillus sp. KSM-2001: 0.4-0.6
X0.4-0.6 μm, oval or circular, central quasi-terminal Bacillus sp. KSM-2002: 0.4-0.8X0
．． 4-0.8 μm 9-circular or di-β-circular, central quasi-terminal Bacillus sp. KSM-2003:Q, 5-0.8X0.
8-1.2 μm, oval or cylindrical, central subterminal Bacillus sp., KSM-2005: 0.5-0.8
X0.8-1.2 μm, oval, central quasi-edge (e) Durham stain positive for all strains (f) acid-fast negative for all strains (g) Growth form on broth agar plate Bacillus sp. KSM- 2001: Grows at pH 7.0 and forms circular, medium convex, and smooth wave-like colonies. The size of the colony is 1.0 to 4.0 m in diameter, and the surface is smooth and has a dewdrop-like pale yellow color. The colonies are translucent and oily.

Bacillus sp. KSM-2002: Forms circular, convex circular, and smooth wave-shaped colonies. The size is 1.0 in diameter
~5.0in The surface is smooth, dewdrop-like, and pale yellow to milky white. It is also a translucent and oily colony.

Bacillus sp. KSM-2003: Grows in RiH7,O and forms circular, table-shaped, and smooth wave-shaped colonies. The surface of the colony is smooth, dewdrop-shaped, and pale yellow in color.

It is also a translucent and oily colony.

Nolayer Chills Sp. KSM-2005: pH 7.0
It grows in round, medium convex, or smooth wavy colonies. The surface of the colony is smooth, dewdrop-shaped, and pale yellow in color. It is also translucent and has finger-shaped colonies.

(h) Growth on a gravy agar slope Bacillus sp. KSM-2001: At pH 7.0, it shows weak band-like growth and forms milky white, translucent colonies. It grows in a wave band shape at pH 10.0.

Bacillus sp. KSM-2002: shows weak band-like growth at pH 7.0, forming milky white, translucent colonies. It grows in a wave band shape at pH 10.0.

Bacillus sp. KSM-2003: shows weak band-like growth at pH 7.0, forming milky white, translucent colonies. It grows in a wave band shape at pH 10.0.

Bacillus sp. KSM-2005: pH70*
10.0, it grows in a wave band shape and forms milky white, translucent colonies.

(i) Broth liquid culture All bacteria grow slightly at pH 7.0, but no bacterial membrane is formed.

0) Meat juice gelatin puncture culture Bacillus sp. KSM-2001: Gelatin did not liquefy at pH 7.0.

Bacillus ax subi KSM-2002, 2003, 20
05: Both gelatin is liquefied at pH 7 and 0.

Provisional) Lidomus Milk Both strains grow slightly, but no coagulation of milk or change in lidmus color is observed.

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

Nitrate reduction, denitrification reaction, MR test, vP test, indole production, hydrogen sulfide production, utilization of inorganic nitrogen sources (
Nitric acid) IJum.

Ammonium sulfate, production of pigment, use of citric acid (C
hristensen, Simons' medium), 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: Not decomposed.

(d) Growth pH range Bacillus sp. KSM-2001: pH 8-10.
Growth is good at 0. However, it can grow within the pH range of 7.0 to 11.0.

Padels ΦSP KSM-2002: -pH6,0
Grows at ~11,0.

Bacillus sp. KSM-2003: pH 7.0-1
Grows at 1.0.

Bacillus sp. KSM-2005: pH 7.0-1
Grows at 0,0.

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

Bacillus xsM-zoo2, 2oo3, 2o
os: Good growth at 20°C to 30°C. However, 10℃~3
Can grow within a temperature range of 7℃.

(ω Attitude towards oxygen Both strains can grow under both aerobic and anaerobic conditions.

(h) Production of acids and gases from sugars None of the strains produced acids and gases from the following sugars.

L-arabinose, D-xylose, D-glucose,
D-mannose, D-7 lactose, D-galactose, maltose, sucrose, lactose, Toreno 1
0~su, D-sorbitol, D-mannitol, inositol, glycerol, starch.

(i) Ability to assimilate sugars (j) Resistance to sodium chloride Bacillus sp.

Bacillus nisgi - KSM-2005 grows in the presence of near % sodium chloride.

The mycological properties of the above alkaline protease-producing bacteria are
Bacillus Zazonus, Agriculture Handbook N1427 (1974)
According to the description by J. 1973), the comparison with other strains is as follows.

It is clear that both strains are Durham-positive spore-forming bacteria belonging to the genus Bacillus, but although there are some differences, they are capable of converting glucose, mannitol, etc., but are unable to produce acid. Bacillus
It is considered to be closely related to Bacillus brevis.

However, Bacillus plevis differs from the bacteria of the present invention in that it cannot grow in a medium containing 5% salt. In addition, Pf A/ se zQ, X large (Bacill
steurii), but differs from the bacterium of the present invention in that it grows under anaerobic conditions, does not require NH2Cl in an alkaline medium, and does not convert urea to ammonium carbonate. In addition, Bacillus 70 Idenreich (Bacillus 70 Idenreich), which is located between Bacillus plebis and Bacillus 9 sturi,
udenreichii), but differs from the bacterium of the present invention in that it converts urea and deamines phenylalanine.

Furthermore, among the strains of the present invention, Bacillus sp. KSM-200
1 and -2003 both have starch hydrolyzing ability.
Although Bacillus alcalophilus is considered to be a close relative of Bacillus alcalophilus, these strains differ in that they can grow at pH 7 or higher, whereas Bacillus alcalophilus cannot grow at pH 7.

From the above points, the present inventor has developed the alkaline protease-producing bacterium of the present invention, namely, Bacillus nis♂-KSM-20.
01, KSM-2002, KSM-2003 and KSM
-2005 are all conventionally known. It was determined that this is a new bacterial species of the genus Bacillus, which is different from the species of the genus Bacillus. The strains were deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology as follows.

Bacillus sp. KSM-2001 (@Koken Bacillus No. 9
No. 449) Bacillus sp. KSM-2002 (Feiko Kenzonoyori No. 9450) Bacillus sp. KSM-20
03 (Meiko Kenzonoyori No. 9451) Bacillus sp. K
In order to obtain the novel alkaline protease of the present invention using the above-mentioned strains SM-2005 (Feikoken Bacterial Serial No. 9453), the strain may be inoculated into an appropriate medium and cultured according to a conventional method. It is preferable that the medium contains appropriate amounts of a carbon source and a nitrogen source that can be converted into precious metals.

There are no particular restrictions on the carbon and nitrogen sources, but examples of nitrogen sources include corn gluten meal, soybean flour, cornsteel liquor, casamino acids, yeast extract, Pharmamedia, sardine meal, and meat extract. , peptone, Hizoro, Azono9 War, cornmeal, Nyipeen meal, coffee grounds, cotton oil cake, cultivator,
Examples include Amiflex, Azozolone, Zest, and Azotux. Further, as a carbon source, an assimilable carbon source such as arabinose, xylose, glucose,
Examples include mannose, fusictose, caractose, sucrose, maltose, lactose, sorbitol, mannitol, inositol, glycerin, soluble starch, inexpensive blackstrap molasses, invert sugar, and assimilable monoacids such as acetic acid. In addition, phosphoric acid, Mg2+, Ca2+,
Hn2+, zn2+.

Inorganic salts such as Co'', Na+, K+, and, if necessary, inorganic or organic trace nutrients can be added to the medium as appropriate.

Collection and purification of the target substance, protease, from the culture thus obtained can be carried out in accordance with common enzyme collection and purification methods.

That is, the bacterial cells are separated by centrifugation or filtration of the culture, and the bacterial cells and culture F solution are separated by conventional separation methods such as salting-out method, isoelectric focusing method, solvent precipitation method (
The protease of the present invention is obtained by precipitating the protein with methanol, ethanol, indelonol acetone, etc.) or concentrating it by ultrafiltration (for example, Diaflow membrane YC1 manufactured by Amicon). In the salting-out method, for example, ammonium sulfate (30 to 70% saturated fraction), and in the solvent precipitation method, for example, after precipitating the enzyme in 75tIi ethanol,
It is also possible to make this into a freeze-dried powder by filtration, centrifugation, and desalting.

Desalination methods include dialysis or Sephadex G-25
General methods such as gel filtration method using etc. are used.

The enzyme solution obtained in this way can be used as it is, but it can also be further purified and crystallized by a known method for use. Further purification of the enzyme can be achieved by adsorption chromatography, e.g. hydroxyanoq-tight chromatography, DEAE-Sephadex or DEAE-Sephadex.
Fractional purification may be carried out by appropriately combining ion exchange chromatography such as AE-cellulose and molecular sieve Dull chromatography such as Sephadex or Biodal.

The alkaline protease of the present invention thus obtained has the following physicochemical properties. In addition, in the following, alkaline protease produced by Bacillus sp.
01, the product produced by the KSM-2002 strain to alkaline protease, -2002, the product produced by the KSM-2003 strain to alkaline protease, -2003, and K
The alkaline protease produced by strain SM-2005 is referred to as -2005.

(a) Substrate specificity The activities of each enzyme against urea-denatured hemoglobin were made the same, and the activities against keratin, casein, myoglobin, and bovine serum albumin were measured. Add 1% of each substrate (12% for urea-denatured hemoglobin) to 100 mM borate buffer (pH 10,5), and add each enzyme solution I
U was added and the reaction was carried out at 25° C. for 10 minutes (60 minutes in the case of using keratin as a substrate). Table 1 shows the relative activity of each enzyme when the activity of API-21 for each substrate is set as 1.

As is clear from these results, the alkaline protease of the present invention has superior keratin decomposition properties compared to the enzymes API-21, Alcalase, Savinase, etc., which are currently commonly used as enzymes for detergents.

(b) Optimum reaction pH Urea-denatured hemoglobin with a final concentration of 2% was added to each buffer solution (100 mM), and each enzyme solution of 1.0X10~'A, U was added, and the reaction was performed at 25°C for 10 minutes. . optimal p
The initial reaction rate at each pH was expressed relative to the value of H as 100%. The results are shown in Figures 1-A to 1-D. The buffers used and their pH ranges are as follows.

Acetate buffer pH4.0~6.0 Phosphate buffer pH6.0~&0Borate buffer pH&
o-10.0 carbonate buffer pH 9-11.0 sodium phosphate buffer pH 10.5~I ZO As is clear from these figures, alkaline protease has a -2
The optimum pH for 001 and Ni-2002 is 9.5-10.5
, the optimum pH of -2003 for alkaline protease is &5
~11.5, the optimum pH of -2005 for alkaline protease was 8.5-10.0.

(c) pH stability The same buffers used in (b) above (100mM)
Add 2 x 10~' A, U enzyme solution and heat at 5°C.
I left it for 24 hours. This treated solution was diluted 5 times with 0.5 M borate buffer, and then reacted with urea-denatured hemoglobin as a substrate at pH 10.5, and the residual activity was measured. The enzyme activity before treatment was taken as 100%, and the activity of samples treated in each pH range was compared. This result is the second
-A to 2-D.

As is clear from these figures, alkaline protease -2001 and -2002 were stable in the pH range of 4 to 12, and alkaline protease -2003 and -2005 were stable in the pH range of 5 to 12. .

(d) Optimum reaction temperature I - Shown in Figure D.

As is clear from these figures, the optimal temperature for alkaline protease is -2001 and -2005 at 40°C, and the optimum temperature for alkaline protease -2002 and -2003 is 30°C.
Each enzyme had its optimum temperature in the range of 5 to 40°C, and all enzymes exhibited 50% or more of their peak activity in the low temperature range of 20 to 30°C.

(e) Influence of surfactants and builders 2 x 1
0''-'A, U, and alkaline proteases were added to 50mM borate buffer (pH & 0) in which various surfactants or builders at predetermined concentrations were dissolved, and the mixture was left at 25°C for 1 hour. After diluting the sample 5 times, the residual activity of υ was measured using the Anson-hemoglobin method.The enzyme activity obtained in the same manner without the addition of surfactant or builder was taken as 100, and the residual activity was expressed as a relative value.The results are shown in Table 2.

As is clear from the results, alkaline protease ni-2001 and ni-2002 were stable against all the surfactants and builders tested. In addition, alkaline protease K-2003 is used for LAS, SAS and ED.
It was somewhat unstable to TA, but very stable to other surfactants and builders. Furthermore, although -2005 is slightly inferior to the former alkaline protease, it can be said to be relatively stable.

(f) Effect of enzyme inhibitors Common enzyme inhibitors, namely, 9-chloromercury penzoate (PCMB), diisopropyl fluoridate (DFP), annano-9ine, leupeptin,
To investigate the effects of pepstatin and chymostatin on the alkaline protease of the present invention, each inhibitor was prepared in 100mM borate buffer (pH 8.0) (100mM for DFP only) and lithium-hydrochloride to give a predetermined concentration. buffer pH 7,0) and 4 x 10~'A
,U.

After adding alkaline protease, the mixture was treated at 25°C for 1 hour. Thereafter, after diluting 10 times with the same buffer, the residual activity of υ was measured by the Ann-Hemoglopin 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.

All enzymes in Table 3 are inhibited by DFP and chymostatin, suggesting that they are Seri/proteases.
Its inhibition rate is very small compared to conventional serine proteases, and one of the characteristics of the alkaline protease of the present invention is that it has strong resistance to these inhibitors.

(2) Effect of metal ions Table 4 shows the results of investigating the effects of metal ions on enzyme activity. Each metal salt was adjusted to 5mM by 0.
1 M borate buffer (pH 8,0) plus each alkaline protease 4 x 10 = A, U.

After the addition, treatment was performed at 25°C for 10 minutes. Thereafter, the mixture was diluted 10 times with the same buffer, and the enzyme activity was measured by the annon-hemoglobin method. Using a system without metal salts as a control,
The influence of each metal ion was expressed as a relative value when its activity was taken as 100.

Table 4 below: Table 4. As a result, all proteases were strongly inhibited by Cu''+, but were very stable and unaffected by other metal ions.

(h) Molecular Weight The molecular weight of each alkaline protease was measured by Dull permeation chromatography using TOYO, e-L HW-55. In this gel filtration, the eluate is 0.1
10 m containing M potassium chloride, 5mM calcium chloride
M) 'J Sous acid buffer (pH 7,0) was used, and bovine serum albumin (MW: 6a00) was used as a standard protein.
0), ovalbumin (MW: 4 to 000), chymotridecinogen A (MW: 25,000), and ribonuclease A (MW: 1a,700) were used. The results are shown in Table 5.

Table 5 [Effects of the Invention] The alkaline protease produced by the alkaline protease-producing bacterium of the present invention shows high activity in alkaline areas such as soft soil, and this activity is more than 50% of the maximum activity even in the low temperature range. It has activity. Furthermore, this alkaline protease has excellent decomposition power for keratin, etc., and is effective against various components mixed in detergent compositions and washing, such as surfactants, builder components, and metal ions. Even in its presence, there is almost no decrease in its effect. Therefore, the protease of the present invention is extremely excellent as a protease to be incorporated into various detergents.

〔Example〕

Next, the present invention will be explained in more detail with reference to Examples.

Example 1 Isolation and collection of protease-producing bacterial strains: (1) Approximately 12' of a soil sample collected in Haga District, Tochigi Prefecture
The suspension was suspended in Jk sterilized physiological saline 1o-, left at 80°C for 20 minutes, and heat treated. 0.1 ml of the heat-treated supernatant was inoculated onto a keratin halo agar medium, and cultured under cover 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 Thicarzeximethylcellulose 1 Thirynic acid di-
Potassium 0.1% Magnesium sulfate heptahydrate 0.02% agar
1.5% (It) 10% of the above medium composition that has been separately sterilized
Add 0.01 yu, 0.1 ti, liter sodium carbonate,
After adjusting the final pHt to 7.0.9.0 and 10.5, a lithographic medium was prepared.

After culturing, those that produced a halo around the grown colonies were selected and purified two to three times using the same medium to obtain uniform protease-producing bacteria. Out of 300 soil samples, 12,011 strains were obtained from plate culture at each pH.

(Ill) These strains obtained in (i) above were inoculated into the keratin liquid medium shown below, and incubated aerobically at 30°C for 48 hours.
A shaking culture was performed for hours.

Glucose 0.2% Keratin (wool) o, 5 Ti Yeast extract 0.05 Potassium typhosphate 0.1 Magnesium thiosulfate (7 hydrate) 0.02% Sodium carbonate (separately sterilized) o, 4% pH 9, 0 After the cultivation period was completed, a strain with remarkable ability to decompose keratin was selected, and alkaline protease producing bacteria were obtained.

The four treasury stocks thus obtained were named Bacillus sp. KSM-2001, 2002, 2003 and 2005, respectively.

Example 2 Bacillus sp. KSM-2 obtained according to Example 1
001 (FERM, P-9449), KSM-2002
(FERM, P-9450), KSM-2003 (FERM
RM, P-9451) and 8M-2005 (FERM
, P-9453) was inoculated into the following liquid medium, and cultured with shaking aerobically at 30° C. for 48 hours to produce alkaline protease.

Glucose 2% Fish extract 1% Soybean flour 1% Magnesium sulfate 0.02% Potassium phosphate 0.1% pH 10.0 After culturing, the obtained culture was centrifuged (3000r
pm: 10 minutes) to remove the bacteria, and the resulting culture supernatant was used as an enzyme solution to measure its protease activity. Alkaline protease activity was measured by the following two methods.

(a) Anson-hemoglobin method using urea-denatured hemoglobin as a substrate: According to the method of Anson (Anson, M, L:
J, Ger.

Serum hemoglobin is denatured and adjusted to pH 10 with caustic soda.
, 5. This substrate solution (22% as hemoglobin)
] to 0.5d and enzyme solution 0.1a/(1,0xlO-1,
0x10 μA, U, ) was added and the reaction was carried out at 25°C for 10 minutes, and 4.9% trichloroacetic acid 1,0~ was added to stop the reaction. After the reaction, centrifugation (3000r
pm for 10 minutes) and the protein decomposition product contained in the supernatant was subjected to the Folin-Lowry method (0, H, Lowry et al., J. Biol. Chem 193265 (195
1)).

IA, U, is 1 m per minute under the above reaction conditions.
The amount of enzyme that releases mole of tyrosine was taken as the amount of enzyme.

(et al.) Method using keratin as a substrate: Add 0.5 mK of a solution of 0.5% wool keratin suspended in 20 mM carbonate buffer and 0.1-mK of enzyme solution,
The reaction was carried out for 02 hours, and 49% trichloroacetic acid aqueous solution 1.
0~ was added to stop the reaction. The following operations were the same as in (a), and the amount of protein degradation products was measured. Note that IKU was defined as the amount of enzyme that releases immole of tyrosine in 1 minute under the above reaction conditions.

The zolotease activity of the enzyme solution obtained in this example was
This is the table of contents.

Table 6 Example 3 Ammonium sulfate was added to each enzyme solution obtained in Example 2 to saturation of 75%. The resulting precipitate was centrifuged (800
After dialysis against ion-exchanged water (0 rpm 15 minutes), the enzymatic properties (physical and chemical properties) were as described above.

Example 4 Cleaning formula #: A cleaning test was conducted according to JIS K3371.

The cleaning system is a commercially available phosphorus-free heavy detergent (no enzyme added).
After preparing it with 4'pH water to 0.133%,
Each zolotease solution of -2001, -2002 and -2003 was added to alkaline protease at 0, OI AU
/l, respectively.

The test cloth was the collar of a dress shirt (worn for 3 days), where dirt on human skin tends to adhere, and was cut to about 11 x 13 cIII so that pairwise comparisons could be made, and washed with a washing system with or without enzymes. Immersion was performed at 30°C for 1 hour.

After the immersion was completed, washing was performed at 20° C. and 120 rpm for 10 minutes using a tergotometer (manufactured by Jyugen Seisakusho).

After rinsing and drying, the pairs of collar cloths (15 sets) were compared and the degree of stain removal was visually judged by three experienced judges. The evaluation method is 5 points if the stain is almost completely removed, 1 point if the stain is almost completely removed, and the total evaluation score for the 15 collar cloths is calculated, and the evaluation score is based on the cleaning system without enzymes added. The ratio when 100 was expressed as the detergency index. The results are shown in Table 7.

Table 7 Example 5 Enzyme concentration t"0.016 kU for alkaline protease -2002 and -2005 zolotease solutions
Example 4 except that immersion was carried out at 10°C for 2 hours.
A cleaning test at still temperature was conducted in the same manner as above. The results are shown in Table 8.

According to the results in Table 8 and above, the alkaline protease of the present invention:
It is clear that it is an excellent enzyme for round cleaners that not only has detergency at normal washing temperatures, but also exhibits its effectiveness at very low temperatures.

[Brief explanation of the drawing]

Figures 1-A to 1-D are diagrams showing the influence of pH on the activity of alkaline proteases -2001 to -2005 of the present invention, respectively. Figures 2-A to 2-D are diagrams showing the influence of pH on the stability of alkaline proteases of the present invention -2001 to -2005, respectively. Figures 3-A to 3-D are diagrams showing the influence of temperature on the activity of -2001 to -2005 alkaline proteases of the present invention. that's all

Claims (1)

[Claims] 1. An alkaline protease having the following physical and chemical properties. (1) Action: Acts on urea-denatured hemoglobin, keratin, casein, myoglobin, and bovine serum albumin. (2) Substrate specificity High activity against urea-denatured hemoglobin, keratin, casein, myoglobin, and bovine serum albumin. It has particularly high activity against keratin. (3) Influence of metal ions Strongly inhibited by Cu^2^+. 2. The alkaline protease (K-2001) according to claim 1, which further has the following properties. (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, it has a relative activity that is 50% or more of the activity at the optimum pH. (5) pH stability It is extremely stable at pH 4-11 and does not deactivate, and at pH 4-12.
5 also maintained an activity of 50% or more. (6) Optimal temperature The working temperature ranges over a wide range from 10 to 70°C, and the optimum temperature is 40°C. Moreover, even in the range of 18 to 48°C, it has 50% or more of the activity at the optimum temperature. (7) Molecular weight of about 17,000 (according to gel filtration method) 3. The alkaline protease (K-2002) according to claim 1, which further has the following properties. (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, it has a relative activity that is 50% or more of the activity at the optimum pH. (5) pH stability Extremely stable at pH 7.0 to 10.5, does not deactivate, pH 4
-12, the activity was maintained at 80% or more. (6) Optimal temperature The working temperature ranges over a wide range of 10-70°C, and the optimum temperature is 35-40°C. Moreover, even in the range of 18 to 45°C, it has 50% or more of the activity at the optimum temperature. (7) Molecular weight of about 67,000 (according to gel filtration method) 4. The alkaline protease (K-2003) according to claim 1, which further has the following properties. (4) Working pH and optimum pH The working pH range is 5 to 12 or higher. The optimum pH is 8.5 to 11.5, and even in the range of 7 to 12, the relative activity is 50% or more of the activity at the optimum pH. (5) pH stability Extremely stable at pH 5-10.5, does not deactivate, pH 4-1
Even at 2.5, the activity was maintained at 50% or more. (6) Optimal temperature The working temperature ranges over a wide range from 10 to 70°C, and the optimum temperature is 35°C. Moreover, even in the range of 18 to 48°C, it has 50% or more of the activity at the optimum temperature. (7) Molecular weight of about 53,000 (according to gel filtration method) 5. The alkaline protease (K-2005) according to claim 1, which further has the following properties. (4) Working pH and optimum pH The working pH range is 5 to 12 or higher. Optimum pH is 8.5
~10.0, and even in the range of 7.5 to 12 or more, it has a relative activity that is 50% or more of the activity at the optimum pH. (5) pH stability Extremely stable at pH 5-12, does not deactivate, pH 4.5-1
Even at 2.5, the activity was maintained at 50% or more. (6) Optimal temperature The working temperature ranges over a wide range from 10 to 70°C, and the optimum temperature is 40°C. Moreover, even in the range of 22 to 55°C, it has 50% or more of the activity at the optimum temperature. (7) Molecular weight: approximately 40,000 (according to gel filtration method) 6. A microorganism that belongs to the genus Bacillus and produces alkaline protease K-2001 having the following properties. (1) Action: Acts on urea-denatured hemoglobin, keratin, casein, myoglobin, bovine serum albumin, etc. (2) Substrate specificity High activity against urea-denatured hemoglobin, keratin, casein, myoglobin, and bovine serum albumin. It has particularly high activity against keratin. (3) Influence of metal ions 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, it has a relative activity that is 50% or more of the activity at the optimum pH. (5) pH stability It is extremely stable at pH 4-11 and does not deactivate, and at pH 4-12.
5 also maintained an activity of 50% or more. (6) Optimal temperature The working temperature ranges over a wide range from 10 to 70°C, and the optimum temperature is 40°C. Moreover, even in the range of 18 to 48°C, it has 50% or more of the activity at the optimum temperature. (7) Molecular weight: approximately 17,000 (according to gel filtration method) 7. A microorganism that belongs to the genus Bacillus and produces alkaline protease K-2002 having the following properties. (1) Action: Acts on urea-denatured hemoglobin, keratin, casein, myoglobin, and bovine serum albumin. (2) Substrate specificity High activity against urea-denatured hemoglobin, keratin, casein, myoglobin, and bovine serum albumin. It has particularly high activity against keratin. (3) Influence of metal ions 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, it has a relative activity that is 50% or more of the activity at the optimum pH. (5) pH stability Extremely stable at pH 7.0 to 10.5, does not deactivate, pH 4
-12, the activity was maintained at 80% or more. (6) Optimal temperature The working temperature ranges over a wide range of 10-70°C, and the optimum temperature is 35-40°C. Moreover, even in the range of 18 to 45°C, it has 50% or more of the activity at the optimum temperature. (7) Molecular weight: approximately 67,000 (according to gel filtration method) 8. A microorganism that belongs to the genus Bacillus and produces alkaline protease K-2003 having the following properties. (1) Action: Acts on urea-denatured hemoglobin, keratin, casein, myoglobin, and bovine serum albumin. (2) Substrate specificity High activity against urea-denatured hemoglobin, keratin, casein, myoglobin, and bovine serum albumin. It has particularly high activity against keratin. (3) Influence of metal ions Strongly inhibited by Cu^2^+. (4) Working pH and optimum pH The working pH range is 5 to 12 or higher. The optimum pH is 8.5 to 11.5, and even in the range of 7 to 12, the relative activity is 50% or more of the activity at the optimum pH. (5) pH stability Extremely stable at pH 5-10.5, does not deactivate, pH 4-1
Even at 2.5, the activity was maintained at 50% or more. (6) Optimal temperature The working temperature ranges over a wide range from 10 to 70°C, and the optimum temperature is 35°C. Moreover, even in the range of 18 to 48°C, it has 50% or more of the activity at the optimum temperature. (7) Molecular weight: approximately 53,000 (according to gel filtration method) 9. A microorganism that produces alkaline protease K-2005, which belongs to the genus Bacillus and has the following properties. (1) Action: Acts on urea-denatured hemoglobin, keratin, casein, myoglobin, and bovine serum albumin. (2) Substrate specificity High activity against urea-denatured hemoglobin, keratin, casein, myoglobin, and bovine serum albumin. It has particularly high activity against keratin. (3) Influence of metal ions Strongly inhibited by Cu^2^+. (4) Working pH and optimum pH The working pH range is 5 to 12 or higher. 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 Extremely stable at pH 5-12, does not deactivate, pH 4.5-1
Even at 2.5, the activity was maintained at 50% or more. (6) Optimal temperature The working temperature ranges over a wide range from 10 to 70°C, and the optimum temperature is 40°C. Moreover, even in the range of 22 to 55°C, it has 50% or more of the activity at the optimum temperature. (7) Molecular weight approximately 40,000 (by gel filtration method)