JPH0763366B2 - Novel protease - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel protease that acts in a wide pH range, and more specifically, it can be widely used as a washing enzyme suitable for Japanese laundry conditions. It relates to a protease that can.

[Conventional technology]

Although the incorporation of protease into detergents has been performed for a long time, the pH of the current detergents is all on the high alkaline side. Those that act stably in the active agent have been desired and developed.

Typical examples are alcalase, savinase,
Esperase (manufactured by Novo Industry), maxatase (manufactured by Gist Procardis), etc. are commercially available and used in many cleaning agents.

However, since the active region of these proteases is on the high temperature side, and Japanese washing is performed near room temperature, it is required to provide a protease having a stronger activity expressing ability than the conventional protease even in the low temperature region. On request
API-21 (manufactured by Showa Denko) has recently been developed.

Other desired properties of proteases include the ability to decompose insoluble proteins such as keratin well, and it is also known that proteases having such ability contribute to washing (Motokawa Minamoto: Fiber Magazine, Vol. 26, Vol.
322 (1985)).

[Problems to be solved by the invention]

Basically, the properties required of conventional proteases for detergents are that they have an optimum reaction pH on the alkaline side, are active even at low temperatures, and are excellent in degrading insoluble proteins such as keratin. Performance,
In addition, as long as the protease has an optimum activity in a wide pH range, it can be used as a heavy or light detergent, and thus the development of such an enzyme has been desired.

[Means for solving problems]

Therefore, the present inventors, in addition to the basic performance of ~,
As a result of conducting an intensive search for a novel protease having an optimal activity in a wide pH range, Bacillus sp. KSM-2004 obtained from soil in Haga-gun, Tochigi Prefecture
It was found that the protease produced by (Bacillus sp. KSM-2004) satisfies the above conditions, and has completed the present invention.

The Bacillus sp. KSM-2004 exhibits the following mycological properties.

(A) Morphological properties (a) Cell shape and size Bacilli 0.5-0.8 × 2.0-5.0 μm (b) Polymorphism Not observed.

(C) Motility No flagella and no mobility (d) Spores 0.4-0.8 × 0.4-0.8 μm oval or circular middle quasi-edge (e) Gram stain positive (f) Acid-negative (B) Various media (A) Growth state on broth agar plate It grows at pH 7.0 and forms a round, convex, smooth-wave colony. The size is φ1.0-5.0 mm, the surface is smooth and has a dewdrop-like pale yellow color. It is an opaque and oily colony.

(B) Growth on broth agar slope It shows normal growth at pH 7.0, and is milky white, translucent and spreads in a wide band.

(C) Meat juice liquid culture It grows slightly at pH 7.0 but does not form pellicle.

(D) Meat broth gelatin stab culture All liquefy gelatin at pH 7.0.

(E) Litmus milk Liquefaction of milk and bleaching of litmus in deep areas are observed.

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

Nitrate reduction, denitrification reaction, MR test, VP test, indole formation, hydrogen sulfide formation, inorganic nitrogen utilization (sodium nitrate, ammonium sulfate), pigment formation, oxidase, OF test.

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

Starch hydrolysis, casein degradation, tyrosine hydrolysis, catalase, urease (weakly positive).

(C) Utilization of citric acid Negative in Coerser's medium and positive in Christensen's and Simmons' medium.

(D) Good growth in the growth pH range of pH 6 to 10.

(E) Good growth in the growth temperature range of 20 to 30 ℃, but can grow at 10 ℃ to 40 ℃.

(F) Attitude toward oxygen Aerobic. Cannot grow under anaerobic conditions.

(G) Generation of acid and gas from sugar (I) Tolerance to sodium chloride Can grow in the presence of 5% sodium chloride.

Grow slightly in the presence of 7% sodium chloride.

The above mycological properties of Bacillus sp. KSM-2004,
"Barge Aise Manual of Day Terminator Bacteriology 8th Edition (1975)" and "The Genus Bacillus, Agricultural Handbook No.4"
27 (1973) ”and the comparison with other strains is as follows.

KSM-2004 is a Gram-positive aerobic bacillus, and has sporulation ability, and thus is clearly belonging to the genus Bacillus. And KSM-2004 has no motility,
That is, it has no flagella, can grow on a Sabouraud agar medium, has a starch hydrolyzing ability, and produces acid from glucose, arabinose, xylose, etc.
circulans) was determined to be a fungus. But,
KSM-2004 hydrolyzes tyrosine, but Bacillus circulans is different in that it cannot hydrolyze it. In addition, Bacillus circulans is generally said to react very weakly to casein, gelatin and litmus milk or not at all, but this strain reacts well to these substrates. The difference is also in the points.

As is clear from the above description, this strain is Bacillus
It is similar to Circulans but differs in some respects from this and also from other known strains. Therefore, the present inventors determined that this strain was a new strain of the genus Bacillus, and deposited it with the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology as Micromachine Research Institute No. 9452 (FERM P-9452).

In order to obtain the novel protease of the present invention using Bacillus sp. KSM-2004, the strain may be inoculated into an appropriate medium and cultured according to a conventional method. It is preferred that the medium contains appropriate amounts of assimilable carbon source and nitrogen source.

The carbon source and the nitrogen source are not particularly limited, but examples thereof include corn gluten meal, soybean flour, corn steep liquor, casamino acid, yeast extract, pharmamedeia, sardines, meat extract, peptone as the nitrogen source. , Hypro, Aji Power, Corn Meal, Soy Bean Meal, Coffee Meal, Cottonseed Oil Meal, Cultivator, Amifrex and Adipron, Zest, Azicus. Further, as a carbon source, a carbon source that can be assimilated,
Examples thereof include arabinose, xylose, glucose, mannose, sucrose, maltose, soluble starch, lactose, molasses and assimilable organic acids such as acetic acid. In addition, phosphoric acid, Mg ²⁺ , Ca ²⁺ , Mn ²⁺ , Zn ²⁺ , Co ²⁺ , Na ⁺ , K
Inorganic salts such as ⁺ 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.

That is, cells are separated by centrifuging or filtering the culture, and the cells and the culture filtrate are separated by a conventional separation means such as salting out method, isoelectric focusing method, solvent precipitation method ( The protein of the present invention is obtained by precipitating the protein with methanol, ethanol, isopropanol, acetone or the like, or by concentrating the protein with ultrafiltration (for example, Diaflow membrane, manufactured by Amicon). For example, ammonium sulfate (30-70% saturated fraction) is used in the salting-out method, and solvent precipitation is performed using
After precipitating the enzyme in 75% ethanol, it is possible to obtain a freeze-dried powder by filtering, centrifuging, or desalting. As a desalting method, a general method such as dialysis or a gel filtration method using Sephadex G-25 is used.

The protease solution thus obtained may be used as it is, or may be purified and crystallized by a known method and used. To purify the protease, 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 and fractionated. It should be purified.

The protease of the present invention thus obtained has the following physicochemical properties. (A) Optimal reaction pH Urea-modified hemoglobin (substrate) was added to various pH buffers (100 mM) so that the final concentration was 2.2%, and the reaction was carried out at 25 ° C for 10 minutes. The initial reaction rate for the substrate at the optimum pH was defined as 100%, and the initial reaction rate at each pH was expressed relatively. The results are shown in FIG. As is clear from Fig. 1, the optimum reaction pH of the protease produced by this bacterium
Has a wide range of pH from 6.5 to 10.5.

The buffer solutions used and their pH ranges are as follows.

Acetate buffer pH4-6 Phosphate buffer pH6-8 Borate buffer pH8-10 Carbonate buffer pH9-11 Sodium phosphate buffer pH10.5-12 (b) pH stability About 2 x 1 in the same buffer (100 mM)
0 ^-4 AU of protease was added and the mixture was allowed to stand 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 to measure the residual activity. 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 FIG. As is clear from FIG. 2, the protease of the present invention is extremely stable in a wide range of pH 5-12.

(C) Optimum reaction temperature Under each temperature, a protease of about 5 × 10 ⁻⁵ AU was used, and the reaction was carried out at pH 10.5 using urea-modified hemoglobin as a substrate. The optimum temperature of the protease of the present invention was 40 ° C.
The maximum activity was 55% or more even at around 20 ° C.
The results are shown in FIG.

(D) Substrate specificity The specificity of the protease of the present invention for various substrates was compared with other commercially available alkaline proteases. The substrate used was urea-modified hemoglobin, keratin, casein, myoglobin, bovine serum albumin, and the concentration in the reaction solution was
Hemoglobin was 2.2% and the others were 1.0%. Add these substrates to 0.1M borate buffer at pH 10.5 and add about 1 x each enzyme.
10 ⁻⁴ AU was added and the reaction was carried out at 25 ° C. for 10 minutes (60 minutes for keratin). The results are shown in Table 1. The numerical value is
The amount of each enzyme was set to 1 × 10 ^-4 AU (unifying the activity against urea-modified hemoglobin), this was reacted with each substrate, and the standard of activity for each substrate was defined as the activity of API-21. Enzyme activity was expressed as its relative value.

From the above results, it was revealed that the protease of the present invention has excellent keratin degrading ability and high substrate specificity.

(E) Molecular weight The molecular weight of this protease was determined to be 25,000 using Sephadex G-100 gel perchromatography.
It was. As the eluent, 0.1 M potassium chloride and 5 mM
10 mM Tris-HCl buffer containing calcium chloride (pH 7.
0) was used. The standard proteins are bovine serum albumin (MW: 68,000) and ovalbumin (MW: 45,00).
0), chymotrypsinogen A (MW: 25,000) and ribonuclease A (MW: 13,700).

(F) Effect of surfactants and builders on protease stability 50 mM borate buffer solution (pH8.pH) containing about 2 × 10 ^-4 AU protease in which various kinds of surfactants or builders were dissolved at a predetermined concentration.
0) and left at 25 ° C. for 1 hour. After that, it was diluted 5-fold with the same buffer, and the residual activity was measured using urea-modified hemoglobin as a substrate. The results are shown in Table 2.

From these results, it is considered that the protease of the present invention is fairly stable in the surfactant or builder.

(G) Effect of enzyme inhibitor 4 × 10 ^-3 AU of protease was added to 0.1M borate buffer solution (pH 8.0) containing a predetermined concentration of a general protease inhibitor, and the mixture was incubated at 25 ° C. for 1 hour. Time treatment was performed (when diisopropylfluoridate (DFP) was used, treatment was performed with 0.1 M Tris-HCl buffer, pH 7.0). After the treatment, it was diluted 10 times with 0.1 M borate buffer (pH 8.0), and the enzyme activity was measured by the Anson hemoglobin method. As a control, a system that had been treated in the same manner without the addition of an inhibitor was prepared, and the effect of the inhibitor was expressed by the relative value when the activity was set to 100. The results are shown in Table 3.

Since this protease is slightly inhibited by DFP, it is considered that it has serine at the active center, but it is more resistant to DFP than general serine enzymes.

(H) Effect of metal ion 0.1M borate buffer (p of each metal salt added to 5 mM)
After adding alkaline protease 4 × 10 ^-3 AU to H8.0),
The treatment was carried out at 10 ° C for 10 minutes. Then, the enzyme activity was measured by the Anson hemoglobin method after diluting 10 times with the same buffer solution. Using a system without addition of metal salt as a control,
Relative value when 0 is shown The effect of each metal ion is shown. The results are shown in Table 4.

From this result, it was confirmed that the protease of the present invention is very stable to various metal ions and that the presence thereof does not have any influence.

〔The invention's effect〕

The protease of the present invention exhibits optimum activity in a wide pH range as described above and has activity even in a low temperature range. Moreover, it has an excellent decomposing power for keratin and the like, and
Even in the presence of various components such as surfactants, builder components, metal ions, etc., which are mixed in the detergent composition or mixed during washing, the action thereof is hardly reduced. Therefore, the protease of the present invention is extremely excellent as a protease to be added to heavy and light detergents.

〔Example〕

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

Reference Example 1 Separation and collection of protease-producing strains: (i) About 1 g of a soil sample collected in Haga-gun, Tochigi Prefecture was suspended in 10 ml of sterilized physiological saline, and the suspension was allowed to stand at 80 ° C for 20 minutes for heat treatment. 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% Potassium phosphate 0.1% Wool keratin 1% Carboxymethylcellulose 1% Magnesium sulfate heptahydrate 0.02% Agar 1.5% (ii) The above medium composition was separately sterilized 10% Add sodium carbonate 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. From 300 soil samples, 120 strains were obtained by plate culture at each pH.

(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.

Glucose 0.2% Keratin (wool) 0.5% Yeast extract 0.05% Potassium potassium phosphate 0.1% Sodium carbonate (separate sterilization) 0.4% pH 9.0 During or after culturing, strains with remarkable keratin degrading power were selected. Bacillus sp. KSM-2004 was obtained from 120 strains obtained from the keratin halo plate medium by this method.

Example 1 Bacillus sp. KSM-2004 (F obtained in Reference Example 1
ERM P-9452) was inoculated into the following liquid medium, and shake culture was carried out aerobically at 30 ° C. for 48 hours.

Glucose 2% Fish meat extract 1% Soybean flour 1% Magnesium sulfate heptahydrate 0.02% Potassium dihydrogen phosphate 0.1% Sodium carbonate (separate sterilization) 0.4% pH 9.0 After culturing, the resulting culture is centrifuged (3,000 Rotation; 10
The bacteria were removed by subjecting the obtained culture supernatant to an enzyme solution and the protease activity thereof was measured. The activity of 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., 2
2, 79 (1938) denatured bovine serum hemoglobin with urea, was pH10.5 at caustic soda. To 0.5 ml of this substrate solution (2.2% as hemoglobin), 0.1 ml of enzyme solution (0.1 x 10
^{-4 to} 1.0 × 10 ^-4 AU) was added, 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, and centrifuged (3000 rpm min), the supernatant protein decomposition product Fuorin-Lowry method contained in (OHLowry et, J.Biol.Chem. 193, 265 (1951 )) to by connexion quantitative did. 1 AU was defined as the amount of enzyme that liberates 1 mmole of tyrosine per minute under the above reaction conditions.

(B) Method using keratin as a substrate 0.5 ml of 0.5% wool keratin suspended in 20 mM carbonate buffer was added with 0.1 ml of enzyme solution 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. In addition, 1KU is, under the above reaction conditions,
It was defined as the amount of enzyme that liberates 1 mmole of tyrosine per minute.

The protease activity of the enzyme solution obtained in this Example was 5
It is as shown in the table.

Example 2 Ammonium sulfate was added to the 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 K3371. The cleaning system is
0.133% of commercial phosphorus-free heavy detergent (without addition of enzyme)
And then added with the protease solution of the present invention at 0.01 AU /.
The test cloth is the waist collar part (worn for 3 days) where dirt on human skin is likely to adhere.
After cutting to about 13 cm, it was immersed in a cleaning system with or without addition of enzyme at 30 ° C. for 1 hour. After the immersion, use a targotometer (Kamishima Seisakusho) at 20 ℃ 120r.p.
m. Washing was carried out for 10 minutes. After rinsing and drying, a pair of collar cloths (15 sets) were compared and the degree of stain removal was visually judged by three skilled judges. The judgment method is
The total evaluation score of 15 pieces of collar cloth was calculated with 5 points when the stains were almost completely removed and 1 score when the stains were hardly removed, and the evaluation score of the cleaning system without addition of enzyme was set to 100. The ratio in each case was expressed as a detergency index. The results are shown in Table 6.

From the above results, it is clear that the protease of the present invention is an excellent detergent enzyme that can sufficiently exert its action.

[Brief description of drawings]

FIG. 1 is a drawing showing the effect of pH on the activity of the protease of the present invention. FIG. 2 is a drawing showing the effect of pH on the stability of the protease of the present invention. FIG. 3 is a drawing showing the effect of temperature on the activity of the protease of the present invention.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Okamoto Akihiko 1-229-8 Nanachiza-cho, Koshigaya-shi, Saitama

Claims (1)

[Claims] 1. A novel protease having the following physicochemical properties. (1) Action It acts on urea-modified hemoglobin and keratin. It also has some effect on casein. (2) Substrate specificity It has high activity against urea-modified hemoglobin and keratin. It also has some activity against casein. The activity against myoglobin and bovine serum albumin is very weak. (3) Working pH and optimum pH The working pH range is 4.5 to 12.0. The optimum pH is 6.5 to 10.5
It has a relative activity of 50% or more of the activity at the optimum pH even in the range of 5.5 to 11.5. (4) 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 4.0 to 12.5. (5) Optimum temperature The working temperature is in a wide range from 10 to 70 ℃, and the optimum temperature is
40 ° C. Moreover, it has 50% or more of the activity at the optimum temperature even in the range of 20 to 50 ° C. (6) Molecular weight about 25,000 (by gel filtration method) (7) Effect of metal ions It is very stable against various metal ions. (8) Effects of surfactants and detergent builders: sodium linear alkylbenzene sulfonate, sodium alkyl sulfate, sodium polyoxyethylene alkyl sulfate, sodium α-olefin sulfonate, α-sulfo fatty acid ester, sodium alkane sulfonate,
Soap, sodium dodecylsulfate, sodium tripolyphosphate, zeolite, ethylenediaminetetraacetic acid hardly inhibit the activity.