JP3026111B2 - Novel alkaline protease and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel alkaline protease K-16 and a method for producing the same. More particularly, the present invention relates to an alkaline protease which has extremely excellent stability to surfactants and is useful as a detergent-containing enzyme. The present invention relates to protease K-16 and a method for producing the same.

[0002]

2. Description of the Related Art Proteases have been incorporated into detergents, and many alkaline proteases are currently used as detergent enzymes. Representative examples thereof include alcalase, savinase, and esperase (manufactured by Novo Industries). However, many of these alkaline proteases have a problem that their stability in a surfactant solution is poor. Was.

On the other hand, Ya enzyme (Japanese Patent Application Laid-Open No. 61-280278) and the like have been reported as being stable to surfactants.
This has a problem that, since the active region is on the high temperature side, it is not compatible with the Japanese washing style in which washing is generally performed at around room temperature.

[0004] Also, API-21 (Japanese Patent Publication No. 60-55118) has been developed as a protease having a higher activity expression ability than conventional proteases in a lower temperature range, but also in a surfactant solution. Was not yet fully satisfactory.

[0005]

Therefore, it has been desired to newly provide an alkaline protease having high stability in a surfactant and having an excellent function as a detergent-containing enzyme.

[0006]

Means for Solving the Problems In order to solve the above problems, the present inventors have conducted extensive searches to obtain alkaline protease-producing bacteria from soil in Japan. As a result, they found that microorganisms belonging to the genus Bacillus collected from soil in Haga-gun, Tochigi Prefecture produce a novel alkaline protease having excellent stability in a detergent-containing system,
The present invention has been completed.

That is, the present invention provides a novel alkaline protease K-16 and a method for producing the same.

The microorganism producing the alkaline protease K-16 of the present invention belongs to the genus Bacillus and includes, for example, those having the following mycological properties.

(A) Morphological properties (a) Cell shape and size: Bacillus 0.8-1.0 μm × 2.2
２５25 μm (b) Polymorphism: None. (C) Motility: having periflagellate and motile. (D) Spores (size, shape, position): 1.0 to 1.2 μm x 1.
4 to 2.2 μm, oval, median margin, swelling of sporangia slightly. (E) Gram staining: positive (f) Acid-fast: negative (g) Growth form on gravy agar plate: round, leaf-like, smooth surface, pale yellow, translucent colony. (H) Growth on gravy agar slope: irregular foliate, smooth, pale yellow, translucent colonies slightly rough on the surface. (I) Liquid broth culture: good growth, turbidity, no pellicle. (J) Broth gelatin stab culture: liquefaction with good growth. (K) Litmus milk: peptone, but no coagulation of milk, no change in litmus.

(B) Physiological properties (a) Nitrate reduction: positive (b) Devagination: negative (c) MR test: negative (d) VP test: positive (e) Indole formation: negative (f) sulfide Hydrogen generation: negative (g) Starch hydrolysis: positive (h) Utilization of citric acid: positive (i) Utilization of inorganic nitrogen source: Utilizes nitrate, but does not use ammonium salt. (J) Pigment formation: negative (k) Urease: negative (l) Oxidase: positive (m) Catalase: positive (n) Growth temperature range: 55 ° C or less (o) Growth pH range: pH 6.6 to 10.3 Can grow. (P) Attitude to oxygen: aerobic (q) OF test: oxidized (O-type) (r) Resistance to sodium chloride: grows in the presence of 10% sodium chloride. (S) Acid production and gas production from sugar

[0011]

[Table 1]

Regarding the above mycological properties, "Bergey's Manual of Systematic Bacteriology (Williams & Wilkins, 1984)"(Bergey's Manual).
s Manual of Systematic Bacteriology), this strain was found to be Bacillus subtilis ( Ba
cillus subtilis ). However, while Bacillus subtilis does not grow at all at pH 10 medium, this strain grows well at pH 10.
In addition, there is a difference that Bacillus subtilis cannot grow at 55 ° C, while the present strain can grow at 55 ° C.

As is clear from the above results, it is appropriate that this strain belongs to Bacillus subtilis.
At the differ in some respects, and because also differs from other known strains, the present strain Bacillus sp (Baci
llus sp.) KSM-K16, and deposited with the Institute of Microbial Industry and Technology of the National Institute of Advanced Industrial Science and Technology as Microbial Laboratories Deposit No. 11418.

In order to produce the alkaline protease K-16 of the present invention, which is extremely stable to a surfactant, using this strain, the cells may be inoculated into an appropriate medium and cultured in a conventional manner. .

As the medium to be used, any medium which can be used for culturing a normal microorganism and which can be used for the present strain can be used. It is preferable that the source be contained in an appropriate amount.

The carbon source and the nitrogen source are not particularly limited. Examples of the nitrogen source include corn gluten meal, soy flour, corn steep liquor, casamino acid, yeast extract, pharma media, sardine meal, and meat. Extracts, peptone, hypro, adippower, corn meal, soybean meal, coffee grounds, cottonseed oil lees, cultivators, amiflex and adipron, zest, azix and the like. As the carbon source, a carbon source that can be assimilated, for example, arabinose, xylose, glucose, mannose, fructose, galactose, sucrose, maltose, lactose, sorbitol, mannitol, inositol, glycerin, soluble starch, molasses, and invert sugar And assimilating organic acids such as acetic acid. In addition, phosphoric acid, Mg ²⁺ , Ca ²⁺ , Mn ²⁺ , Z
Inorganic salts such as n ²⁺ , Co ²⁺ , Na ⁺ , and K ⁺ , and if necessary, inorganic and organic trace nutrients can be appropriately added to the medium.

Collection and purification of the alkaline protease K-16, which is the target substance, from the culture thus obtained can be carried out according to general means for collecting and purifying enzymes.

That is, the cells are separated by centrifugation or filtration of the culture, and the cells are separated from the cells and the culture filtrate by a usual separation means such as salting out, isoelectric precipitation,
The protein is precipitated by a solvent precipitation method (methanol, ethanol, isopropyl alcohol, acetone, etc.), or is subjected to ultrafiltration (for example, Diaflow membrane Y).
C, Amicon) to obtain the alkaline protease K-16 of the present invention. In the salting out method, for example, ammonium sulfate (30-70% saturated fraction), and in the solvent precipitation, for example, 75%
After precipitating the enzyme in% ethanol, it can be filtered, centrifuged, or desalted to give a lyophilized powder. Here, as a desalting method, a general method such as dialysis or gel filtration using Sephadex G-25 is used.

The enzyme solution thus obtained can be used as it is, or can be further purified and crystallized by a known method. To further purify the enzyme, for example, adsorption chromatography such as hydroxyapatite chromatography, DEAE-Sephadex, DEAE-cellulose, CM-cellulose and CM-cellulose
The separation and purification may be carried out by appropriately combining ion exchange chromatography such as biogel and molecular sieve gel chromatography such as Sephadex or biogel.

The alkaline protease K-16 of the present invention thus obtained has the following enzymatic properties. In the following, the measurement of enzyme activity was performed as follows.

1 ml of 50 mM boric acid-NaOH buffer (pH 10) containing 1% casein was mixed with 0.1 ml of the enzyme solution,
After reacting at 10 ° C for 10 minutes, 2 ml of a reaction stop solution (0.123 M trichloroacetic acid-0.246 M sodium acetate-0.369 M acetic acid)
Was added and left at 30 ° C. for 20 minutes. Next, the mixture was filtered through a filter paper (manufactured by Whatman, No. 2), and the proteolytic product in the filtrate was measured by an improved method of the Foreign-Lowry method.

1P. U. Is the amount of enzyme that releases 1 mmole of tyrosine per minute under the above reaction conditions.

(1) Action It acts on various proteins under highly alkaline conditions.

(2) Substrate Specificity The specificity of alkaline protease K-16 for various substrates was compared with other commercially available proteases. Substrates used were casein, urea-modified hemoglobin, animal hair keratin,
Degradation activity against these was measured with elastin. 50
1% of each substrate (2.2% for urea-denatured hemoglobin) was added to mM boric acid-NaOH buffer (pH 10.0), and each enzyme solution was 0.5 × 10 5
^-4 P.E. U. (3.5 × 10 ^-4 PU for elastin) was added, and the reaction was carried out at 40 ° C. for 10 minutes. Table 2 shows the activity of each enzyme when the activity of alkaline protease K-16 in each substrate was taken as 100.

[0025]

[Table 2]

As is clear from these results, the alkaline protease K-16 of the present invention can satisfactorily decompose water-soluble and water-insoluble proteins. Compared with B, it shows an excellent effect particularly on elastin.

(3) Optimum pH Casein was added to various pH buffers (50 mM) to a final concentration of 0.91%, and alkaline protease K-16 was added.
5.2 × 10 ^-5 P.E. U. In addition, the reaction was performed at 40 ° C. for 10 minutes, and the activity was measured. The activity at each pH was expressed relative to the activity at the optimum pH as 100. The results are shown in FIG. As is clear from FIG. 1, the alkaline protease K-16 of the present invention was used.
Has an optimum pH of 11.0 to 12.3. The various buffers used and their pH ranges are as follows. Acetate buffer pH 3.9 to 5.7 Phosphate buffer pH 6.6 to 8.3 Carbonate buffer pH 9.2 to 10.9 Phosphate-NaOH buffer pH 10.9 to 12.7 KCl-NaOH buffer pH 10.9 to 12.6

(4) pH stability 7.9 × 1 in the same buffer (20 mM) used in (3)
^0-3P . U. Of alkaline protease K-16,
It was left at 25 ° C. for 48 hours. This treatment solution was diluted 40-fold with a 50 mM boric acid-NaOH buffer (pH 10.0), and the activity was measured. The relative activity at each pH was determined with the enzyme activity before treatment as 100%. The results are shown in FIG. As is apparent from FIG. 2, the alkaline protease K-16 of the present invention has a stable region of pH 6.0 to 12.0 in the absence of Ca ²⁺ ,
^{In the} presence of ²⁺ , its stable range was pH 5.0-12.0.

(5) Optimal temperature 50 mM boric acid-NaOH containing 0.91% casein as a substrate
3.1 × 10 ^-5 P.S. in buffer (pH 10.0). U. Was added and the reaction was carried out at each temperature for 10 minutes. The relative activity at each temperature was determined with the activity at 40 ° C as 100%. The results are shown in FIG. As is apparent from FIG. 3, the optimum temperature of the present invention the alkaline protease K-16 is a 55 ° C. in Ca ²⁺ absence was 70 ° C. in 5 mM Ca ²⁺ presence.

(6) Heat resistance 1.6 × 10 5 in 20 mM boric acid-NaOH buffer (pH 9.5)
^-3P . U. Of alkaline protease K-16, and heat-treated at each temperature for 10 minutes. After cooling with ice, 50 mM boric acid-NaOH
It was diluted 5-fold with a buffer (pH 10.0). Then, the activity was measured using 0.91% casein as a substrate. Untreated activity of 10
The relative activity at each treatment temperature was determined as 0%. FIG. 4 shows the results. As is apparent from FIG. 4, Ca ²⁺ 50 ° C. in the absence, under 5MMCa ²⁺ present in the heat treatment conditions until 60 ° C., was maintained activity of 90% or more.

(7) Molecular Weight The molecular weight of alkaline protease K-16 was examined by sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis. Marker kits for low molecular weight (Bio-Rad), ie, phosphorylase b (molecular weight: 97,400), bovine serum albumin (molecular weight: 6)
6,200), ovalbumin (molecular weight: 42,700), carbonic anhydrase (molecular weight: 31,000), soybean trypsin inhibitor (molecular weight: 21,500), and lysozyme (molecular weight: 14,400). According to this method, the molecular weight of the alkaline protease K-16 of the present invention was determined to be 28,000 ± 1,000.

(8) Isoelectric point The isoelectric point of alkaline protease K-16 was examined by isoelectric focusing. Serverlight 9-11 was used as the amphoteric carrier for the column. According to this method, the isoelectric point of the alkaline protease K-16 of the present invention was found to be 10.5 or more.

(9) Effects of Metal Ions The effects of various metal ions on the alkaline protease K-16 of the present invention were examined. First, 20mM
Various metal salts were added to boric acid-NaOH buffer (pH 9.5) at a concentration of 1 mM, and 3.9 × 10 ^-3 P.S. U. Was added and left at 30 ° C. for 20 minutes. Then, 50 mM boric acid-
After diluting 5-fold with a NaOH buffer (pH 10.0), the residual activity was measured. The residual activity was expressed as a relative value to the enzyme activity similarly treated without addition of a metal salt. The results are shown in Table 3. As is clear from these results, it is found that the activity of the alkaline protease K-16 of the present invention is inhibited by Hg ²⁺ and Cu ²⁺ . Also, from the results of the above (5) and (6)
It can be seen that Ca ²⁺ improves thermal stability.

[0034]

[Table 3]

(10) Effect of Inhibitor The effect of a general enzyme inhibitor on the alkaline protease K-16 of the present invention was examined. Various inhibitors were added to a 10 mM phosphate buffer (pH 7.0) to a predetermined concentration, and the alkaline protease K-16 7.9 ×
^10-3P . U. Was added and left at 30 ° C. for 20 minutes. Thereafter, the treatment liquid was diluted 20 times with ion-exchanged water, and the residual activity was measured. The residual activity was expressed as a relative value with respect to the enzyme activity similarly treated without addition of the inhibitor. The results are shown in Table 4. As is clear from the results, the alkaline protease K-16 of the present invention is inhibited in activity by the serine protease inhibitors diisopropylfluorophosphate (DFP), phenylmethanesulfonyl fluoride (PMSF) and chymostatin. This shows that the protease has a serine residue in the active center.

[0036]

[Table 4]

(11) Effect of surfactant 6.6 × 10 ^-2 U. Was mixed with 5 ml of a 0.1 M Tris-HCl buffer (pH 9.
0, containing 10% ethanol) and allowed to stand at 40 ° C. for 4 hours, and then diluted with 50 mM boric acid-NaOH buffer (pH 10.0).
After 0-fold dilution, the residual activity was measured. The activity at the treatment time of 0 minutes was defined as 100%, and the residual activity was expressed as a relative value. Table 5 shows the results. As is clear from these results, the alkaline protease K-16 of the present invention exhibited high stability even when various surfactants were present at high concentrations (1 to 10%).

[0038]

[Table 5]

[0039]

As described above, the alkaline protease K-16 of the present invention exhibits an excellent action on insoluble proteins,
In addition to exhibiting sufficient activity in a wide temperature range, it has extremely high stability in a surfactant, and thus is useful as an enzyme for adding a detergent.

[0040]

Next, the present invention will be described in more detail by way of examples.

Example 1 Isolation and collection of protease-producing strains: (1) About 1 g of a soil sample was sterilized with 10 ml of physiological saline.
And heat-treated at 80 ° C. for 20 minutes. 0.1 ml of the heat-treated supernatant was inoculated into a keratin halo agar medium and 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% Animal hair keratin 1% Carboxymethylcellulose 1% Potassium phosphate 0.1% Magnesium sulfate heptahydrate 0.02% Agar 1.5%

(2) Separately sterilized 10% sodium carbonate (1%) was added to the above medium composition to adjust the final pH to 10.5, and a plate medium was prepared. After the cultivation, those having halos around the grown colonies were selected, and 2-3 times in the same medium.
Purification was repeated to obtain a uniform protease-producing bacterium.

(3) These strains obtained in the above (2) were inoculated into the following liquid medium, and cultured with shaking at 30 ° C. aerobically for 48 hours. Glucose 2.0% Polypeptone S 1.0% Yeast extract 0.05% Potassium phosphate 0.1% Magnesium sulfate heptahydrate 0.02% Sodium carbonate (separate sterilization) 1.0% pH 10.5 After the culture is completed, the obtained culture is centrifuged (3000 rpm) ; 10
Min) to remove the bacteria, and the resulting culture supernatant was used as an enzyme solution.

(4) A crude enzyme sample was prepared from the enzyme solution obtained in the above (3) by freeze-drying, and the storage stability at 40 ° C. in a commercially available liquid detergent was evaluated. Among them, Bacillus sp. KSM-K16 strain was obtained as one strain producing an enzyme having the highest stability.

Example 2 Culture of cells and purification of alkaline protease K-16: (1) The alkalophilic bacterium Bacillus sp. KSM-K16 obtained in Example 1 was transformed into the following liquid medium (3.0 l).
And inoculated at 30 ° C. aerobically for 48 hours with shaking.
Alkaline protease K-16 was produced. Glucose 2.0% Fish extract 1.0% Soy flour 1.0% Magnesium sulfate 0.02% Potassium phosphate 0.1% pH 10.0

(2) After completion of the culture, 3 l of the obtained culture was centrifuged (10,000 rpm; 5 minutes) to remove the bacteria, and the supernatant was freeze-dried. After dissolving 2 g of the lyophilized powder in 10 ml of ion-exchanged water (crude enzyme solution), the solution was dissolved in a 10 mM Tris-HCl buffer (2 mM Ca ²⁺ added, pH 7.5) using a dialysis membrane.
) Against dialysis overnight (26 ml of dialysis solution (activity 3.1)).
5P. U. / Ml, specific activity 1.97P. U. / Mg protein). Next, the solution was applied to a column filled with a CM-52 cellulose carrier equilibrated with a 10 mM Tris-hydrochloric acid buffer (2 mM Ca ²⁺ added, pH 7.5), and the column was washed with the same buffer. The alkaline protease K-16 was eluted with the same buffer containing sodium chloride. When the active fractions were collected, the total amount was 15 ml and the activity was 1.12 P.L. U. / Ml,
The specific activity is 5.75P. U. / Mg protein. The solution was dialyzed overnight against 50 mM Tris-HCl buffer (10 mM Ca ²⁺ , 0.2 M sodium chloride added, pH 8.0), and then concentrated by ultrafiltration (Amicon, fractional molecular weight 5,000). The gel was subjected to gel filtration chromatography on Sephadex G-50 (Pharmacia) equilibrated with 50 mM Tris-HCl buffer (10 mM Ca ²⁺ , 0.2 M sodium chloride added, pH 8.0), and developed with the same buffer. I let it. The active fraction thus obtained had a total volume of 11.5 ml and an activity of 0.9 P.S. U. / Ml, specific activity 6.03P. U. / Mg protein. The solution was dialyzed against ion-exchanged water overnight, and then, the active was 0.56 P.S. U. / Ml,
Specific activity 5.60P. U. / Mg protein solution.

[Brief description of the drawings]

FIG. 1 is a drawing showing the effect of pH on the activity of alkaline protease K-16 of the present invention.

FIG. 2 is a drawing showing the effect of pH on the stability of alkaline protease K-16 of the present invention.

FIG. 3 is a drawing showing the effect of temperature on the activity of alkaline protease K-16 of the present invention.

FIG. 4 is a drawing showing the effect of temperature on the stability of alkaline protease K-16 of the present invention.

──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Mikio Takaiwa 859-4 Hiraicho, Tochigi City, Tochigi Prefecture (72) Inventor Tohru 2606-6, Akabane, Kaigamachi, Haga-gun, Tochigi Prefecture (72) Inventor Shuji Kawai Tochigi Prefecture 308 Takebayashi-cho, Utsunomiya-city (72) Susumu Ito Inventor 3441-64, Higashimine-cho, Utsunomiya-city, Tochigi (56) References JP-A-1-270884 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) BIOSIS (DIALOG) WPI (DIALOG)

Claims (3)

(57) [Claims] 1. An alkaline protease K-16 having the following enzymatic properties: (1) Action It acts on various proteins under highly alkaline conditions. (2) have good activity against substrate specificities water soluble protein sequence to a water-insoluble protein. (3) Optimum pH When casein is used as a substrate and the reaction is carried out at 40 ° C. for 10 minutes, the optimum pH is 11.0 to 12.3. (4) pH stability When casein is used as a substrate and treated at 25 ° C., it is extremely stable in the range of pH 5.0 to 12.0 in the presence of Ca ²⁺ . (5) Optimum temperature When the reaction is carried out at pH 10.0 using casein as a substrate, the optimum temperature is 55 ° C. (6) Heat resistance When heat-treated at pH 9.5 and 50 ° C. for 10 minutes, it has a residual activity of 90% or more. (7) Molecular weight 28,000 ± 1,000 (Sodium dodecyl sulfate (SDS)-
(By polyacrylamide gel electrophoresis) (8) Isoelectric point 10.5 or more (by isoelectric focusing) (9) Effect of metal ion When casein is used as a substrate, the activity is inhibited by Hg ²⁺ and Cu ²⁺ Is done. In addition, Ca ²⁺ increases thermal stability. (10) Effect of Inhibitor Ethylenediaminetetraacetic acid, p-chloromercury benzoic acid, and antipain do not inhibit the activity. The activity is inhibited by diisopropylfluorophosphate, phenylmethanesulfonyl fluoride and chymostatin. (11) Influence of surfactants Surfactants such as sodium linear alkyl benzene sulfonate, sodium polyoxyethylene alkyl sulfate, sodium dodecyl sulfate, sodium α-olefin sulfonate, sodium alkane sulfonate and α-sulfo fatty acid ester are high. Even at concentrations, the enzyme is extremely stable. 2. The alkaline protease K-16 according to claim 1, wherein a microorganism which belongs to the genus Bacillus and produces the alkaline protease K-16 is cultured, and the enzyme is collected from the obtained culture. Production method. [Claim 3] belongs to the genus Bacillus, the microorganism Bacillus sp to produce alkaline protease K-16 (Ba
cillus sp.) KSM-K16.