JPH03191781A - Alkali protease and productive microorganism thereof - Google Patents

Abstract

PURPOSE:To obtain the subject enzyme having excellent stability in a liquid detergent component and stably reactional even at a high temperature by specifying action of alkali protease, optimum pH, range of stable pH, optimum temperature, heat resistance and molecular weight. CONSTITUTION:The objective alkali protease has hollowing properties: (1) action: hydrolyze protein. (2) optimum pH: at 10.5-12.0, in a case of reaction at 30 deg.C for 10min using casein as a substrate. (3) range of stable pH: in a case of treatment at 30 deg.C for 24hr is pH6-10 and in a case of treatment at 5 deg.C for 24hr is pH5-11. (4) optimum temperature: at 70 deg.C in a case of reaction at pH10.0 using casein at a substrate. (5) heat resistance: a temperature reducing activity to a half value in a treatment at pH10.0 for 10min is about 65 deg.C. (6) molecular weight: molecular weight determined by SDS polyacrylamide gel electrophoresis is 46000+ or -2000.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention has excellent stability against anionic detergent components and operates stably even at high temperatures. This invention relates to a novel alkaline protease and microorganisms that produce it.

[Problems with conventional technology] In recent years, attempts have been made to improve detergency by adding enzymes to detergents, and currently, protease and amylase are being used.

An enzyme-containing detergent containing cellulase, lipase, etc. is applied on top. Among them, protease.

It has become indispensable as a detergent ingredient because it absorbs 10-40% of the organic dirt that adheres to clothes, efficiently breaks down protein stains that cannot be removed by detergent ingredients alone, and improves cleaning power.

As detergent proteases, enzymes known as alcalase, esperase, savinase, etc. are conventionally used. Although these enzymes have sufficient stability as enzymes for formulating solid detergents, they are insufficiently stable in liquid detergents and are quickly deactivated at high temperatures, so they are not suitable for use in laundry, especially in Europe and in the cleaning industry. The activity cannot be fully expressed under the conditions of high temperature and high concentration of detergent.

Therefore, there is a need for a protease that has excellent stability in liquid detergents and can withstand use under high temperature and high concentration detergents.

[Means for solving problems]

An object of the present invention is to provide an alkaline protease that has excellent stability in liquid detergent components and acts stably even at high temperatures.

In order to achieve the above-mentioned objective, the present inventors conducted screening from soil using LAS resistance characteristics as a marker.
The present inventors have discovered that an enzyme produced by a strain of the genus Bacillus isolated from soil in Tama, Ota Ward, satisfies the above conditions, and have completed the present invention.

As described below, the strain of the present invention is judged to be a bacterium related to Bacillus firmus based on its mycological properties, but it differs from this in several respects and also from other known strains. Therefore, the present inventors identified this strain as a new bacterium, named it Bacillus 5D521, and deposited it with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Microbiology Research Institute No. 11162.

<Mycological properties> The new bacterial species 5D521 of the present invention, which is closely related to Bacillus firmus, exhibits the following mycological properties.

(A) Morphological properties (a) Cell shape and size Bacillus 0.3-0.6X1.3-2.0pm (b) Cell pleomorphism not observed (c) Motile flagella, Has mobility.

(d) Sporangium bulge, elliptical, subvertical 0.3-0.4 um Xo, 4-0.5 p m (e)
Positive for Durham staining (f) No acid-fastness B) Growth status on various media (a) Growth status on broth agar medium Grows at pH 7 and forms one round, flattened, gold-rimmed colony. The surface of the colony is smooth, shiny, and cream-colored.

(b) Growth on a gravy agar slant Grows in a wave band shape at pH 7, forms shiny cream-colored colonies, and does not form 0 pigment.

(Q) Meat juice liquid culture It grows well at pH 7, but does not form a bacterial membrane.

(d) Meat juice gelatin puncture culture Liquefy gelatin at pH 7.

(e) Ridmus milk Liquefaction of milk is observed at pH 7.

pH is neutral at the end of culture.

(C) Physiological properties (a) Nitrate reduction: negative (b) 3151 nitrogen reaction: negative (c) MR test: negative (d) VP test: negative VP Brosno pH 7,2 (e) Hydrogen sulfide formation: negative (f) Hydrolysis of starch: Positive (g) Utilization of citric acid: Koser, Christ
It is not used in the culture medium of S. ensen.

(h) Utilization of inorganic nitrogen source: Sodium nitrate.

Utilize ammonium sulfate.

(i) Formation of dye No two dyes are formed.

(j) Urease: negative (k) Oxidase: positive (1) Catalase: positive (m) Growth temperature range = Good growth at 20-30°C, but growth possible at 15-45°C.

(n) Growth pH range: Good growth at pH 7-10.

It does not grow at pH below 6.8 and above pH 10°8.

(o) Attitude towards oxygen: aerobic. Unable to grow under anaerobic conditions.

Production of acids and gases from Cp>N species: Table 1 (+: Produces -: Does not produce Table 1, blank space below) Production of acids and gases from sugars is useful in comparing the properties of bacteria Therefore, we examined it in detail in direct comparison with type culture. Bacillus firmus IFO for comparison
3330 was used.

The initial pH of the culture medium used in the test was set to 9.0, which is good for growth in both plots.

(q) O-F test: Fermentation (r
) Tolerance to sodium chloride: Grows under 7% NaCl.

The above mycological properties of Bacillus 5D521 are described in "Virgin's Manual of Obstructive Bacteriology, 8th edition (1975) J and "The Genus Bacillus, Agriculture Handbook No. 42.
7 (1973) J. Comparison with other strains is as follows.

SD521 is a Gram-positive aerobic bacillus and has spore-forming ability, so it is clear that it belongs to the genus Bacillus. 5D521 is positive for catalase, negative for vP reaction, does not grow at 65°C, has starch decomposition ability, does not generate gas from glucose, does not use citric acid, etc. Bacillus fi
rmus) was identified as a related bacterium.

However, 5D521 does not grow at pH 6 or 8, but Bacillus firmus does, and while 5D521's sporangia swell, Bacillus firmus' sporangia rarely swell. Regarding production of acid gas from Bacillus firmus, 5D521 produces acid from sugars such as maltose, glycerol, trehalose, starch, etc., but Bacillus firmus does not. There is.

As is clear from the above description, the present strain is judged to be a bacterium related to Bacillus firmus based on its mycological properties, but it differs from this and other known strains in several respects. Therefore, the present inventors obtained the present strain Bacillus 5D.
521 was determined to be a new strain related to Bacillus firmus.

Method for Obtaining Protease> The protease produced by the Bacillus strain 5D521 is referred to as 5DP521.

The protease 5DP52 of the present invention was produced using the 5D521 strain.
1 can be obtained by inoculating the strain into an appropriate medium and culturing it according to a conventional method.

The medium used in the present invention may be any medium as long as it allows the bacteria of the genus Bacillus of the present invention to proliferate and produce alkaline protease.For example, the carbon source may include glucose, maltose, sucrose, and soluble starch. As nitrogen sources such as pon, organic nitrogen sources such as soybean meal, rice cake, bran cake, cornstarch liquor, and inorganic nitrogen sources such as ammonium sulfate are used. In addition, inorganic salts such as phosphates, potassium salts, and magnesium salts are added.

The culture in the present invention is carried out under aerobic conditions, for example, by aeration stirring method or shaking culture method.The culture temperature may be any temperature of 20 to 40°C, but preferably 30°C to 37°C for the best growth. , the initial pH is pH 9-10, but the pH during culture is 8.
．． 5 to 10 are preferable, respectively, and the culture time is 16 to 60.
The cultivation time may be approximately 1 hour, and the culture may be terminated when the protease activity reaches its maximum.

The target protease can be isolated and purified from the thus obtained culture solution in accordance with a general enzyme separation and purification method. That is, a supernatant or filtrate can be obtained from which bacterial cells and medium solids are removed by centrifugation or filtration. Salting-out method, solvent precipitation method, and spray drying method involve adding soluble salts and hydrophilic organic solvents to these separated liquids to precipitate proteins.

The protease of the present invention is obtained by a freeze-drying method or the like.

Furthermore, it can be purified and used in combination with purification methods such as ion exchange chromatography and gel filtration chromatography.

The activity of the alkaline protease of the present invention thus obtained is measured by the method shown below.

Measuring method of titer > M/20pH 10.0 A
Add 50 μl of an appropriately diluted enzyme solution to 500 μl of tkin s-Pan tin boric acid buffer, and incubate at 30° C. for 3 to 5 minutes.

500 μl of 2% Hammersten casein solution at pH 10.0 was added to the solution, and 10 minutes later, 2 ml of TCA solution was added to stop the reaction.

After being left at 30°C for 10 minutes or more, it was filtered using No. 2 filter paper (Toyo Filter Paper). 0 in 1 ml of filtrate
．． 5 ml of 4M soda carbonate, 6-fold dilution of phenol reagent 1
After adding ml of the solution and allowing it to stand at 30°C for 20 minutes to develop color, the absorbance at 660 nm is measured.

The enzyme unit is a protease that, when reacted with casein as a substrate at 30°C and pH 10°O, produces a decomposed product in the TCA-soluble fraction that exhibits a color at 660 nm, equivalent to 1 mole of tyrosine, per second. 1 katal
).

Physicochemical Properties of Enzyme> The physicochemical properties of 5DP521 will be explained in detail below.

(1) Action The action of the two enzymes is casein and BSA.

Hydrolyzes proteins such as OVA, hemoglobin, and keratin.

(2) Substrate specificity The substrate specificities of the dual enzymes are shown in Table 2.

Table gJ2 shows the activity when undenatured casein is used as a substrate as 100%, and 1 decomposition rate is expressed as a relative value.

Conditions: Substrate denaturation conditions 100°C 10 minutes reaction conditions
pH: 10,030°C Substrate concentration: 1% Enzyme concentration: Approximately 1 nkatal/ml From this table, it can be seen that this enzyme has high specificity for hemoglobin and keratin.

(3) Optimal pH/stable pH; Next, the optimal pH for one enzyme
A graph of H and stable pH range is shown in FIG.

The buffer is Brittton-Robinson broad range buffer pH 4-12.

When examining the optimum pH, add the enzyme solution to approximately 1 nkatal/m in each pH If solution containing 1% casein.
1 and reacted at 30°C for 10 minutes to measure the activity (1-a).

To check the stable pH, add enzyme to a buffer solution of each pH at a concentration of about 200 nkatall/ml and incubate at 5°C.
After incubating at 30°C for 24 hours, the activity was measured (1-b, 1-c). The residual activity at each pH was determined, setting the activity before incubation as 100%.

As can be seen from Figure 1, this enzyme has a pH of 10.5 to 1.
The optimum pH is 2.0, and the stable pH range is 5℃.
H5-11, pH 6-10 at 30°C.

(4) Optimal temperature and temperature stability; Furthermore, the optimal temperature and temperature stability of this enzyme are shown in FIG. When determining the optimum temperature, use a pH 10.0 containing 1% casein as a substrate.
(r)M/20E')W11a'a The solution was incubated for 5 minutes at warm water temperature, and then the enzyme was added and allowed to react at each temperature for 10 minutes. Relative activity was determined at each temperature, setting the activity at 30°C as 100 (2-a).

Temperature stability was measured by the following method. Approximately 20 nkatal/ml of enzyme was added to M/20 borate buffer pH 10.0 once, heat treated at each temperature for 10 minutes, cooled on ice, and the activity was measured (2-b). 1
The residual activity after heat treatment for 0 to 90 minutes was measured (2-C).

As can be seen from Figure 2 (a-c'), this enzyme was heated at 70°C.
It has an optimum temperature nearby. In addition, the temperature at which the activity is halved by heat treatment at pH 10, 0, and 10 minutes is around 70°C;
More than 60% of the activity remains even after heat treatment at ℃ for 90 minutes.

Savuinase, Alcalase, and Esperase, which are conventionally contained in detergents, have an optimum temperature of 60-65°C under these conditions, and their residual activity after 10 minutes of treatment at 65°C is 30% or less, so the protease of the present invention It can be seen that this product has higher activity at high temperatures and is more stable than conventional products.

(5) Effects of metal ions: Next, the effects of various metal ions on this enzyme were investigated. M/20 borate buffer pH 10
Various metal ions were added to a concentration of 1 mM to an enzyme solution of about 20 nkatal/ml that had been diluted and prepared with . The results are shown in Table 3 as relative activities, with the activity at 0 time without the addition of metal salts being taken as 100%.

Table 3 shows that the activity of this enzyme is inhibited by the addition of copper sulfate.

Furthermore, the conditions and method for investigating the influence of calcium ions on temperature stability are as follows.

Contains 1mM calcium chloride or EDTA.

About 20% of this enzyme was added to M/20 borate buffer pH 10.0.
Add to 0 n k / ml and heat at 60°C for 10
The mixture was heat-treated for a minute, and water-cooled f&residual activity was measured. The results are shown in Table 4, taking the activity value before heat treatment as 100%.
As can be seen from Table 4, the stability of this enzyme is increased by calcium ions.

Table 3 (6) Effect of inhibitors: Next, the conditions and methods for examining the effects of various inhibitors on this enzyme are as follows.

M/20 boric acid loose solution pH 10.0, about 20% of this enzyme
nkatal/ml, various inhibitors were added, and residual activity was measured after incubation at 30°C for 30 minutes. The results are shown in Table 5.
Expressed as relative activity expressed as %.

As shown in this table, this enzyme is significantly inhibited by PMSF, which indicates that it is a serine protease.

Table 5 The product was added to a liquid detergent containing about 25% anion, about 10% nonion, and 50% water at a concentration of about 200 nkatall/ml, stored at 40°C for one month, and the activity was measured. The results are shown with the activity immediately after adding the enzyme to the washing solution taken as 100.

As can be seen from Figure 3, when this enzyme was stored at 40°C during washing, the approximate half-life was 21B, and the residual activity after 4 weeks was 35%.
erase, Alcal-a s e, S a
It can be seen that the storage stability is much better than that of VINASE.

(7) Molecular weight: Next, the molecular weight of one enzyme is determined by SDS.
It was measured by polyacrylamide electrophoresis.

Using the same method, the molecular weight of one enzyme was 46,000±2,00
It was calculated as 0.

(7) Stability in liquid detergent: Furthermore, the stability of this enzyme in liquid detergent is shown in Figure 3. 2% lees, 2% maltose, 0.
500 liters of a liquid medium containing 2% magnesium sulfate, 0.05% magnesium sulfate, and 1% sodium carbonate was placed in a 1 m3 culture tank, steam sterilized, and cultured in advance with 5D52.
One strain was inoculated and cultured with aeration and stirring at 35°C for 26 hours. This culture solution was treated with a sterilizing membrane, and the protease activity of the obtained supernatant was 85 nkatal/ml.

Example 2; <5DP521 Enzyme Preparation Method> The microbial culture solution obtained in Example 1 was treated with a sterilizing membrane, and then the enzyme was concentrated using a UF membrane. This concentrated solution was subjected to ammonium sulfate weir fractionation to reach 35% saturation.

Further, the obtained precipitate was dissolved in ion-exchanged water and dialyzed.

The solution was then vacuum lyophilized to concentrate the enzyme. The enzyme preparation was added to 75% PG/128mM NaCl/1000p.
Dissolved in p1000pp 1'L OOOnkata
An enzyme sample of l/ml was obtained. The enzyme properties of the sample were examined and found to be as described above.

Example a; <5DP521 enzyme preparation method> The ammonium sulfate fraction prepared according to Example 2 was dialyzed and subjected to DEAE Cellulofine A-200 column chromatography, 0M Toyopearl, and high performance gel filtration chromatography column Shodex WS. -803 was used for separation and purification. When this sample was subjected to SDS polyacrylamide gel electrophoresis, a single band was detected, and its molecular weight was as described above.

[Brief explanation of drawings]

Figure 1 shows the optimum pH range and pH stability of this enzyme. Figure 2 shows the temperature profile and temperature stability, and Figure 3 shows the storage stability in liquid detergent.

Claims (1)

[Claims] 1) Alkaline protease having the following properties (1) Action: Hydrolyzes proteins. (2) Optimal pH: When reacted at 30° C. for 10 minutes using casein as a substrate, the optimal pH ranges from 10.5 to 12.0. (3) Stable pH range: The stable pH range when treated at 30°C for 24 hours is from pH 6 to 10, and the stable pH range when processed at 5°C for 24 hours is from pH 5 to pH 11. (4) Optimal temperature: When reacting with casein as a substrate at pH 10.0, the optimal temperature is 70°C. (5) Heat resistance: The temperature at which the activity is halved after treatment at pH 10.0 for 10 minutes is approximately 65°C. When heat treated at 70°C for 10 minutes at pH 10.0, 4
0% or more activity remains, and 60% or more activity remains after treatment at pH 10.0 and 60°C for 90 minutes. (6) Molecular weight: The molecular weight measured by SDS polyacrylamide electrophoresis is 46,000±2,000. 2) A microorganism that belongs to the genus Bacillus and produces a protease having the physicochemical properties of claim 1.