KR790000956B1 - Preparation of bacillopeptidase "c" - Google Patents

Abstract

Bacillopetidase C(I) having antiinflammatory activity was prepd. by fermentation of bacillus No. 794(ATTC 21964) (II). II was cultured in a liq. medium contg. starch, soybean cake, wheat bran, and inorg. salts at pH 7-9 and 28-40≰C for 4 hrs, and then the broth was pruified by DEAE-Sephadex column chromatg. to give I after dialyzation with (NH4)2 so4. I was completely inactivated by both diisopropylfluoro phosphate and EDTA, and is used as a digestant, detergent, and softener of meat.

Description

Process for preparing peptidase C with basil

Figure 1 is a graph showing the peptidase C activity pH range in Basil.

2 is a graph showing the safe pH range.

3 is a graph showing the active temperature range.

4 is a graph showing the safe temperature range.

5 is a graph showing the separation in 8 ml column of DEAE sifadex A-50.

The present invention relates to a method for producing peptidase C with a novel protease, bacillin, by Bacillus sp., More specifically, Bacillus no. It relates to a method of culturing 794 strains (Baccillus No. 794, unpublished turbidity repair No. 1522) to produce peptidase C with a new protease, Basil, which has extremely high anti-inflammatory activity, and to collect the same.

In the past, proteases were used as fire extinguishing agents, but in recent years, they have been used extensively as anti-inflammatory agents, succulents, washing agents, and food processing agents. As the demand for proteases increases, extensive research has been conducted, and new useful enzymes are continuously found in various proteases, particularly alkaline proteases.

The present inventors have searched for other protease-producing bacteria without being satisfied with the activity and productivity of the known alkali protease, and confirmed that a strain of the genus Bacillus produced a protease that was completely different from the known alkali protease. The invention has been completed.

Since the alkaline protease obtained in the present invention has been found to be a novel protease that is completely different in physicochemical properties from the conventional alkali protease and has an extremely high anti-inflammatory action and other protease activity, it was named as a novel protease, Basillo peptidase C.

Conventional alkaline proteases are completely deactivated at about 10 ^-3 moles of diisopropyl fluoro phosphate (hereinafter referred to as DFP), an inhibitor of serine enzymes, while E. ditea (inhibitor of neutral protease) EDTA) is hardly affected.

However, the novel protease, Basillopeptidase C, in the present invention is not only recognized as a completely new enzyme in that it is completely deactivated by both PD and EDI. It is very special in that it shows remarkable inhibition rate in anti-inflammatory activity test by Rat edema method.

The strain used in the present invention is a newly separated strain in the soil by the present inventors, etc. Undeposited deposit repair No. Although deposited as 1522, its bacteriological properties are as follows.

(1) morphological properties

1. Shape: Bacillus

2. Size: 0.7-0.9 × 2.5 ~ 3.5μ

3. Mobility: Yes. Have hair growth

4. Spore: Formed. Spherical 1.3-1.6μ Terminal

5. Gram Dye: Changed (±)

(2) Findings of culture

1. Growth conditions: pH 7-9 is optimal, good growth at 28-40 ℃. Under 50 ℃ growth

Not. Aerobic.

2. Colony shape: It grows well in aquatic agar, and has smooth surface and glossy. opacity. The color is pale yellowish brown.

3. Appropriate angel slope: Normally equal to or better than agar slope.

4. Soybean House Agar Slope: Same as above, showing creamy appearance.

5. Saline addition broth culture: Grows at 5% saline.

6. Gummy liquid culture: The whole liquid is uniformly turbid and does not form a biofilm.

7. Gummy gelatin puncture: 22 ℃, liquefied in layers in 1 week.

8. Litmas Milk: Slightly painted.

9. Glucose Asparagine Agar: Does not grow.

(3) physiological properties

1. Hydrolysis of Starch: Negative

2. Generation of indole: voice

3. Production of Acetyl Methylcarbinol: Negative

4. Catarases: positive

5. Liquefaction of gelatin: positive

7. Reduction of nitrates: negative

8. pH of Glucose Broth: 7.8-8.0

9. Ureases: negative

10. Reduction of methylene blue: positive

11. Gas evolution from nitrates under basic conditions: negative

12. Growth Factors: Requires thiamine.

13. Growth in glucose broth under basic conditions: negative

The bacterium, Bacillus, was compared to the known bacterium described in the Bergis Manual of Deterministic Bacteriology, 757, 1957 (Bergey's Manual of Determinative Bacteriology, 7th Edition). It is thought to be close to brevis or Bacillus spericus, but it is different from spericus in terms of magnetization of casein or gelatin, but in the form of spores, growth in 5% saline medium and demand of chiamin There is no known species that is completely different from brevis.

As a result, the strain used in the present invention is a new strain Bacillus No. belonging to the genus Bacillus. It was named 794.

In the present invention, the production of a novel protease, Basillopeppeptase C, is achieved by culturing a new protease, bacillopeptidase-producing bacterium belonging to the genus Bacillus, representing a strain of Bacillus No. 794, in a medium.

The culture method is generally advantageous to solid culture or liquid culture according to the fermentation method of the general microorganism. As a medium for cultivation, synthetic, semi-synthetic or natural medium is used.Carbohydrates such as glucose, sucrose, maltose, gluconic acid, starch or starch, suitable hydrolysates are used as carbon sources, and peptone, meat extract and cornstack solution as nitrogen sources. Soy flour, dry yeast, gluten, casein decomposed products, urea, ammonium sulfate, ammonium nitrate and the like are used alone or in combination. In some cases, an appropriate amount of carbonates, sulfates, hydrochlorides, salts or antifoaming agents such as magnesium, manganese and calcium may be added. Incubation temperature is preferably carried out at 25-40 ℃.

The incubation time should be appropriately determined by the incubation temperature, the size of the culture volume, the culture type, etc., but the production of new protease and basillopeptidase C is generally completed in 40-100 hours. In this way, for separating and collecting the protease produced during the culture, various enzyme purification methods known in the art can be used.

다든가, 메타놀, 에타놀, 이소프로파놀, 아세톤 등의 수용성 유기 용매를 가하는 침전법 또는 인산칼슘, 알루미나, 벤토나이트, 이온교환수지, 이온교환세파덱스 등에 의한 흡착, 용리법 등에 의하여 본 효소를 정제분리하고 얻어진 조효소는 전술한 정제법에 의하여 전기 영동적으로 단일의 순수 결정으로 얻었다.That is, the culture solution is salted out by using a known method to remove the cells and using a water-soluble inorganic salt such as ammonium sulfate, sodium sulfate, magnesium sulfate in the filtrate.

The enzyme is purified and separated by precipitation method of adding water-soluble organic solvent such as methanol, ethanol, isopropanol, acetone, adsorption by calcium phosphate, alumina, bentonite, ion exchange resin, ion exchange cephadex, elution method, etc. The obtained coenzyme was electrophoretically obtained as a single pure crystal by the above-described purification method.

Next, the physicochemical properties of the novel protease and bacillopeptidase C obtained by the present invention are as follows.

(1) action

As shown in FIG. 1, the enzyme exhibits activity in the range of pH 6-11 when milk casein is used as a substrate, and the optimum pH is 9-9.3.

(2) substrate specificity

Results of digestion test using various proteins are shown in Table 1.

(3) Optimum pH and stable pH range

When the substrate is milk casein, the effect of pH on the activity of the enzyme is shown in FIG. The optimum pH is 9.3, with half the activity at pH 6.5-7 or 10.5-11. In addition, FIG. 2 shows the effects of various pH on enzyme stability. In other words, the residual activity after treatment at 40 ° C. for 60 minutes at various pHs of M / 50 calf buffer solution (dodite reagent cyclohexyl aminopropanesulfonic acid) was observed. As can be seen in FIG. 2, it exhibits 90% or more of residual activity in the range of pH 7-10.5 and is stable in a wide range.

(4) measuring the titer

The measure of titer is by Anson-Hagihara's method. That is, take 1.0 ml of enzyme solution (diluted so that the ΔOD value when measuring and controlling the absorbance of 660 mμ using M / 50 Caps buffer solution is finally in the range of 0.3-0.9) and 5 ml of casein solution of 0.6%. After 10 minutes at 40 ° C, add 5.04 of 0.44M Trichlor acetic acid solution, mix it sufficiently, stop the enzyme reaction, stop at 30 ° C for 30 minutes, filter with filter paper, and filter the filter paper with 0.55M sodium carbonate solution. 5 ml and 1 ml of porin reagent are added, and the mixture is left at 40 ° C. for 30 minutes and the absorbance of 660 mμ is measured. As mentioned above, as a measuring method or an enzyme titration method, 1 unit (1pu) of enzyme power which keeps the peptide which shows the absorbance equivalent to 1 (gamma) tyrosine for 1 minute in reaction liquid at pH 9.0 and 40 degreeC.

(5) Optimal temperature and stable temperature range

Figure 3 shows the temperature curve. As can be seen in the figure is the optimum temperature of the action of the enzyme near 45 ℃ and shows more than 80% activity at 35 ° -55 ° C. The temperature stability is completely stable below 40 ° C. and 50% residual activity at 60 ° C. as shown in FIG. 4.

(6) deactivation conditions by pH and temperature

Almost all activity is lost below pH 6 or above 12, and completely lost at pH 4 or 12 at 40 ° C., 1 hour treatment. In addition, the pH 7 significantly lost stability at 60 ° C and completely lost activity at 65 ° C. In addition, even at pH 9.5, the activity is completely lost at 70 ℃.

(7) Influence of Inhibitors

First, the influence of various inhibitors is shown in Table 2. Values are expressed as residual activity. Alkaline proteases that are known are serine enzymes that contain serine in the active group structure and are specific inhibitors thereof. It is generally known that it is inhibited by DM and poteroinhibitors, but not by this diet, ortho-phenanthroline, while the neutral protease is a metal enzyme as a DNA and ID inhibitor. It is known that the behavior against is far from the opposite.

TABLE 2

As mentioned above, this enzyme indicates that it is a serine enzyme having an active center of serine of alkaline protease, and it is markedly inhibited by this DTI, and the fact that the secondary metal is a novel enzyme that does not exist in the past plays an important role in some form. This became clear. T enzyme was used as a neutral protease and A enzyme was used for comparison as an alkaline protease.

T Enzyme: Neumolyse-Yamadosei Co., Ltd. neutral protease, crystal

A enzyme: Alkaline protease II-Sega Chemical Co., Ltd., three times Bacillus subtilis bar amylo Fungariticus crystal

(8) Purification Method

The coenzyme powder is dissolved in a 10% solution in water, and stirred at 5 ° C. for several hours to completely extract the enzyme. Calcium acetate is added to the final concentration of about 1% and the pH is adjusted to 7.5-8.0 to remove the precipitated fractions and the supernatant is obtained. Separately, ammonium sulfate was added to a final concentration of 0.6 saturation, and left to stand for several hours, followed by recovery of the precipitate by centrifugation.

Next, M / 200 containing calcium acetate of M / 500 and pH 7.5 tris hydrochloric acid buffer solution are dissolved again, and the same buffer is used as an external solution for desalting. This enzyme was eluted by adsorption on the column chromatograph of D. Sephadex A-50, equilibrated with copper buffer, and the copper buffer containing 5-7 times the volume of the bed, and then the copper buffer containing 0.1M saline. do. Under the same conditions, the activity is almost the highest by performing chromatography, and this enzyme is concentrated to 10-15 times, so that this enzyme can be obtained as a crystal.

(9) physical and chemical properties

As mentioned above, the physicochemical properties of the protease and bacillopeptidase C obtained in the present invention have become clear, and it is clear that the protease is different from the protease known in the prior art.

In other words, the enzyme is completely inactivated by diisopropyl fluorophosphate or inactivated by this DTI, regardless of the conventional alkali protease classification criteria. It is completely inactivated and the isoelectric point of many alkaline proteases is above pH8. The isoelectric point of this enzyme is pH6.0, and the enzyme has specificity in that it is remarkably superior to the alkali protease known for its anti-inflammatory effect. It was recognized as a protease and was named Basillopeptidase C.

This novel protease, basileropeptidase C, is extremely useful as an anti-inflammatory agent and has many uses, such as fire extinguishing agents, leather and meat softeners and detergents due to its high titer of protease activity.

Next, the experimental results for the anti-inflammatory action of a novel protease, Vasillo peptidase C, will be described.

Four rats (group 130-150 g body weight) were injected intraperitoneally with 0.5 mg of each enzyme sample solution per mouse. After 1 hour, 0.05 ml of 1% carrageenin solution was injected subcutaneously into the rat's hind paw. As a result of determining the effect of each enzyme by measuring the degree of swelling, the swelling inhibition rate of trypsin was 33%, α-chymotrypsin was 25% and bromeraine was 37%. The effect was shown.

The edema inhibition rate by the mouse foot edema method (LP) which used carrageenan as a base agent is shown.

Example 1

20 L of medium containing 1% starch, 2% soybean meal, 1% bran, 0.5% potassium phosphate, 0.5% magnesium sulfate is placed in a 30 L fermentation tank and autoclaved at 120 DEG C for 20 minutes. Bacillus no. 200 ml of the culture medium in which 794 strains (unpublished depository repair No. 1522) were inoculated and incubated at 30 ° C. for 3 days at 100% aeration and 300 rpm. Immediately after the end of the culture, the cells were centrifuged to remove the cells and 15 ml of the filtrate was obtained. The mixture was concentrated under reduced pressure to 3 L, and 27 g of calcium acetate was added thereto to adjust the pH to 8.0. The precipitate formed was filtered off and then ammonium sulfate was added and centrifuged until 0.6 saturation was recovered. At the end of fermentation, 80% of the activity was recovered. The solution was re-dissolved in 300 ml of a solution containing M / 500 calcium acetate in a buffer of M / 200 tris hydrochloric acid pH7.5 and dialyzed using the same buffer as an external solution. Separately, the enzyme solution was adsorbed onto a column of 3 L Di Sephadex A-50 buffered with the above buffer and washed with about 10 L of the same buffer. Subsequently, the same solution elutes with a solution containing 0.1 M saline to elute the enzyme. About 70% of the titer recovery rate was obtained in the 3L eluate, and 0.8 g of the crystal was obtained by performing chromatography under the same conditions and concentrating 10 times.

Example 2

2 liters of the culture filtrate obtained in the same manner as in Example 1 was salted with ammonium sulfate without dialysis, and dialyzed, followed by dia-sepadex chromatography, and the obtained eluent was concentrated 8 times to obtain 60 mg of crystals.

Claims (1)

Bacillus No. belonging to the genus Bacillus 794 (Unpublished Deposit No. 1522) Orient the strain (microorganisms that produce bacillopeptidase C), and generate and collect bacillopeptidase C in the medium. Manufacturing method.

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