JP2882652B2 - Alkaline protease and its producing microorganism - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a novel alkaline protease having excellent stability to anionic detergent components and acting stably even at high temperatures, and a microorganism producing the same.

[Problems of the prior art] In recent years, attempts have been made to increase the detergency by adding enzymes to detergents. At present, enzyme-containing detergents containing protease, amylase, cellulase, lipase and the like have been put on the market. Among them, proteases have become indispensable as detergent components because they represent 10 to 40% of the induced dirt attached to clothing and efficiently decompose protein dirt that cannot be removed by detergent components alone and increase detergency. I have.

As proteases for detergents, enzymes known as alcalase, esperase, sabinase and the like have been used. Although these enzymes have sufficient stability as solid detergent blending enzymes, their stability in liquid detergents is insufficient, and their deactivation at high temperatures is rapid. Under the conditions of high temperature and high concentration of detergent, the activity cannot be sufficiently exhibited.

Therefore, a protease that has excellent stability in a liquid detergent and can withstand use under a high-temperature and high-concentration detergent is desired.

[Means for Solving the Problems] It is an object of the present invention to provide an alkaline protease having excellent stability in a liquid detergent component and acting stably even at a high temperature.

The present inventors, in order to achieve the above object, as a result of screening from soil using LAS resistance characteristics as a marker,
The present inventors have found that the enzyme produced by one strain of the genus Bacillus isolated from the soil of Tamagawa, Ota-ku satisfies the above conditions, and have completed the present invention.

As described below, the strain of the present invention is judged to be Bacillus pharmus related bacterium based on mycological properties, but differs in some respects and also differs from other known strains. Accordingly, the present inventors have identified this strain as a novel strain, named it Bacillus SD521, and deposited it in the Microbial Industry Research Institute of the National Institute of Advanced Industrial Science and Technology under No. 11162 of the Microbial Laboratory.

<Mycological properties> The new strain SD521, which is closely related to Bacillus pharmus of the present invention, has the following mycological properties.

(A) Morphological properties (a) Cell shape and size Bacillus 0.3-0.6 μm × 1.3-2.0 μm (b) Cell polymorphism Not observed (c) Motility Flagella and motile.

(D) Spores Spore swelling, oval, sub-edge 0.3-0.4μmX0.4-0.5μm (e) Gram staining positive (f) No acid resistance (B) Growth on various media (a) Gravy agar medium Growing at pH 7 and forming round, flat, whole-edge colonies. The surface of the colonies is smooth, shiny and creamy.

(B) Growth on gravy agar slope Growing in a band form at pH 7, forming glossy cream-colored colonies. No pigment is formed.

(C) Broth liquid culture Growing well at pH 7, but no pellicle formation.

(D) Broth gelatin puncture culture Gelatin is liquefied at pH7.

(E) Litmus milk Liquefaction of milk is observed at pH 7.

 PH neutral at the end of culture.

(C) Physiological properties (a) Nitrate reduction: negative (b) Denitrification reaction: negative (c) MR test: negative (d) VP test: negative VP Pros pH 7.2 (e) Hydrogen sulfide production: Negative (f) Starch hydrolysis: positive (g) Use of citric acid: Not used in Koser and Christensen's medium.

(H) Use of inorganic nitrogen source: Use sodium nitrate and ammonium sulfate.

(I) Dye formation: No dye is formed.

(J) Urease: negative (k) Oxidase: positive (l) Catalase: positive (m) Growth temperature range: good growth at 20-30 ° C, but 15-4
Can grow at 5 ℃.

(N) pH range of growth: good growth at pH 7-10, no growth at pH 6.8 or lower and pH 10.8 or higher.

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

(P) Production of acid and gas from saccharides: Table 1 (+; produced-; not produced) *) Since the production of acid and gas from sugar is useful in comparing the properties of bacteria, it was examined in detail in comparison with direct type culture. For comparison, Bacillus Pharmaceutical IFO3
330 was used.

The initial pH of the medium used for the test is that both bacteria have good growth
The pH was adjusted to 9.0.

(Q) OF test: Fermentation (r) Resistance to sodium chloride: grows under 7% NaCl.

The bacteriological properties of Bacillus SD521 described above are described in "Barges Manual of Detergent Bacteriology Eighth Edition (1975)" and "The Genus Bacillus, Agriculture Handbook No.427 (1973)".
According to the description, the following is a comparison with other strains.

SD521 is a Gram-positive aerobic bacillus and has a spore-forming ability, so that it clearly belongs to the genus Bacillus. And SD521 is catalase positive, V
A bacterium similar to Bacillus firmus because it has a negative P reaction, does not grow at 65 ° C, has starch resolution, does not generate gas from glucose, and does not use citric acid. Was identified.

However, although SD521 does not grow at pH 6.8, Bacillus
Farmers grow, SD521 spores swell, but Bacillus pharma rarely swells. Compared to culture, SD521 produces acids from sugars such as maltose, glycerol, trehalose, and starch, but does not produce Bacillus pharmas.

As is clear from the above description, this strain is judged to be Bacillus pharmus related bacterium based on mycological properties, but differs in some respects and also differs from other known strains. Therefore, the present inventors have determined that this strain Bacillus SD
521 was determined to be a new strain related to Bacillus farmus.

<Method for Obtaining Enzyme> The protease produced by the Bacillus SD521 strain is referred to as SDP521. In order to obtain the protease SDP521 of the present invention using the strain SD521, the strain may be cultured in a conventional manner by inoculating the strain into an appropriate medium.

The medium used in the present invention is not particularly limited as long as the bacterium belonging to the genus Bacillus of the present invention can proliferate and produce an alkaline protease.Examples of the carbon source include glucose, maltose, sucrose, and soluble starch. As the nitrogen source, an organic nitrogen source such as soybean meal, refuse, bran, and corn steep liquor, and an inorganic nitrogen source such as ammonium sulfate are used. In addition, phosphates,
Inorganic salts such as potassium salts and magnesium salts are added.

The culture in the present invention is performed under aerobic conditions, for example, by aeration and agitation or shaking culture. The cultivation temperature may be any of 20 to 40 ° C, but is preferably 30 to 37 ° C, which has the best growth. The initial pH is preferably 9 to 10, and the pH during culture is preferably 8.5 to 10, respectively. The culture time is about 16 to 60 hours, and the culture may be terminated when the protease activity reaches the maximum.

The desired protease can be separated and purified from the culture solution thus obtained in accordance with a general method for separating and purifying enzymes. That is, it is possible to obtain a supernatant or a filtrate from which cells and medium solids have been removed by a centrifugation method or a filtration method. The protease of the present invention is obtained by a salting-out method, a solvent precipitation method, a spray-drying method, a freeze-drying method, or the like, in which a soluble salt or a hydrophilic organic solvent is added to these separated liquids to precipitate proteins. Furthermore, purification can be performed by combining 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 following method.

<Method for measuring titer> 50 μl of an appropriately diluted enzyme solution is added to 500 μl of Atkins-Pantin borate buffer at M / 20 pH 10.0, and pre-incubated at 30 ° C. for 3 to 5 minutes. To the solution was added 500 μl of a 2% Hamerstencasein solution having a pH of 10.0, and after 10 minutes, 2 ml of a TCA solution was added to stop the reaction.

After leaving at 30 ° C. for 10 minutes or more, the mixture was filtered using No. 2 filter paper (Toyo Filter Paper). 5 ml of 0.4 M sodium carbonate and 1 ml of a 5-fold diluted phenol reagent were added to 1 ml of the filtrate, and the mixture was added at 30 ° C.
After allowing the color to develop for 20 minutes, the absorbance at 660 nm is measured.

In addition, when the enzyme unit is reacted at 30 ° C. and pH 10.0 using casein as a substrate, the proteinase activity which produces a decomposed product having a color development of 660 nm corresponding to 1 mol of tyrosine per second in the TCA-soluble fraction per second is 1%. Qatar (1katal).

<Physicochemical properties of enzyme> The physicochemical properties of SDP521 will be described in detail below.

(1) Action: The action of this enzyme hydrolyzes proteins such as casein, BSA, OVA, hemoglobin, and keratin.

(2) Substrate specificity: Table 2 shows the substrate specificity of the present enzyme.

The activity when using native casein as a substrate is defined as 100%, and the degradation rate is indicated as a relative value.

Conditions: Substrate denaturation conditions 100 ° C 10 minutes Reaction conditions pH 10.0 30 ° C Substrate concentration 1% Enzyme concentration Approx. 1nkatal / ml From this table, it can be seen that this enzyme has high specificity for hemoglobin and keratin.

(3) Optimum pH and stable pH;
FIG. 1 shows a graph of the H region. Buffer is Britton-Robins
The on range buffer is pH 4-12.

To determine the optimal pH, each sample containing 1% casein
The enzyme solution was added to the pH buffer at a concentration of about 1 nkatal / ml, reacted at 30 ° C. for 10 minutes, and the activity was measured (FIG. 1 (a)).

To determine the stable pH, add the enzyme to the buffer at each pH.
The activity was measured after adding at 200 nkatal / ml and incubating at 5 ° C. and 30 ° C. for 24 hours (FIG. 1 (b),
(C)). Assuming the activity before incubation as 100%, each p
The residual activity at H was determined.

As can be seen from FIG. 1, the enzyme has an optimum pH of pH 10.5 to 12.0, and has a stable pH range of 5 to 11 at 5 ° C and pH at 30 ° C.
H6 to 10.

(4) Optimum temperature and temperature stability; FIG. 2 shows the optimum temperature and temperature stability of the present enzyme. To determine the optimum temperature, a M / 20 borate buffer at pH 10.0 containing 1% casein as a substrate was incubated at each temperature for 5 minutes, and then the enzyme was added and reacted at each temperature for 10 minutes. . The relative activity at each temperature was determined with the activity at 30 ° C. as 100 (FIG. 2 (a)).

Temperature stability was measured by the following method. M / 20 borate buffer
Add the enzyme to a concentration of about 20 nkatal / ml at pH 10.0, and add 10
After heat treatment for minutes, and cooling on ice, the activity was measured (FIG. 2 (b)). Similarly, the residual activity after heat treatment at 60 ° C. for 10 to 90 minutes was measured (FIG. 2 (c)).

As can be seen from FIG. 2 ((a) to (c)), the present enzyme has an optimum temperature around 70 ° C. The temperature at which the activity is reduced by half at pH 10.0 for 10 minutes is around 70 ° C, and 90 ° C at 60 ° C.
Even after the heat treatment, 60% or more of the activity remains.

Savinases, alcalases and esperases conventionally formulated in detergents have an optimum temperature of 60 to 65 ° C under these conditions, and the residual activity after treatment at 65 ° C for 10 minutes is 30% or less. It can be seen that the protease has higher activity at high temperatures and is more stable than the conventional product.

(5) Effect of metal ions; Next, the effects of various metal ions on the present enzyme were examined. Various metal ions were added to an enzyme solution of about 20 nkatal / ml diluted and prepared with M / 20 borate buffer pH 10.0 to a concentration of 1 mM, and incubated at 40 ° C. for 24 hours, and then the activity was measured. The results are shown in Table 3 as relative activities, where the activity at 0 time was 100% without the addition of a metal salt.

Table 3 shows that the addition of copper sulfate inhibits the activity of the enzyme.

Furthermore, the effect of calcium ions on temperature stability was investigated. The conditions and method are as follows.

Add this enzyme to M / 20 borate buffer (pH 10.0) containing 1 mM calcium chloride or EDTA to a concentration of about 20 nkatal / ml, heat-treat at 60 ° C for 10 minutes, cool on ice, and measure residual activity did. The results are shown in Table 4 with the activity value before the heat treatment being 100%. As can be seen from this table, the stability of this enzyme is increased by calcium ions.

(6) Influence of inhibitors: Subsequently, the effects of various inhibitors on the present enzyme were examined. The conditions and method are as follows.

M / 20 borate buffer pH 10.0, about 20 nkatal / ml of this enzyme
, Various inhibitors were added, the mixture was incubated at 30 ° C. for 30 minutes, and the residual activity was measured. The results are shown in Table 5 in terms of relative activity, where the value without inhibitor was 100%.

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

(7) Stability in liquid detergent: FIG. 3 shows the stability of the present enzyme in liquid detergent. Each enzyme has about 25 anions
%, About 10% nonionic, about 50% moisture in liquid detergent
It was added to a concentration of 200 nkatal / ml and stored at 40 ° C. for one month, and the activity was measured. The results were shown with the activity immediately after adding the enzyme to the liquid washing as 100.

As can be seen from FIG. 3, when the enzyme is stored at 40 ° C. during washing, the approximate half-life is 21 days and the residual activity after 4 weeks is
Esperase, A which was 35%
It can be seen that the storage stability is much better than lcalase and Savinase.

(7) Molecular weight: Subsequently, the molecular weight of the present enzyme was measured by SDS polyacrylamide gel electrophoresis.

According to the same method, the molecular weight of this enzyme was calculated to be 46,000 ± 2,000.

[Examples] Example 1; <culturing method of SD521> Soybean meal 2%, maltose 2%, dipotassium phosphate 0.1%
2%, magnesium sulfate 0.05% and sodium carbonate 1%
500 liters of a liquid medium containing 1 μm in a 1 m ³ culture tank, steam sterilized, and inoculated with the previously cultured SD521 strain.
The culture was carried out at 5 ° C. for 26 hours with aeration and stirring. Protease activity of the supernatant obtained by subjecting this culture to a bacteria elimination membrane was 85 nkatal / m
l.

Example 2 <SDP521 enzyme preparation method> The microorganism 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 salting out at 35% saturation.

Further, the obtained precipitate was dissolved in ion-exchanged water and dialyzed. Subsequently, the solution was freeze-dried in vacuo to concentrate the enzyme.
The enzyme preparation was dissolved in 75% PG · 128 mM NaCl · 1000 ppm CaCl ₂ to obtain an enzyme sample of 14,000 nkatal / ml. When the enzyme characteristics of the sample were examined, it was as described above.

Example 3 <SDP521 Enzyme Preparation Method> The ammonium sulfate fraction prepared according to Example 2 was dialyzed and subjected to DEAE Cellulofine A-200 column chromatography, CM Toyopearl and high-performance gel filtration chromatography columns Showdex WS-803. And purified. When SDS polyacrylamide gel electrophoresis was performed using the sample, it was detected as a single band, and its molecular weight was as described above.

[Brief description of the drawings]

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

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1:07) (72) Inventor Koji Suzuki 2-24-25 Tamagawa, Ota-ku, Tokyo Showa Denko KK (72) Inventor Yasufumi Yasuda 5-1 Okawacho, Kawasaki-ku, Kawasaki-shi, Kanagawa Prefecture Showa Denko KK Engineering Research Center (58) Field surveyed (Int. Cl. ⁶ , DB name) C12N 9/54 BIOSIS ( DIALOG) WPI (DIALOG)

Claims (3)

(57) [Claims] An alkaline protease having the following properties: (1) Action: Hydrolyzes a protein. (2) Optimum pH: When casein is used as a substrate and reacted at 30 ° C. for 10 minutes, it has an optimum pH of 10.5 to 12.0. (3) Stable pH range Stable pH when treated at 30 ° C for 24 hours
The range is pH 6 to 10, and the stable pH range when treated at 5 ° C. for 24 hours is pH 5 to 11. (4) Optimum temperature: When casein is used as a substrate and reacted at pH 10.0, it has an optimum temperature of 70 ° C. (5) Heat resistance: The temperature at which the activity is reduced to half after treatment at pH 10.0 for 10 minutes is about 65 ° C. When heat treated at 70 ° C. for 10 minutes at pH 10.0, activity remains at least 40%, and at 90 ° C. for 90 minutes, activity remains at 60% or more. (6) Molecular weight: The molecular weight measured by SDS polyacrylamide gel electrophoresis is 46,000 ± 2,000. 2. A Bacillus SD521 strain, FER, which produces a protease having physicochemical properties according to claim 1.
MP-11162. 3. A Bacillus SD52 according to claim 2.
2. The method for producing a protease having physicochemical properties according to claim 1, wherein one strain, FERM P-11162, is cultured, and the protease is separated and purified from the culture broth.