JPH0763368B2 - Heat-resistant alkaline protease and method for producing the same - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a thermostable alkaline protease and a method for producing the same, and more particularly to a novel thermostable alkaline protease produced from a novel microorganism belonging to the genus Bacillus and a method for producing the same.

[0002]

2. Description of the Related Art Alkaline protease is an enzyme that specifically hydrolyzes peptide bonds of proteins.
It is widely used in the textile, leather and other industries. Further, it is known that such an alkaline protease exerts an enzymatic activity in alkaline and is produced by an alkalophilic microorganism.

Alkaline proteases produced from the above-mentioned alkali-philic microorganisms include Japanese Patent Publication No. 62-3
There is NF-1 (see Table 4) disclosed in Japanese Patent No. 1911. Further, as disclosed in JP-A-62-160451, this NF-1 is well used for recovering silver from photographic film because it decomposes gelatin well among proteins, so It has been reported that the enzyme can be repeatedly used at a much higher recovery rate than the recovery of silver using an alkaline protease produced from a sex microorganism.

[0004]

The above-mentioned NF
In the gelatin decomposition reaction by -1, the higher the reaction temperature, the more N
It has been found that the gelatin decomposing power of F-1 is increased and the silver recovering power is increased. However, this NF-1 lacks thermal stability in a high temperature range and its activity decreases, so that the reaction temperature range is There is a problem of being limited. The present invention aims to solve such problems, and an object thereof is to provide a heat-resistant alkaline protease having excellent heat resistance and high proteolytic reactivity even in a high temperature range, and a method for producing the same.

[0005]

The heat-resistant alkaline protease according to the present invention is characterized by having the following characteristics in order to achieve the above-mentioned object. (a) Molecular weight: about 30,000 (measured by SDS-polyacrylamide gel electrophoresis), (b) UV absorption spectrum of aqueous solution: maximum absorption is 275
~ 280 nm, (c) Extinction coefficient (E1%, 1 cm, 280 nm): 7.1, (d) Solubility: Soluble in water, insoluble in methyl alcohol, ethyl alcohol, acetone and ether, (e) Casein as substrate Optimum pH: 12 to 13, (f) pH stability: Stable at pH 5 to 12 when kept at 40 ° C for 1 hour in the presence of 2.5 mM calcium ion, (g) Casein as substrate Optimum temperature: 85 ℃ in the presence of 10mM calcium ion, (h) Thermal stability: It is stable up to 60 ℃ at 10 minutes at pH10, 50% residual activity at 80 ℃, and 90 ℃ at 90 ℃. Complete inactivation, (i) Stabilization and inhibition: Calcium ion stabilized, not inactivated by PCMB (parachloromercuric benzoic acid) or EDTA (ethylenediaminetetraacetic acid), and inactivated by DFP (diisopropylchlorophosphate). Activity, (j) Specific activity: 4080 U / mg .

The method for producing a heat-resistant alkaline protease according to the present invention comprises culturing strain 18-1 belonging to the genus Bacillus in an alkaline medium, accumulating the heat-resistant alkaline protease having the above-mentioned properties in the medium, and producing the heat-resistant alkaline protease. It is characterized by collecting. The aforementioned B18-1 strain is the Journal of Fermentation a
nd Bioengineering, vol.72, No.4 (1991) "Utilization of a Thermostable AlkalineProt
ease from an Alkalophilic Thermophile for the Reco
B1 in "Very of Silver from Used Xray-Film."
A strain which is described as "8 '" and is announced as "B18'" in the "Fermentation and Properties of Thermostable and Alkaline-Resistant Proteases from Bacillus sp. B18 '" by the inventors at the Japanese Society of Fermentation on November 8, 1991 Is.

The B18-1 strain is a new strain isolated from the soil collected at the Beppu hot springs by the inventors and has the following mycological properties. All the culture media used were adjusted to pH 10 with the addition of 1% sodium carbonate. (a) Morphology: A bacillus having a size of 0.6 to 0.9 × 2.4 to 3.0 µm, which is singular or long-chained and has flagella and is motility. It also forms spores of 1.0-1.2 x 1.2-1.4 µm. (b) Gram stainability: positive (c) anti-acidity: negative (d) Growth condition in medium: Growth condition when cultured in each medium containing 1% sodium carbonate at 45 ° C for 1 to 2 days.
Shown in. The color of the bacterium is white at the beginning of culture,
It becomes yellowish white in the latter stage of culture.

[Table 1] (e) Physiological properties: 1. Growth conditions: pH; grows in alkaline, not in neutral or acidic. Temperature: Does not grow above 20 ° C or above 62 ° C. 2. Reduction of nitrate: Reduce. 3. Denitrification reaction: Does not grow under anaerobic conditions. 4. MR test: Not clear due to alkaline medium. 5. VP test: Negative 6. Indole formation: No formation. 7. Hydrogen sulfide formation: No formation. 8. Hydrolysis of starch: It is hydrolyzed. 9. Utilization of citric acid: Use in Christensen medium and Coser medium. 10. Use of nitrates: Use. 11. Use of ammonium salt: Use. 12. Urease activity: Not clear due to alkaline medium. 13. Oxidase activity: positive 14. Catalase activity: positive 15. Attitude toward oxygen: aerobic 16. 6.0-F test: Does not grow under anaerobic conditions. 17. Whether or not acid is produced from sugars: Because of the alkaline medium,
The change in indicator due to acid formation is unclear and unknown. 18. Liquefaction of gelatin: Liquefaction. 19. Sodium chloride tolerance: Grow in medium containing 5% sodium chloride. Generally, the pH value of the bacterial species belonging to the genus Bacillus is 5 to 8 as a suitable growth condition. On the other hand, the B18-1 strain as described above is an aerobic spore bacterium,
It can be said that it belongs to the alkalophilic Bacillus genus, which is clearly distinguished from general Bacillus, because it hardly grows in neutral and acidic media and grows very well in alkaline media.

Further, as the alkaliphilic Bacillus strain similar to the B18-1 strain used in the present invention, the B21-2 strain and the Japanese Patent Publication No. 55-46711 disclosed in Japanese Examined Patent Publication No. 62-31911. No. disclosed in the publication. 4 shares
No. 48-2794 is disclosed. 221 strain and No. No. 58 disclosed in Japanese Patent Publication No. 56-4236. 6 strains, API-21 strain disclosed in JP-A-58-134990 and the like are known. In addition, unlike these strains, as strains that can grow in a relatively high temperature range, No. 2 disclosed in Japanese Patent Application Laid-Open No. 2-255087 can be used. A
There is H-101 strain. However, B used in the present invention
The No. 18-1 strain is capable of growing in a relatively high temperature range. The maximum growth temperature of the AH-101 strain is 55 ° C, whereas it is a thermophilic strain that can grow at a high temperature of 62 ° C. Since the AH-101 strain does not have a nitrate reducing action, but has a reducing action, the B18-1 strain used in the present invention is No. AH-1
It can be clearly distinguished from the 01 strain. In addition, from the difference in the addition amount of sodium carbonate that is suitable in the medium for culturing the B18-1 strain, which will be described later, the B18-1 strain is No. A
It can be clearly distinguished from the H-101 strain.

As described above, B18 belonging to the genus Bacillus
The -1 strain is distinguished from known strains and is recognized as a new strain of the genus Bacillus. Further, as the strain used in the present invention, B18- belonging to the genus Bacillus if it can produce the thermostable alkaline protease which is the object of the present invention.
Needless to say, the strain is not limited to one strain, and any of natural and artificial mutant strains of the B18-1 strain may be used. The strain B18-1 has been deposited at the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology under the deposit number 12563 (FERM P-12563).

Next, a method for culturing a microorganism which produces a heat-resistant alkaline protease according to the present invention, a method for collecting a heat-resistant alkaline protease, a method and a unit for measuring the activity of a heat-resistant alkaline protease, and properties of the heat-resistant alkaline protease will be described. In the following, the thermostable alkaline protease according to the present invention is abbreviated as "enzyme of the present invention".

(1) Method for Culturing Microorganism The medium for culturing the microorganism used in the present invention, for example, strain B18-1 cannot be applied to cultivate ordinary neutral Bacillus genus, and the pH is 10 It is necessary to set the degree. When culturing under such conditions, glucose, starch and the like as the carbon source of the medium, polypeptone, soybean flour, corn steep liquor, meat extract, yeast extract, organic nitrogen sources such as casein are preferably used as the nitrogen source. In addition to these carbon sources and nitrogen sources, salts such as phosphates are added as needed. Since the B18-1 strain is an alkalophilic bacterium, sodium carbonate is further added to make the medium alkaline. In particular, in the production of the enzyme of the present invention, it is optimal to add 1.5% sodium carbonate in the medium, and as an example of the composition of the medium, 1% polypeptone,
A culture solution containing 1% soybean flour, 2% glucose, 0.1% dipotassium hydrogen phosphate and 1.5% sodium carbonate is used. In addition, while the preferable amount of sodium carbonate added to the medium for culturing the B18-1 strain is 1.5%, No.
Add 0.5% sodium carbonate to AH-101 strain
(pH 9.5) is preferable, and also from this point, the B18-1 strain has N
o. It can be clearly distinguished from the AH-101 strain.

When the culture temperature is high, it is advantageous for the growth of the B18-1 strain, but when it is too high, the self-digestion of the enzyme of the present invention produced by the B18-1 strain becomes severe and the enzyme production efficiency decreases. Therefore, the optimum temperature is about 40 ℃. When the B18-1 strain was cultured at 40 ° C. using the above culture solution, the production of the enzyme of the present invention started after 16 hours, and
After 24 hours, the maximum amount of production is reached
It reaches U / ml, but after that it gradually deactivates.

(2) Method for collecting the enzyme of the present invention As described above, the B18-1 strain is cultured, and the enzyme group containing the enzyme of the present invention is secreted and produced in this culture solution. After completion of the culture, the culture solution containing these enzyme groups is centrifuged, filtered, or the like to remove bacterial cells. further,
To this culture filtrate, a salting-out agent such as ammonium sulfate or an organic solvent such as alcohol or acetone is added to precipitate the enzyme group. Then, the precipitate is dehydrated by filtration, centrifugation and the like and dried to obtain a crude enzyme powder. Subsequently, the crude enzyme powder is purified by a known separation and purification means such as an adsorbent, an ion exchanger, a gel filtration agent, etc. to obtain a highly pure enzyme of the present invention.

(3) Method for measuring enzyme activity of the present invention and 5 ml of borate buffer (pH 11.5) containing 0.6% of unit casein, 1 ml of a test solution (solution containing the enzyme of the present invention) was added, and the mixture was added at 30 ° C. After reacting for 10 minutes, 5 ml of trichloroacetic acid mixture (0.11 M trichloroacetic acid, 0.22 M sodium acetate, 0.33 M acetic acid) was added, and the mixture was allowed to stand at 30 ° C for 20 minutes to precipitate unreacted casein and then filtered. After removing the precipitate, the tyrosine produced by the above reaction in the filtrate is quantified by measuring the absorbance at 275 nm. One unit (U) of enzyme means the amount of enzyme that produces 1 μg of tyrosine per minute under the above reaction conditions.

(4) Properties of the enzyme of the present invention The characteristics of the enzyme of the present invention are as follows. (a) Molecular weight: about 30,000 (measured by SDS-polyacrylamide gel electrophoresis) (b) Ultraviolet absorption spectrum of aqueous solution: maximum absorption is 275
~ 280 nm (c) Absorption coefficient (E1%, 1 cm, 280 nm): 7.1 (d) Solubility: Soluble in water, insoluble in methyl alcohol, ethyl alcohol, acetone and ether (e) Optimal pH and pH stability : Activity and pH of the enzyme of the present invention
The relationship with is shown in FIG. The buffer used for the activity measurement contained 2.5 mM calcium ions, and boric acid-sodium hydroxide buffer at pH 8.5 to 9.5 and borax-sodium hydroxide at pH 10.0 to 13.0. This is a buffer solution, and the relative activity at each pH is shown in FIG. 1 with the activity value at the pH showing the highest activity as 100%. In addition, FIG. 2 shows the pH stability of the enzyme of the present invention in the presence of calcium ions, and also shows the stability of the enzyme in the presence of 2.5 mM calcium ions at 40 ° C.
The pH stability of the heat-resistant alkaline protease activity is shown when it is kept warm for 1 hour. The buffers used for activity measurement each contained 2.5 mM calcium ion, and acetate buffer was used at pH 4.5, Tris-HCl buffer was used at pH 8.0, and pH 10.0 and pH 12.0. Is a borax-sodium hydroxide buffer solution, and the activity was measured after incubation at 40 ° C. for 1 hour using these buffer solutions. In addition, in FIG. 2, the residual activity at each pH is relatively shown, with the activity value at the pH showing the highest activity being 100%. Therefore, as is clear from FIGS. 1 and 2, the enzyme of the present invention had an optimum pH of 12 to 13 when casein was used as a substrate, and was incubated at 40 ° C. for 1 hour in the presence of 2.5 mM calcium ion. In some cases pH 5-12
Then it is stable.

(F) Range of suitable temperature for action The relationship between the activity of the enzyme of the present invention and temperature is shown in FIG. The activity was measured using casein as a substrate and a borate buffer solution (pH 10.0) containing 10 mM calcium ions as a buffer.
Was used for 10 minutes after the reaction. Note that, in FIG. 3, the relative activity at each temperature is shown with the activity value at 30 ° C. being 100%, and as is clear from FIG. 3, the enzyme of the present invention shows the maximum activity at 85 ° C.

(G) Thermostability FIG. 4 shows the thermostability of the enzyme of the present invention, and the thermostability was measured by measuring pH 10 containing 10 mM calcium chloride.
The enzyme solution was heated for 10 minutes each using the borate buffer solution, and the activity was measured. In addition, in FIG. 4, the activity value at the temperature showing the highest activity is 100%.
The residual activity at each temperature is shown relative to. As is clear from FIG. 4, the enzyme of the present invention is
It is stable up to 60 ° C at pH 10, has 50% residual activity at 80 ° C, and completely deactivates at 90 ° C.

(H) Stabilization and inhibition: Stabilized by calcium ion ⁺ , not inactivated by PCBM (parachloromercuric benzoic acid) or EDTA (ethylenediaminetetraacetic acid), and inactivated by DFP (diisopropylchlorophosphate). To do. (i) Specific activity: 4080 U / mg

(J) Amino acid composition: 6N of the enzyme of the present invention
After hydrolysis with hydrochloric acid for 24 hours, the amino acid composition was determined by an amino acid analyzer. Table 2 shows the number of each amino acid group when the molecular weight is 30,000. For comparison, the number of each amino acid group is set forth in JP-A-2-255087. AH-1
The amino acid composition of the alkaline protease produced from strain 01 is also shown.

[Table 2]

(K) Amino acid sequence (N-terminal) Table 3 shows 20 amino acids from the N-terminal of the enzyme of the present invention.
The amino acid sequence up to the residue, and for comparison is disclosed in
No. 255087. The amino acid sequence of the alkaline protease produced from the AH-101 strain is also shown.

[Table 3]

Next, the enzyme of the present invention having the above-mentioned characteristics is compared with another conventionally known enzyme (alkaline protease) and shown in Table 4 below.

[Table 4] As is clear from Table 4, the enzyme of the present invention is different from any other known enzyme in its characteristics and is recognized as a novel enzyme. In addition, among the enzymes listed in Table 4, the No. 12 enzyme (the amino acid composition and the amino acid sequence are shown in Tables 2 and 3 and produced from the No. AH-101 strain) is No. 13 in particular. Is considered to be the most similar to the enzyme of the present invention. However, the No. 12 enzyme has a molecular weight of 29,000, whereas the No. 13 enzyme of the present invention has a molecular weight of 30,000, and also in the amino acid analysis shown in Tables 2 and 3. These enzymes No. 12 and No. 13 are clearly distinguished from each other because the compositions of these enzymes are different and the amino acid sequences from the N-terminus are different. Therefore, it is recognized that the enzyme No. 13 of the present invention is a novel enzyme.

[0022]

The enzyme of the present invention is stable up to 60 ° C. when treated at pH 10 for 10 minutes in the presence of calcium ions.
It has excellent heat resistance as evidenced by the residual activity of 50% at ℃. In addition, it is stable at pH 5 to 12 when kept at 40 ° C for 1 hour in the presence of 2.5 mM calcium ion, and is optimal when casein is used as a substrate.
As is clear from the pH value of 12 to 13, it exhibits activity in alkaline conditions. Therefore, the enzyme of the present invention exhibits high activity in a high temperature range and alkalinity.

The above-mentioned enzyme is obtained by culturing a microorganism belonging to the genus Bacillus, for example, a new strain B18-1 in an alkaline medium and accumulating the enzyme in the medium and collecting the enzyme. It is manufactured by

[0024]

EXAMPLES Next, specific examples of the thermostable alkaline protease which is the enzyme of the present invention and the method for producing the same will be described with reference to the drawings and tables. First, 50 g of soybean powder, 50 g of fish meat extract, 50 g of glucose, 5 g of potassium phosphate and 2.5 g of yeast extract were dissolved in 4.5 l of water as a culture solution and sterilized at 120 ° C. for 20 minutes, and 50 g of anhydrous sodium carbonate as an alkaline solution. Dissolved in 450 ml of water,
Sterilize at 120 ° C for 20 minutes. These culture solutions and an alkaline solution were mixed in a 5 liter jar, and the alkaline culture solution was inoculated with the strain B18-1 which had been cultured overnight, and the mixture was incubated at 40 ° C.
Shake culture for 20-24 hours. 5 such cultures
The cells were removed by centrifugation and filtration to obtain a total of 14 l of the culture filtrate containing the enzyme group containing the enzyme of the present invention. The enzyme activity of this culture filtrate was measured and found to be about 864 U / ml.

Next, 14 l of this culture filtrate was concentrated using an ultrafiltration membrane to obtain 1.9 l of concentrated culture solution (yield 96%). Ammonium sulfate was added to this concentrated culture solution to 0.7 saturation to give 1 After standing overnight, the precipitated enzyme protein was filtered through Celite to obtain a crude enzyme. This crude enzyme was dissolved in 20 mM Tris-HCl buffer (pH 8.8) containing 50 mM sodium chloride, passed through a DEAE-Toyopearl 650M (manufactured by Tosoh Corporation) column equilibrated with the same buffer after dialysis, and colored substance Impurities such as are adsorbed and removed on the column. The active fraction passed through this column to obtain 1.5 l of effluent containing the active fraction, and the yield was 61%.

Further, this fraction was concentrated by ultrafiltration or salting out, dissolved in a 10 mM phosphate buffer (pH 7.6), dialyzed, and equilibrated with the same buffer CM-Toyopearl (TOSO). The enzyme of the present invention is adsorbed when it is passed through a column. The column on which the enzyme of the present invention is adsorbed is 0 to 0.2 M
When the gradient elution was performed with the sodium chloride solution up to, the elution peaks were at 0.05M and 0.1M.
Enzyme activity was found in this latter. Fractions showing this enzyme activity were collected and subjected to SDS-polyacrylamide gel electrophoresis to obtain a single band, and a highly pure enzyme of the present invention was obtained. The final yield was about 40%. The enzyme of the present invention obtained by the above-described production method acts on proteins such as casein, gelatin, gluten, and hemoglobin to hydrolyze peptide bonds, especially in a high temperature range and alkaline.

[0027]

INDUSTRIAL APPLICABILITY The enzyme of the present invention is excellent in thermostability and exhibits high activity in alkalinity, so that it can be widely used in industries such as foods, fibers and leather.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between the activity of the enzyme of the present invention and pH.

FIG. 2 is a graph showing the pH stability of the enzyme of the present invention in the presence of calcium ions.

FIG. 3 is a graph showing the relationship between the activity of the enzyme of the present invention and temperature.

FIG. 4 is a graph showing the thermostability of the enzyme of the present invention.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Tsutomu Katada 4-chome, Kashima 4-chome, Yodogawa-ku, Osaka City, Osaka Prefecture, Japan 6-23, Japan Chemical Machinery Manufacturing Co., Ltd. (56) Reference JP-A-2-255087 (JP, A)

Claims (2)

[Claims] 1. A heat-resistant alkaline protease having the following characteristics. (a) Molecular weight: about 30,000 (measured by SDS-polyacrylamide gel electrophoresis), (b) UV absorption spectrum of aqueous solution: maximum absorption is 275
~ 280 nm, (c) Extinction coefficient (E1%, 1 cm, 280 nm): 7.1, (d) Solubility: Soluble in water, insoluble in methyl alcohol, ethyl alcohol, acetone and ether, (e) Casein as substrate Optimum pH: 12 to 13, (f) pH stability: Stable at pH 5 to 12 when kept at 40 ° C for 1 hour in the presence of 2.5 mM calcium ion, (g) Casein as substrate Optimum temperature: 85 ℃ in the presence of 10mM calcium ion, (h) Thermal stability: It is stable up to 60 ℃ at 10 minutes at pH10, 50% residual activity at 80 ℃, and 90 ℃ at 90 ℃. Complete inactivation, (i) Stabilization and inhibition: Calcium ion stabilized, not inactivated by PCMB (parachloromercuric benzoic acid) or EDTA (ethylenediaminetetraacetic acid), and inactivated by DFP (diisopropylchlorophosphate). Activity, (j) Specific activity: 4080 U / mg . 2. A heat-resistant alkaline protease characterized by culturing a B18-1 strain belonging to the genus Bacillus in an alkaline medium, accumulating a heat-resistant alkaline protease having the following characteristics in the medium, and collecting the heat-resistant alkaline protease. Manufacturing method. (a) Molecular weight: about 30,000 (measured by SDS-polyacrylamide gel electrophoresis), (b) UV absorption spectrum of aqueous solution: maximum absorption is 275
~ 280 nm, (c) Extinction coefficient (E1%, 1 cm, 280 nm): 7.1, (d) Solubility: Soluble in water, insoluble in methyl alcohol, ethyl alcohol, acetone and ether, (e) Casein as substrate Optimum pH: 12 to 13, (f) pH stability: Stable at pH 5 to 12 when kept at 40 ° C for 1 hour in the presence of 2.5 mM calcium ion, (g) Casein as substrate Optimum temperature: 85 ℃ in the presence of 10mM calcium ion, (h) Thermal stability: It is stable up to 60 ℃ at 10 minutes at pH10, 50% residual activity at 80 ℃, and 90 ℃ at 90 ℃. Complete inactivation, (i) Stabilization and inhibition: Calcium ion stabilized, not inactivated by PCMB (parachloromercuric benzoic acid) or EDTA (ethylenediaminetetraacetic acid), and inactivated by DFP (diisopropylchlorophosphate). Activity, (j) Specific activity: 4080 U / mg .