KR950009838B1 - Protease from streptomyces thermonitrificans - Google Patents

Abstract

The thermo-stable protease (I) from Streptomyces thermonitrificans ATCC 23385 (II) is pepared by (1) culturing (II) at 45-55 deg.C with yeast extract 2g, Na glutamate 6.7g, galactose 30g, MgSO4 0.1g, FeCl2 0.01g, MnCl2 0.01g, ZnCl2 0.01g, KH2PO4 4g, K2HPO4 1.55g/l, pH 7.5 and (2) centrifuging the culture broth to get the supernatant, (3) precipitating (2) with acetone, (4) loading (4) through the columns of cationic exchange, phenyl-Sepharose and gel filtration sequentially. The mw of (I) is 25-30 kd. The inhibitors for (I) are elastinal, 3,4-dichloroisocoumarine and aprotinin. (I) is useful for cheese making, synthesis of artificial sweetner and leather industry.

Description

Heat-stable proteolytic enzymes derived from Streptomyces thermonitrificans

Figure 1 is a photograph showing the results of the electrophoresis of the protein dehydrogenase purified from Streptomyces thermolypicans according to the present invention.

2 is a graph showing the activity of each temperature of the proteolytic enzyme purified according to the present invention.

The present invention relates to a novel heat stable proteolytic enzyme derived from Streptomyces thermonitrifican-s (ATCC 23385) and a purification method thereof.

Proteolytic enzymes are currently used in various industrial applications. For example, it is used in the production of cheese, the synthesis of artificial sweeteners and part of the manufacturing process of leather products, and is used as an additive to enhance the cleaning action of synthetic detergents, and the range of its use is very diverse. In particular, thermally stable proteolytic enzymes have greater potential for industrial applications compared to mesophilic proteolytic enzymes that are easily lost at high temperatures.

To date, many types of enzymes have been found in thermophilic bacteria that grow at temperatures above physiological temperatures. Enzymes obtained from these thermophilic microorganisms have been studied steadily because they are more thermally stable than enzymes having the same function obtained from mesophilic microorganisms and are very useful in many industries requiring high temperature treatment. Enzymes were purified and their specificity to the substrate was studied in detail.

In general, microorganisms having an optimal culture temperature of 50 to 65 ° C. or more are referred to as ultra high temperature microorganisms. It is known that microorganisms belonging to the genus Thermus are extremely hot and are found mainly in hot spring areas, and some bacteria of the genus Bacillus also belong to ultra high temperature microorganisms.

Bacillus thermoproteolyti

Thermolysin isolated from cus) is one of the most studied proteolytic enzymes (see Endo, S., Hakko Kogaku Zasshi 40, 346-353, 1962). Thermolysine is a zinc metalloprotease (Znmetalloprotease) with a molecular weight of about 34,5000 Daltons and an optimum temperature of enzyme activity of about 80 ° C (Sidler, W. and Zuber, H. Eur. J. Appl. Microbiol. Biotechnol , 10, 197-209, 1980). The genus Bacillus has been a particularly intensive study for obtaining pyrolytic proteases, where neutral proteolytic enzymes isolated from the culture of B. stearothermophilus NCIB 8924 are stable to temperatures up to 65 ° C. Enzyme isolated from the culture of Bacillus stearothermophilus NRRL B-3880 showed stability to temperatures up to 70 ° C. (Sidler, W. and Zuber, H., Eur. J. Appl. Microbiol. Biotechnol. 4 , 255-266, 1977). Neutral proteolytic enzymes purified from Bacillus caldolyticus, a bacterium that can grow up to 100 ° C in hot spring areas, remained stable at temperatures up to 80 ° C (Heinen, UJ and Heinen, W. Arch). Microbiol. 82, 1-23, 1972).

In addition to the genus Bacillus, proteolytic enzymes have been purified from other thermophilic bacteria, for example thermomomcholine (Gaucher, G. M. and Stevenson, isolated from Malbranchea pulchella var. Sulfurea).

KJ Methods in Enzymology 45, 415-433, 1976), thermase (Hausdof, G. et al., Int. J. Peptide Protein Res. 15 isolated from Thermoactinomyces vulgaris) , 420-429, 1980) and alkaline proteolytic enzymes isolated from Streptomyces rectus (see Mizysawa, K. and Yoshida, FJ Biol Chem. 248, 4417-4423, 1973).

A number of proteolytic enzymes have also been purified from the genus Thermos. For example, in Thermos aquaticus YT-1, aqualysin I with a molecular weight of 28,500 daltons showing high activity in alkaline conditions and aqualysin II showing high activity at neutral pH were purified (Matsuzawa , H., et al., Agric. Biol. Chem. 47, 25-28, 1983). These proteolytic enzymes are all thermally stable enough to exhibit maximum activity at high temperatures, for example 80 to 90 ° C. In addition, proteolytic enzymes having a molecular weight of 31,000 Daltons and a maximum active pH of 7.8 were purified from fermentation broth of Thermos caldophilus GK24 obtained from the hot spring area (Taguchi, H. et al., J. Biochem. ( Tokyo) 93, 7-13, 1983). Enzymes isolated from these summer genera are all characterized as serine proteolytic enzymes.

Accordingly, the present inventors succeeded in identifying and purifying the heat-stable proteolytic enzyme from the culture medium of Streptomyces thermomonipicans, which is one of the thermophilic bacteria, while studying proteolytic enzymes on various thermophilic bacteria. .

Streptomyces thermomonipicans is a family of thermophilic fungi (Actinomycete), first classified by Desai and Dhala, and is known to have an optimum incubation temperature of about 50 ° C (Desai, AJ and Dhala). , SA Anronie van Leeuwenhoek 33, 137-144, 1967). To date, no proteolytic enzymes purified from Streptomyces thermomonipicans have been reported, but the molecular weight is approximately 21,500 Daltons from Streptomyces rectus var. Proteolyticus belonging to this genus. Heat-stable serine proteolytic enzymes with a pH of 10.7 have been purified (see Mizusawa, K. and Yoshida, FJ Biol. Chem. 248, 4417-4423, 1973). The proteolytic enzyme isolated from Streptomyces thermomonic pecanans, a product of the present invention, was identified as a novel proteolytic enzyme completely different from the proteolytic enzyme.

Hereinafter, the present invention will be described in detail.

After confirming the presence of proteolytic enzymes, specifically, entoproteases, in the fermentation medium of Streptomyces thermomonipicans (see Horikoshi, K. Agric. Biol, Chem. 35 1407-1212 (1971)), appropriate culture conditions After culturing in a centrifugation and centrifugation, only the culture was taken and precipitated with acetone to obtain a precipitate. The precipitate is dissolved and purified sequentially using cation exchange chromatography, phenyl-sepharose chromatography and gel filtration chromatography.

Sodium Dodecyl Sulfate (SDS) -electrophoresis of the proteolytic enzyme thus obtained revealed a molecular weight of about 28.000 Daltons, showing the highest enzymatic activity at 60 ° C. in a buffer solution of pH 8.0. In addition, this enzyme is a chymotrypsin type that cleaves a site next to an aromatic amino acid and cuts the substrate N-Suc (succinyl) -Ala-Ala-Pro-Phe-pNA (paranitroanilide) in the ↑ position. Nitroaniline was formed to give a yellow half, and the enzyme reaction was induced by APMSF (4-amidinophenyl methanesulfonylfluoride), aprotinin, 3,4-dichloroisocomarin and elastinal. Inhibited, but not by 1,10-orthophenanthrosine, MEM (N-ethylmarimide, pepstatin, leupeptin).

Hereinafter, the present invention will be described in detail with reference to Examples.

The following examples are provided only to specifically explain the present invention, but do not limit the scope of the present invention.

Example 1

[Cultivation of Streptomyces thermomonipicans]

The protease secreted from the strain was produced using a medium of the composition as shown in Table 1 below. First, for initial culture, 150 ml of the medium was added to a 1,000 ml flask, and 1% of the medium composition was inoculated with the strain grown on the agar plate. Then incubated for 12 hours while shaking at 300 rpm in a 50 ℃ shaker.

Then, 3 L of the medium was added to a 5 L fermenter for production culture, and 150 ml of the flask culture obtained above was inoculated. Then incubated for 24 hours at 50 ℃

TABLE 1

Fermentation medium composition of Streptomyces thermomonipicans

The medium was adjusted to pH 7.5 and sterilized at 121 ° C. for 10 minutes.

Example 2

[Purification of protease]

The proteolytic enzyme was separated and purified from the culture solution obtained in Example 1 by the following process.

<Step 1> Centrifugation

6 l of the culture culture shaken in Example 1 was centrifuged at 8,500 rpm for 30 minutes using a centrifuge (JA-10 rotor of Backman) to separate and remove the cells and only the supernatant was taken.

<Step 2> Acetone Sedimentation

The supernatant obtained by centrifugation was transferred to a beaker, and acetone (Junsei, Japan) cooled to 4 ° C. was added to a final concentration of 60% (v / v) while stirring, and then left at 4 ° C. for 40 minutes to form a precipitate. I was. Subsequently, the supernatant was discarded by centrifugation at 8,500 rpm for 30 minutes using a centrifuge (JA-10 rotor, Beckman) to obtain a precipitate, and then dissolved in 700 ml of distilled water. The solution was dialyzed in 20 mM sodium acetate buffer (pH 5.0) using a dialysis membrane.

<Step 3> S-Sepharose Chromatography

The protein solution dialyzed using the dialysis membrane was added to an S-Sepharose column (5 × 10 cm, Pharmacia) equilibrated with 20 mM sodium acetate buffer (pH 5.0) and washed with the buffer. Sodium chloride was added to the buffer at a concentration of 0 to 350 mM, and a total of 2 liters of buffer was added at a rate of 200 ml / hour, and solutions containing proteolytic enzymes were collected in tubes at a fraction of 11.7 ml each. Each fraction was measured separately according to the method described in Example 3 to collect the 950ml fraction showing the activity separately.

<Step 4> Phenyl Sepharose Chromatography

Sodium chloride was added to a final concentration of 2M to 950 ml of the solution containing the proteolytic enzyme obtained by the S-Ceraros chromatography, and then the solution was equilibrated with 2M sodium chloride buffer in 20 mM sodium acetate (pH 5.0). Proteolytic enzymes were adsorbed by addition to a phenyl-sepaphos column (5 × 5 cm, Pharmacia). The column was washed again with 20 mM sodium acetate buffer, and then 1 liter of 20% ethylene glycol buffer in 20 mM sodium acetate was added at a rate of 100 ml / hour, and a solution containing proteolytic enzymes was collected in a tube of 10 ml each. . Each fraction was measured separately according to the method described in Example 3, and 300 ml of fractions showing activity were collected separately.

<Step 5> S-200 Gel Filtration Chromatography

300 ml of the solution containing the proteolytic enzyme isolated from the phenyl-sepharose chromatography was concentrated using a YM 10 membrane (amicon), and then equilibrated with 200 mM sodium chloride buffer in 50 mM sodium acetate (pH 5.0). To a S-200 gel filtration column (5 × 100 cm, Pharmacia) was added at a rate of 100 ml / hour. A solution containing the proteolytic enzyme to be separated was collected and separated into tubes in 10 ml portions, and 7.6 mg of proteolytic enzymes showing activity were measured by measuring the activity of each proteolytic enzyme according to the method described in Example 3 below. Finally purified pure separation. Taking some of them, sodium dodecyl sulfate-polyacryl-amide gel electrophoresis (SDS-PAGE) was found to have a molecular weight of about 25,000 to 30,000 daltons. The electrophoresis results are shown in FIG. 1 and the standard molecular weights of the left column are 43K, 27.5K, 18.4K, 14.3K, 6.3K and 3.0K.

Example 3

[Method for Measuring Activity of Protease]

The activity of the proteolytic enzymes of the fractions obtained in the chromatography of each step performed in Example 2 was measured as follows. N-Suc (succinyl) -Ala-Ala-Pro-Phe-pNA (pyranitronilide) as a substrate was added to 0.1M Tris-HCl (pH 8.0) containing 0.05 (v / v) Tween 20. The final substrate was added to a concentration of 0.8 mM. The reaction mixture was added to a final volume of 1 ml of the culture solution or column fraction and reacted at 50 ° C. for 10 minutes, followed by mixing by adding 250 μl of 70% acetic acid to mix the mixture. The activity of each solution was confirmed by measuring the absorbance of the solution at a wavelength of 405 nm using a spectrophotometer.

Example 4

[Change of Protease Activity According to Temperature]

Proteolytic enzymes contained in 990 μl of 0.1M Tris-HCl pH 8.0 containing 0.005% (v / v) Tween 20, respectively, were 25, 30, 40, 50, 60, After incubation for 10 minutes at the temperature of 70 ℃ and 80 ℃ 10 μl of N-Suc-Ala-Pro-Phe-pNA (Suc-AAPF-pNA) was added as a substrate to a final concentration of 0.8mM. Each solution was incubated at this temperature for 10 minutes to react and then the reaction was stopped by adding 250 μl of 70% acetic acid and mixing. The absorbance of each solution was measured according to the method described in Example 3, and the results are shown in FIG. As can be seen from FIG. 2, it was found to exhibit the highest activity at 60 ° C.

Example 5

[Specificity of Inhibitors of Protease]

Specificity of the inhibitor was examined to determine the extent to which the purified proteolytic enzyme responds to the inhibitor.

The inhibitors listed in Table 2 were each added to 0.1 M Tris-HCl buffer (pH 8.5) at the concentrations listed in the table to a final volume of 1 ml. After stopping the solution at 50 ° C. for 30 minutes, the substrate Suc-AAPF-pNA used in Examples 3 and 4 was added to a final concentration of 0.8 mM. After the solution was reacted at 50 ° C. for 10 minutes, 250 μl of 70% (v / v) acetic acid was added and mixed to stop the reaction. The absorbance of the solution was measured at 405 nm using an ultraviolet-visible spectrophotometer. On the other hand, the absorbance was measured using the solution which did not add the inhibitor as a control. Table 2 shows the degree to which the inhibitors inhibit the activity of proteolytic enzymes by comparing the absorbances of the samples to which the inhibitors were added based on the control absorbance.

TABLE 2

Effects of Inhibitors on Proteolytic Enzymes of the Invention

Claims (7)

Heat-stable proteolytic enzymes derived from Streptomyces thermonitrificans (ATCC 23385). The proteolytic enzyme of claim 1, wherein the molecular weight is 25,000 to 30,000 Daltons. The proteolytic enzyme according to claim 1, which exhibits maximum activity at a temperature of 50 to 65 ° C. The proteolytic enzyme of claim 1, wherein the enzymatic activity is inhibited by elastinal, 3,4-dichloroisocomarin or aprotinin. Centrifugation of the culture medium incubated in Streptomyces thermomonipicans in the medium to precipitate the supernatant and precipitate with acetone, followed by sequentially performing cation exchange chromatography, phenyl-sepharose chromatography and gel filtration chromatography. A method for purifying heat stable proteolytic enzymes from Streptomyces thermomonipicans. The method of claim 5, wherein the medium comprises yeast extract, monosodium glutamate, galactose, FeCl ₂ , MnCl ₂ and ZnCl ₂ . The method of claim 5 wherein the incubation temperature is 45 to 55 ° C.