JPH05211868A - Production of alkali protease - Google Patents

Abstract

PURPOSE:To provide an alkali protease for carrying out proteolysis in a low- temperature area, capable of utilizing for food processing, industry, detergent, etc. CONSTITUTION:The objective alkali protease is produced by culturing a bacterial strain of the genus Xanthomonas capable of culturing at 10-20 deg.C, concretely, Xanthomonas sp. S-1 (FERM P-12087) in a nutrient medium. The alkali protease has activity even in a low-temperature area, though the optimum temperature is about at 40 deg.C.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing an alkaline protease, and more particularly to a method for producing an alkaline protease which is active in a low temperature region produced by a microorganism belonging to the genus Xanthomonas.

[0002]

2. Description of the Related Art Alkaline proteases are Bacillus spp.
Those produced using microorganisms such as Streptomyces and Aspergillus are known.

This alkaline protease is used for food processing,
It is used in a fairly wide range of fields such as detergents, skin tanning (hair removal), and recovery of silver from films. Recently, it has been desired to produce alkaline proteases that act effectively at low temperatures such as detergents. Is expected to be produced by culturing at low temperature.

[0004]

[Problems to be Solved by the Invention] Currently, there is an alkaline protease (trade name APJ-21: manufactured by Showa Denko) which is commercially available as an alkaline protease having an activity at a lower temperature. Power is not always satisfactory.

[0005]

[Means for Solving the Problems] The inventors of the present invention have conducted extensive studies on the production of an alkaline protease having a sufficient washing effect in a low temperature region, and further on a method of efficiently producing an alkaline protease in a low temperature culture. As a result of further searching for an alkaline protease-producing bacterium, it was found that an alkaline protease having activity in a low temperature region can be obtained from a strain belonging to the genus Xanthomonas, and the present invention has been completed.

That is, in the conventional strains, the enzyme production is generally
Although a culture temperature of 30 ° C or higher is common, the Xanthomonas strain of the present invention is cultured in a temperature range of 10 to 25 ° C, and the production of alkaline protease is maximized at a culture temperature of 15 ° C,
An alkaline protease having activity in the low temperature region can be obtained.

The mycological properties of the isolated strain producing the alkaline protease of the present invention are shown below. A. Morphological properties The following morphological characteristics were observed when cultured on broth agar medium at 30 ° C for 2 days. 1) Cell shape: Bacillus Size: − Colony size: Diameter 0.5mm 2) Motility: Yes 3) Spores: Not formed 4) Gram stain: Negative

B. Physiological properties 1) Reduction of nitrate: − 2) Indole formation: − 3) Arginine dehydrase: − 4) Urease: − 5) β-galactosidase: − 6) Oxidase: − 7) Catalase: + 8) Gelatin Hydrolysis: +9) Hydrolysis of Tween 80: -10) OF test: -11) Growth temperature range: 37 ° C 12) Acid production from glucose: -13) Acid production from maltose: -14) Sugars and Digestibility of organic acids Glucose: + Caproic acid:-Arabinose:-Maleic acid: + Mannitol:-Citric acid: + Maltose: + Phenylacetic acid:-Mannose: + Adipic acid:-Gluconic acid:-

From the above mycological properties, this strain is considered to belong to the genus Xanthomonas. Therefore, this strain was named Xanthomonas sp. S-1 and deposited at the Institute of Microbial Technology, Institute of Industrial Science and Technology.
The deposit number is Microorganism Research Institute Deposit No. 12087.

Examples of the microorganisms used in the present invention include the above-mentioned Xanthomonas sp. S-1 (Deposited by Microtechnology Research Institute, No. 12087), but are not limited to this bacterium and belong to the genus Xanthomonas. All bacteria that produce alkaline protease in the low temperature range can be used in the present invention.

In the present invention, the medium for producing alkaline protease is one that is used for culturing ordinary microorganisms and may be one that can be used for this strain, and carbon sources include starch, dextrin, molasses, and glucose. As the inorganic salt, an alkaline medium is preferably added with salts such as disodium phosphate and magnesium sulfate, and carbonate, and as the nitrogen source, sodium nitrate, urea, organic nitrogen source and the like are used.

The culture temperature is in the range of 10 to 25 ° C, preferably 12 to 17 ° C. The culture pH is in the range of 7.0 to 9.0, preferably pH 8.2 to 8.7. However, the condition is not limited to this. Culturing is usually carried out for 48 to 96 hours, whereby alkaline protease is accumulated in the culture solution.

After the culturing, the crude enzyme solution is obtained by removing the cells and insoluble matter from the culture solution by a general solid-liquid separation means such as centrifugation and filtration. The crude enzyme solution thus obtained is salted out with ammonium sulfate to obtain an alkaline protease. It may be used as it is, or may be further purified by a known purification method such as dialysis, an organic solvent fractionation method, and column chromatography.

The physicochemical properties of the obtained alkaline protease are as follows. a. Action Decomposes various proteins under alkaline conditions. b. Optimum pH and stable pH range Optimum pH is 10.5-12 and stable pH range is 90% relative activity.
When it is set to be not less than%, the pH is 7 to 12. c. Optimum temperature and heat resistance The optimum temperature is 45 ° C, and activity is maintained up to a temperature of 30 ° C. Furthermore, the heat resistance is improved by about 10 ° C. by adding Ca ²⁺ 5 mM.

As is clear from the above, according to the present invention, an alkaline protease active in a relatively low temperature region can be efficiently produced by a low temperature culture than a strain of the genus Xanthomonas.

[0016]

EXAMPLES The present invention will be specifically described below with reference to examples. Example 1 Xanthomonas sp.
sp.) S-1 (Deposited Microorganisms Research Institute No. 12087), 15 ℃
Incubate for 3 days. Next, a liquid medium containing 1% milk casein, 0.2% potassium chloride, 0.02% magnesium sulfate, 1% glucose, 0.4% yeast extract and 1.2% disodium phosphate.
After sterilizing at 120 ° C. for 20 minutes, separately sterilized 0.1 M sodium carbonate buffer (pH 10.5) was added at a volume ratio of 0.25% to prepare a pH 8.5 culture medium. 100 ml of this culture solution was dispensed into a 500 ml shake flask, and 1 platinum loop of the above-inoculated seed was inoculated,
The culture was performed with shaking at each temperature of 10 ° C, 15 ° C, 23 ° C, and 30 ° C for 72 hours. The pH, cell concentration (absorbance value at OD ₈₆₀ nm) and enzyme activity were measured every 24 hours, and the maximum cell concentration value and maximum enzyme activity value at each culture temperature are shown in Table 1 below.

[0017]

[Table 1]

The enzyme activity of alkaline protease was measured by the following method. Add 1.0 ml of appropriately diluted enzyme to 1.0 ml of 2% casein solution (pH 10.5) kept at 30 ℃.
After reacting for 10 minutes, add 4.0 ml of trichloroacetic acid mixed solution to stop the reaction, leave at 30 ° C for 20 minutes, filter with Toyo filter paper No.6, and filter to 1.0 ml of 0.4 M sodium carbonate solution 5.0 ml.
Was added, 1.0 ml of Folin reagent diluted 5 times was added thereto, and the mixture was allowed to stand at 30 ° C. for 20 minutes, and the absorbance at 660 nm was measured. One unit (pu) is defined as the amount of enzyme that liberates 1 μg of tyrosine equivalent per minute under the above conditions.

As is clear from the results shown in Table 1 above, Xanthomonas sp. S-1
In the case of Microorganisms Research Laboratories No. 12087, the maximum activity value of alkaline protease was obtained at a relatively low culture temperature of about 10 to 15 ° C.

Example 2 Six cultures were carried out under the same conditions as in Example 1 except that the culture temperature was set at 15 ° C., and 480 ml of the obtained culture solution was sterilized by centrifugation to give 460 ml of supernatant (350 PU / ml). ) Got. Ammonium sulfate was added to the supernatant to make it 70% saturated, alkaline protease was precipitated, and the salted-out product was recovered by centrifugation. 50 of this salted out
It was dissolved in 5 ml of mM Tris-HCl buffer (pH 8.0), the solution was put into a dialysis membrane, and dialyzed overnight with the buffer to obtain 15 ml of a crude enzyme solution (8,050 PU / ml). Using this enzyme, the physicochemical properties of alkaline protease were investigated below.

(1) Action (decomposition rate of various proteins under alkaline conditions) Measurement conditions pH: 10.5 (10 mM borax-NaOH buffer) Temperature: 30 ° C Reaction conditions: 30 minutes Substrate concentration: 1% Enzyme amount: 30PU / ml Proteolysis rate was measured according to the Anson-Hagiwara modified method, and after reacting with each substrate under the specified conditions, immediately after using Bio-Rad Pr.
The amount of protein was measured using the otein Assay, and the degradation rate was determined by the ratio with the unreacted content. The results are shown in Table 2 below.

[0022]

[Table 2]

(2) Optimum pH and stable pH range The optimum pH is that the enzyme is added to the buffer solution of each pH containing 1% of casein.
It was determined to be 30 PU / ml, reacted at 30 ° C. for 10 minutes, and measured for activity at each pH. Fig. 1 shows the relative activity at each pH when the activity at the optimum pH is 100.

The stable pH range was determined by adding the enzyme to a buffer solution of each pH to 210 PU / ml, incubating at 15 ° C. for 24 hours, and measuring the activity at 30 ° C., pH 10.5. .. Fig. 2 shows the relative activity when the activity at pH 10.5 before incubation is set to 100. The buffer solutions used and their pH ranges are as follows.

PH range Buffer pH 3-7 McIlvaine pH 7-9 Tris-HCl pH 9 Borax-HCl pH 10-12 Borax-HCl

As is clear from FIG. 1 and FIG.
Is 10.5-12. The stable pH range is 90% relative activity.
When above, the pH is 7-12.

(3) Optimum temperature and heat resistance The optimal temperature is determined by adding the enzyme to a buffer solution of pH 10.5 containing 1% casein as a substrate, reacting at each temperature for 10 minutes, and measuring the activity. The relative activity with respect to each temperature when the activity at the optimum temperature is 100 is shown in FIG.

The heat resistance is 50 mM Tris-HCl buffer (pH 8.
0) was added with 210 PU / ml of enzyme, heat-treated at each temperature for 3 hours, ice-cooled, and measured by measuring the activity at 30 ° C, pH 10.5. The activity at pH 10.5 before heat treatment was 100%. FIG. 4 shows the relative activity when the test was performed.

The Ca ²⁺ salt addition (Ca ²⁺ salt amount: 5 mM) shows the improvement in heat resistance by the following Table 3.

[0030]

[Table 3]

As is clear from FIGS. 3 and 4, the optimum temperature is
It is 45 ° C and remains active up to a temperature of 30 ° C. Furthermore,
As shown in Table 3, the heat resistance is about 10 ℃ by adding Ca ²⁺ 5mM.
Improved.

(4) Effect of metal ion Under the following measurement conditions, a certain amount of the enzyme solution was added to each buffer solution, and various metal salts were added at 1 mM.
The residual activity of the enzyme was measured, and the relative activity when the activity without addition of the metal salt was set to 100 is shown in Table 4 below.

Measurement conditions pH: 7.0 (20 mM Tris-HCl buffer) pH: 10.5 (10 mM borax-NaOH buffer) Temperature: 30 ° C. Reaction time: 10 minutes Substrate: 1% casein solution (adjusted with each buffer)

[0034]

[Table 4]

As is clear from Table 4, the enzyme solution according to the present invention is strongly inhibited by trivalent iron ions at pH 7.0 and mercury ions at pH 10.5. The activity was hardly inhibited by the metal ions of.

(5) Effect of inhibitors Under the following measurement conditions, the enzyme solution obtained by the present invention was added to 20 mM Tris-HCl buffer (pH 7.0), and each inhibitor was added at a predetermined concentration to 25 ° C. After treatment for 30 minutes with the enzyme, the residual activity of the enzyme was measured, and the relative activity when the activity without addition of the inhibitor was set to 100 is shown in Table 5 below.

Measurement conditions pH: 7.0 (20 mM Tris-HCl buffer) Temperature: 30 ° C. Reaction time: 10 minutes Substrate: 1% casein solution (adjusted with the above buffer)

[0038]

[Table 5]

As is clear from Table 5, in the enzyme according to the present invention, the inhibition of the activity of the serine protease inhibitor diisopropylfluorophosphate (DFP) and phenylmethanesulfonyl fluoride (PMSF) is due to the metalloprotease inhibitor ethylenediamine. Tetraacetate-2Na
It is presumed that the enzyme according to the present invention is a serine protease having a serine residue at the active center, since it is higher than the inhibition of the activity of (EDTA-2Na) and the SH protease inhibitor by parachloromercury benzoic acid (PCMB). ..

Further, it was revealed that the activity is hardly inhibited by tosylphenylalanine chloromethylketone (TPCK) which is an inhibitor of chymotrypsin of animal serine protease or tosyllysine chloromethylketone (TLCK) which is an inhibitor of trypsin. It was

[Brief description of drawings]

FIG. 1 is a graph showing the optimum pH of the crude enzyme solution obtained in Example 2.

FIG. 2 is a graph showing a stable pH range of the crude enzyme solution obtained in Example 2.

FIG. 3 is a graph showing the optimum temperature of the crude enzyme solution obtained in Example 2.

FIG. 4 is a graph showing the heat resistance of the crude enzyme solution obtained in Example 2.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yoichi Shinmura 5-71-1 Komaba, Abashiri-shi, Hokkaido (72) Inventor Yoko Yamaya 4-chome, Minami 5jo Nishi, Abashiri-shi, Hokkaido

Claims (2)

[Claims] 1. A method for producing an alkaline protease, which comprises culturing a strain of the genus Xanthomonas in a nutrient medium, accumulating the alkaline protease in the culture, and collecting the alkaline protease from the culture. 2. The alkali according to claim 1, wherein the strain of the genus Xanthomonas is Xanthmonas sp. S-1 (Ministry of Industrial Science, Deposit No. 12087). Method for producing protease.