JP2794371B2 - Alkaline protease K-16H - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an alkaline protease K.
More specifically, the present invention relates to an alkaline protease K-16H which has excellent stability to a surfactant and is useful as a detergent-containing enzyme.

[0002]

2. Description of the Related Art Proteases have been widely used in the food / fishery processing industry, leather industry, textile industry, brewing industry, detergent industry, and the like. In practice, many alkaline proteases are currently used as detergent enzymes. Furthermore, in recent years, in particular, detergents for clothes have been promoted to eliminate phosphorus from the viewpoint of environmental issues.However, alkali proteases have been added in order to enhance the detergency which is reduced as a result, and more and more alkaline proteases have been added. Demand is growing.

By the way, in order for proteases to be effectively incorporated during washing, it is not sufficient to simply act under alkaline conditions, and the protease must be stable in a surfactant incorporated in a detergent; and It is required to have the ability to decompose clothing stains, that is, a detergency.

[0004] However, there is no alkali protease that simultaneously satisfies both the stability to surfactants and the excellent detergency required for proteases to be incorporated in such detergents. .

[0005]

DISCLOSURE OF THE INVENTION In view of the above-mentioned circumstances, the present inventors first collected and searched soils throughout Japan to obtain new alkaline protease-producing bacteria. As a result, a strain of the genus Bacillus isolated from soil in Haga-gun, Tochigi Prefecture, produces an alkaline protease that has excellent detergency in detergent formulations and has extremely high stability in surfactants. And filed a patent application (Japanese Patent Application Nos. 3-33116 and 3-33).
117).

[0006] Alkaline protease K-16 produced by the above microorganism is excellent in itself having sufficient stability and detergency for practical use, but is further excellent in stability in a surfactant. It is extremely significant to search for an alkaline protease having a property and a function as a better detergent-containing enzyme.

[0007]

The present inventors have conducted various studies on the above-mentioned alkaline protease K-16. As a result, the enzyme was found to be at least 3% having alkaline protease activity by polyacrylamide gel electrophoresis.
It was found that it was composed of components. As a result of repeated studies to separate and purify these components, the use of various column chromatography has made alkaline protease K-1 as an electrophoretically uniform component.
6H was successfully obtained, and the present invention was completed.

That is, the present invention provides a novel alkaline protease K-16H having the following properties.

(1) Action Simple proteins and complex proteins are hydrolyzed to produce oligopeptides or amino acids. (2) Substrate specificity It has good activity on water-soluble proteins such as casein and hemoglobin and water-insoluble proteins such as keratin and elastin. (3) Optimum pH When casein is used as a substrate and the reaction is carried out in various buffers at 40 ° C. for 10 minutes, the optimum pH is 9.0 to 11.0. Further, it has an activity of 50% or more of the maximum activity at pH 6.0 to 13.0.

(4) pH Stability When Ca ²⁺ is added to various buffers and treated at 55 ° C. for 10 minutes, the pH is extremely stable in the range of 5.0 to 12.0. (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 90% when treated at pH 9.0 and 50 ° C. for 10 minutes, or when treated at 60 ° C. for 10 minutes after adding Ca ^2+.
It has the above residual activity.

(7) Molecular weight 28,000 ± 1,000 (sodium dodecyl sulfate (S
DS)-by polyacrylamide gel electrophoresis). (8) Effect of metal ions When casein is used as a substrate, the activity is inhibited by Hg ²⁺ . In addition, Ca ²⁺ increases thermal stability and pH stability. (9) Effect of inhibitor The activity is not inhibited by ethylenediaminetetraacetic acid, p-chloromercury benzoic acid, and antipain. The activity is inhibited by phenylmethanesulfonyl fluoride and chymostatin. (10) Influence of surfactant Even if a surfactant such as sodium polyoxyethylene alkyl sulfate, sodium α-olefin sulfonate, sodium alkane sulfonate, α-sulfo fatty acid ester is present in a high concentration, it is extremely stable. .

The alkaline protease K-16H of the present invention
Is, for example, Bacillus sp. KS belonging to the genus Bacillus
After inoculating M-K16 (Bacillus sp. KSM-K16; No. 3376, Microtechnical Laboratories) into an appropriate medium and culturing it according to a conventional method, the culture supernatant was subjected to appropriate separation and purification treatments. It can be obtained by attaching. The bacteriological properties of the Bacillus sp. KSM-K16 and the method for obtaining the same are described in Japanese Patent Application Nos. 3-33116 and 3-331.
No. 17, etc.

As the medium used for culturing, any medium can be used as long as it is used for culturing ordinary microorganisms and can be used for the present strain. It is preferable to contain an appropriate amount of a nitrogen source and a nitrogen source.

The carbon source and nitrogen source contained in the medium are not particularly limited. Preferred examples of the nitrogen source include corn gluten meal, soy flour, corn steep liquor, casamino acid, yeast extract, pharmamedia, sardine Examples include meal, meat extract, peptone, hypro, adippower, corn meal, soybean meal, coffee grounds, cottonseed oil grounds, cultivators, Amiflex and adipron, zest and the like.

The carbon source may be a carbon source which can be used, for example, arabinose, xylose, glucose, mannose, fructose, galactose, sucrose, maltose, lactose, sorbitol, mannitol, inositol, glycerin, soluble starch and inexpensive molasses , Invert sugar and the like, and assimilating organic acids such as acetic acid. Furthermore, phosphoric acid, Mg ²⁺ , Ca ²⁺ , M
Inorganic salts such as n ²⁺ , Zn ²⁺ , Co ²⁺ , Na ⁺ , and K ⁺ , and if necessary, inorganic and organic trace nutrients can be appropriately added to the medium.

The collection and purification of the alkaline protease K-16H, which is the target substance, from the culture thus obtained can be carried out in accordance with general methods for collecting and purifying enzymes.

That is, the cells are separated by centrifugation or filtration of the culture, and then separated from the cells and the culture filtrate by a usual separation means, for example, a salting out method, an isoelectric point precipitation method, a solvent precipitation method. (Methanol, ethanol, propyl alcohol, acetone, etc.) to precipitate the protein, or to concentrate by ultrafiltration (for example, Diaflow membrane filter; manufactured by Amicon), or to use Sephadex G-25, polyvinylpyrrolidone. The alkaline protease K-16 can be obtained as a crude enzyme.

Among the above-mentioned separation means, the alkaline protease K-16 is precipitated in, for example, ammonium sulfate (30-70% saturated fraction) in the salting-out method and in 75% ethanol in the precipitation method, and then filtered or filtered. This can be made into a lyophilized powder by centrifugation and desalting. As a desalting method that can be employed here, a general method such as dialysis or a gel filtration method using Sephadex G-25 is used.

Further, to purify and collect the alkaline protease K-16H from the crude enzyme alkaline protease K-16, for example, adsorption chromatography such as hydroxyapatite chromatography, DEAE
-Sephadex, DEAE-cellulose, CM-cellulose, CM-biogel and other ion-exchange chromatography and molecular sieve gel chromatography such as Sephadex and biogel may be appropriately combined for fractional purification. Desirably, DEAE-Biogel, CM-
Good results can be obtained by purifying by appropriately combining ion-exchange chromatography such as biogel and adsorption chromatography such as hydroxyapatite.

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

(Enzyme activity measurement method) Casein (HAMMARSTEN; manufactured by Merck) 1%
( W / v ) in 50 mM borate-NaOH buffer (p
H10.0) 1 ml was kept at 40 ° C. for 5 minutes,
The reaction was started by adding 1 ml of the enzyme solution. 10 at 40 ° C
After reacting for 1 minute, the reaction was stopped (0.123 M trichloroacetic acid-0.246 M sodium acetate-0.369).
M acetic acid) (2 ml) and left at room temperature for about 20 minutes.
The formed precipitate was removed with a filter paper (No. 2 filter paper; manufactured by Whatman), and the acid-soluble protein hydrolyzate in the filtrate was colorimetrically determined by the Foreign-Lowry method. One unit (U) of the enzyme is 1 μm / min under the above reaction conditions.
The amount of the enzyme to release an acid-soluble proteolysate corresponding to ol tyrosine.

(1) Action Hydrolyzes simple proteins and complex proteins to produce oligopeptides or amino acids.

(2) Substrate Specificity The specificity of alkaline protease K-16H for various substrates was compared with other commercially available proteases. The substrates used were casein, hemoglobin, animal hair keratin, elastin, egg yolk and human-derived α-keratin, and the degrading activity thereof was measured. That is, 1% (α) of each substrate was added to a 50 mM boric acid-NaOH buffer solution (pH 10.0).
-0.1% of keratin) was added, and each enzyme solution was 0.04 to 0.1.
043U (0.2200 to 0.215 for elastin)
U) was added and the reaction was carried out at 40 ° C. for 10 minutes. Table 1 shows the relative activity of each enzyme on casein, where the activity on casein was defined as 100.

[0024]

As a result, alkaline protease K-16
H is, in particular, a poorly soluble protein (animal hair ketilan, α-keratin,
Elastin).

(3) Optimum pH 1 ml of various 50 mM pH buffers containing 1% casein
Was added with 0.041 U of alkaline protease K-16H, and the reaction was carried out. As shown in FIG. 1, the activity at each pH was represented by a relative value with the activity at the optimum pH being 100.
From this result, the optimal p of alkaline protease K-16H
H was from 9.0 to 11.0, and had an activity of 50% or more over a wide range from pH 6.0 or more to pH 13.0. The various buffers used and their pH ranges were as follows.

(Buffer used and its pH range) Acetate buffer pH 4.0-5.9 Phosphate buffer pH 6.0-8.1 Carbonate buffer pH 9.3-11.2 Phosphate-NaOH buffer pH11 1.4-12.4 KC1-NaOH buffer pH 12.3-13.2

(4) pH stability 2 mM Ca was added to the various buffers (20 mM) used in (3).
A system to which Cl ₂ was added and a system to which no Cl ₂ was added were prepared,
1.70 U of alkaline protease K-16H was added thereto, and the mixture was allowed to stand at 55 ° C. for 10 minutes. 50m of this treatment liquid
The mixture was diluted 10-fold with M borate-NaOH buffer (pH 10.0), and the enzyme activity was measured. 10 enzyme activity before treatment
The relative activity at each pH set to 0 is shown in FIG. In the figure, the dotted line shows the case without Ca ²⁺ addition, and the solid line shows the case with Ca ²⁺ addition.

As a result, alkaline protease K-16
H is pH 7 under the above-mentioned conditions when CaCl _{2 is} not added.
１１11, 2 mM CaCl ₂ , pH 5
It was extremely stable between 12.

(5) Optimum temperature Using 0.028 U of alkaline protease K-16H at each temperature of 30 ° C. to 80 ° C., 5 mM CaCl 2 was used.
A system to which ₂ was added and a system to which no additive was added were prepared, and the reaction was carried out in the same manner as the standard reaction conditions except for the temperature. FIG. 3 shows the relative activities at each temperature, where the activity under the standard reaction conditions was 100. In the figure, the dotted line indicates the case where Ca ^{2+ was} not added, and the solid line indicates the case where Ca ^{2+ was} not added.
The case of ²⁺ addition is shown. Alkaline protease K-16
The optimal reaction temperature of H is 55 ° C. in the case of CaCl ₂ free system.
And 65 ° C. in the 5 mM CaCl ₂ added system.

(6) Heat resistance A system in which 5 mM CaCl ₂ was added in a 20 mM boric acid-NaOH buffer (pH 9.0) and a system in which 5 mM CaCl ₂ was not added were prepared and kept at 30 to 70 ° C. for 5 minutes. . Then
Add 0.41 U of alkaline protease K-16H,
Heat treatment was performed for 10 minutes. After quenching in ice water, 50m
The residual activity was measured using an enzyme solution that was diluted two-fold with a M borate-NaOH buffer (pH 10.0). FIG. 4 shows the relative activities at each temperature treatment when the enzyme activity treated at 30 ° C. was taken as 100. In the figure, the dotted line shows the case without Ca ²⁺ addition, and the solid line shows the case with Ca ²⁺ addition.

[0032] The alkaline protease K-16H is
In the system without Cl ₂ , up to 50 ° C and 5 mM CaCl ₂
In the addition system, 90% or more activity was maintained up to 60 ° C.
As a result, it was found that this enzyme was stabilized by Ca ²⁺ .

(7) Molecular weight The molecular weight of alkaline protease K-16H is 12.5%
Sodium dodecyl sulfate using acrylamide (SD
S)-Measured by polyacrylamide gel electrophoresis. Molecular weight markers include the Electrophoresis Calibratio Kit
n kit) [phosphorylase b (molecular weight 94,000), bovine serum albumin (molecular weight 67,000), ovalbumin (molecular weight 43,000), carbonic anhydrolase (molecular weight 30,000), soybean trypsin inhibitor (molecular weight 20,100) ) And α-lactalbumin (molecular weight 14,400); manufactured by Pharmacia. By this method, the molecular weight of alkaline protease K-16H was determined to be 28,000 ± 1,000.

(8) Effects of Metal Ions The effects of various metal ions on alkaline protease K-16H were examined. Various metal salts were added to a 20 mM borate-NaOH buffer (pH 9.0) so as to have a concentration of 1 mM, and 1.0 U of the enzyme was added. The mixture was allowed to stand at 30 ° C. for 20 minutes, and then a 50 mM borate-NaOH buffer ( pH 10.0)
Using a 10-fold diluted enzyme solution, the activity was measured.
The activity of a sample similarly treated without addition of a metal salt (control) was 1
Table 2 shows the relative activities when various metal salts were added.
It was shown to.

[0035]

As a result, alkaline protease K-16
H was found to be inhibited by about 50% by Hg ²⁺ . Further, it was sufficiently stable against other metal ions.

(9) Effects of Various Compounds The effects of various compounds on alkaline protease K-16H were examined. That is, a 20 mM phosphate buffer (p
H7.0), various compounds were added to a predetermined concentration, and 1.0 U of alkaline protease K-16H was added. After standing at 30 ° C. for 20 minutes, 50 mM boric acid-
The activity was measured using an enzyme solution diluted 10-fold with a NaOH buffer (pH 10.0). Table 3 shows the relative activity when each compound was added, where the activity of the compound treated in the same manner without adding the compound (control) was taken as 100.

[0038]

* Inhibitor abbreviation EDTA: ethylenediaminetetraacetic acid DTT: dithiothreitol NE M: N-ethylmaleimide HNB: 2-hydroxy-5-nitrobenzylbromide DTNB: 5,5'-dithiobis-2 -Nitrobenzoic acid PCNB: p-chloromercury benzoic acid PMSF: Phenylmethanesulfonyl fluoride

The alkaline protease K-16H was inhibited by phenylmethanesulfonyl fluoride (PMSF) and chymostatin, indicating that serine is involved in the expression of the activity. It was extremely stable with respect to other compounds.

(10) Influence of Surfactant The effect of various surfactants on alkaline protease K-16H was examined. That is, 0.1 M containing 5% ( w / v ) surfactant and 10% ( v / v ) ethanol.
2.0 U of alkaline protease K-16H was added to a Tris-HCl buffer (pH 9.0), and the mixture was allowed to stand at 40 ° C. for 4 hours. This treatment solution was diluted 10-fold with a 50 mM boric acid-NaOH buffer (pH 10.0), and the enzyme activity was measured. Table 4 shows the activity immediately after the addition of the enzyme as 100, and the residual activity as a relative value.

[0042]

The specific surfactants are as follows.

Embedded image

The alkaline protease K-16H exhibited high stability even when various surfactants were present at a high concentration (5%). In addition, the enzyme was very stable in the surfactant even when compared with the stability of a commercially available alkaline protease for detergents to 1.0% ( w / v ) of various surfactants.

[0045]

Next, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

Example 1 Production of Alkaline Protease K-16: Bacillus sp.
No. 76) One platinum loop was inoculated on the following medium A at 30 ° C.
For 24 hours under aerobic shaking to obtain a seed culture.
Next, the obtained seed culture was added to the following medium B at 2% ( v / v).
v ) Inoculation, aerobically shaking culture at 30 ° C. for 48 hours, and 1 liter of culture solution containing alkaline protease K-16.
Got a toll .

Medium A: glucose 2.0% ( w / v ) polypeptone S 1.0 酵母 yeast extract 0.05 一 monopotassium phosphate 0.1 マ グ ネ シ ウ ム magnesium sulfate heptahydrate 0.02 ナ ト リ ウ ム sodium carbonate ( 1.0 〃 (no pH adjustment)

Medium B: glucose 2.0% ( w / v ) fish meat extract 1.0 〃 soy flour 1.0 − potassium phosphate 0.1 マ グ ネ シ ウ ム magnesium sulfate heptahydrate 0.02 ナ ト リ ウ ム sodium carbonate ( 1.0 滅菌 (pH 10.0)

Example 2 Purification of Alkaline Protease K-16H: (1) One liter of the culture solution obtained in Example 1 was centrifuged (1
(000 rpm: 5 minutes) to remove the cells, and the supernatant was concentrated with an ultrafiltration membrane (molecular weight cut off 5,000) and freeze-dried. After dissolving 1.5 g of the obtained powder in 20 ml of ion-exchanged water, dialysis was performed overnight against 10 mM Tris-HCl buffer (containing 2 mM Ca ²⁺ , pH 8.0). Next, the dialysis obtained on a DEAE Bio-Gel A column (2.5 × 16 cm, manufactured by Bio-Rad) previously equilibrated with 10 mM Tris-HCl buffer (containing 2 mM Ca ²⁺ , pH 8.0). The internal solution was adsorbed, and washing and elution were performed with the same buffer.
The non-adsorbed protein part eluted by washing is collected (150 ml),
Concentration was performed by ultrafiltration (YM-5 membrane, manufactured by Amicon).

The concentrated solution (20 ml) was previously added to 1
CM Bio- equilibrated with 0 mM Tris-HCl buffer (containing 2 mM Ca ²⁺ , pH 8.0)
After adsorbing to a Gel column (2.5 × 16 cm; manufactured by Bio-Rad) and eluted with 250 ml of the same buffer, elution was carried out with 0 to 30 mM potassium chloride gradient elution with the same buffer containing 30 mM potassium chloride. (22
5 ml each). Further, similarly, from 30 mM to 100 mM
Was performed (225 ml each). Fractions containing alkaline protease K-16H eluted at a potassium chloride concentration of 20 mM to 30 mM were collected (180 ml), and concentrated by ultrafiltration.

The concentrated solution (7.2 ml) was previously
M Tris-HCl buffer (containing 2 mM Ca ²⁺ , pH
8.0) which had been equilibrated with CM Bio-Ge l
A column (1.6 × 16 cm; manufactured by Bio-Rad) was adsorbed, washed and eluted with 150 ml of the same buffer solution, and then 25 m
M 0 to 25 mM in the same buffer containing potassium chloride
(150 ml each). From the results of electrophoresis of each fraction, fractions containing only alkaline protease K-16H were collected (12.
5 ml), concentrated by ultrafiltration (1 ml), and glycerol was added to a final concentration of 20% ( v / v ).
Stored at -20 ° C until use.

By the above operation, 1.5 g of the crude enzyme powder was obtained.
(Summary of, 79,560U; total protein, 918 mg; specific activity, 86.7U / mg) than, polyacrylamide gel electrophoresis and SDS- Poria click Riruamidogeru uniform alkaline protease by electrophoresis K-16H (total activity,
408 U; purified protein, 3.4 mg; specific activity, 120 U
/ Mg).

[0053]

As described above, the alkaline protease K-16H of the present invention, which is uniformly purified from the crude enzyme alkaline protease K-16, is stable in various surfactants. Accordingly, the enzyme of the present invention are those extremely excellent as enzymes for detergent formulations, are those which can be advantageously used in detergents, and the like.

[Brief description of the drawings]

FIG. 1 Optimum pH of alkaline protease K-16H
Drawing.

FIG. 2 shows the results of a pH stability test of alkaline protease K-16H.

FIG. 3 is a drawing showing the optimal temperature of alkaline protease K-16H.

FIG. 4 is a drawing showing the results of a thermal stability test of alkaline protease K-16H. that's all

────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Susumu Ito 3441-64, Higashimine-cho, Utsunomiya-city, Tochigi (56) References JP-A-1-2477084 (JP, A) JP-A-6-506352 (JP, A) (58) Field surveyed (Int. Cl. ⁶ , DB name) C12N 9/00-9/96 BIOSIS (DIALOG)

Claims (3)

(57) [Claims] 1. An alkaline protease K-16H having the following enzymatic properties. (1) Action It hydrolyzes simple proteins and complex proteins to produce oligopeptides or amino acids. (2) Substrate specificity It has good activity on water-soluble proteins such as casein and hemoglobin and water-insoluble proteins such as keratin and elastin. (3) Optimum pH When casein is used as a substrate and the reaction is carried out in various buffers at 40 ° C. for 10 minutes, the optimum pH is 90 to 11.0. In addition, the maximum activity of 8 in the pH range of 7.0 to 12.0.
It has an activity of 0% or more and has a pH of 6.0 to 13.0.
In the range of 50% or more of the maximum activity value. (4) pH stability When Ca ²⁺ is added to various buffers and treated at 55 ° C. for 10 minutes, the pH is extremely stable in the range of 5.0 to 12.0. (5) Optimum temperature When carrying out the reaction at pH 10.0 using casein as a substrate, the optimum temperature is 55 ° C. (6) Heat resistance When treated at pH 9.0 and 50 ° C. for 10 minutes, or when treated at 60 ° C. for 10 minutes after adding Ca ²⁺ , 90
% Or more of the remaining activity. (7) Molecular weight 28,000 ± 1,000 (by sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis). (8) Effect of metal ion When casein is used as a substrate, the activity is inhibited by Hg ²⁺ . In addition, Ca ²⁺ increases thermal stability and pH stability. (9) Effect of inhibitor The activity is not inhibited by ethylenediaminetetraacetic acid, p-chloromercury benzoic acid, and antipain. The activity is inhibited by phenylmethanesulfonyl fluoride and chymostatin. (10) Influence of surfactant Even if a surfactant such as sodium polyoxyethylene alkyl sulfate, sodium α-olefin sulfonate, sodium alkane sulfonate, α-sulfo fatty acid ester is present in a high concentration, it is extremely stable. . 2. The alkaline protease K-16H according to claim 1, which is produced by a microorganism belonging to the genus Bacillus. 3. The microorganism belonging to the genus Bacillus is Bacillus sp. KSM-K1.
The alkaline protease K- according to claim 2, which is 6.
16H.