KR100478214B1 - Fibrinolytic enzyme derived from Bacillus sp. HLN-21 strain and, isolation and purification method thereof - Google Patents

Abstract

The present invention provides a thrombolytic enzyme having a molecular weight of about 17 KDa and high thrombolytic activity produced by Bacillus sp. The present invention provides a method for isolating and purifying thrombolytic enzymes in large quantities by a simple process while maintaining them.

Description

Thrombolytic enzymes derived from Bacillus HL-21 strains and methods for isolating and purifying them {Fibrinolytic enzyme derived from Bacillus sp. HLN-21 strain and, isolation and purification method

The present invention relates to a novel thrombolytic enzyme and a method for isolating and purifying the same. More specifically, the HLN-21 strain of Bacillus genus isolated and identified from fermented products by mixing fermented straw and soybeans collected from Hampyeong-gun, Jeonnam, Korea ( The present invention provides a method for separating and purifying thrombolytic enzymes in large quantities by a simple process while maintaining thrombolytic enzymes having a molecular weight of about 17 KDa and high thrombolytic activity produced by KFCC-11306).

A thrombus is a gel-like substance called fibrin that is caused by the action of a coagulant repair enzyme in which fibrinogen, which is excessively contained in the blood, coagulates platelets and blood. For example, blood clots may injure blood vessels due to some cause. If the blood clots naturally occur in the body, such as to stop bleeding is always formed. When the blood vessels are recovered, the clots are dissolved by the activity of an enzyme called the glandular system. If the activity balance between the glandular system and the coagulation system is not maintained in this process, the generated blood clot is excessively accumulated in the body, causing atherosclerosis and thrombosis. , Heart disease and high blood pressure can be caused.

Therefore, studies for the removal of blood clots have been continuously conducted. In particular, the pretreatment enzymes that act on the breakdown of fibrin, a major component of blood clots, include urokinase and tissue-type plasminogen activator, t. -PA) and plasminogen activators such as Streptokinase are injected intravenously to activate the fibrinolytic system as the most common method. However, these substances have large side effects such as systemic hemorrhage, high price, and oral administration except eurokinase.

Recently, various studies on more economical and orally administrable thrombosis treatments have been attempted, and new thrombolytic agents for dissolving thrombi in an indirect manner by direct oral administration or activating plasminogen have been developed. Currently, thrombolytic agents that can be administered orally include thrombolytic enzymes isolated from worms (Lumbricus lubellus), hementin from leeches, and salmonases isolated from snake venom (Chung, KH, et al., Thromb. Haemost. 65, 953, 1991). In addition, attempts have been made to discover and develop thrombolytic substances from various living organisms in nature.

Of these, the study of thrombolytic enzymes in fermented foods centered on Korea and Japan is noteworthy in that they are directly available for food. In Japan, strains are isolated from natto, a traditional soybean fermented food, and shiokara, a pickled food, to enhance thrombolytic activity in vivo, orally, by katsuokinase and natto. It has been reported that kinases (nattokinase: Sumi, H., et al., Experientia, 43, 1110, 1987) were purified. Among them, nattokinase is a preenzyme that acts directly on fibrin like plasmin in vivo and shows strong degrading activity and indirectly activates the protease in vivo even after oral administration. The sale of natto as a health food is increasing rapidly (Sumi, H., et al., Acta Haematologica, 84, 139, 1990). In Korea, Bacillus strains with thrombolytic activity were isolated from Cheonggukjang, a traditional fermented food made from soybeans similar to Japanese natto, and purified thrombolytic enzymes from them. Compared to the strains that produce two or three times more than the thrombolytic enzymes and thrombolytic enzymes produced by these strains (Korean Patent No. 229282).

The present inventors also conducted a study to isolate a new thrombolytic enzyme from a food that can be directly ingested, as a result, to isolate the HLN-21 strain of the genus Bacillus having a thrombolytic activity in fermented products mixed with rice straw and soybeans, The present invention was found to be capable of separating and purifying thrombolytic enzymes having a molecular weight of about 17 KDa.

In addition, the process of isolating and purifying enzymes having thrombolytic activity from the culture medium of the conventional strain is very complicated, and because there is a problem in that the yield is not easy to purify in large quantities because it has to go through several steps, this problem is solved. As a result of the study, the high thrombolytic activity of thrombolytic enzymes was prepared by preparing a culture medium having high thrombolytic activity and purifying the enzyme from the culture by a combination of anion exchange column chromatography, dialysis and gel filtration column chromatography. The present invention has been completed by discovering that the enzyme can be separated and purified in large quantities by a simple process while maintaining it.

Accordingly, it is an object of the present invention to provide a novel strain having thrombolytic activity.

Another object of the present invention is to provide a thrombolytic enzyme derived from the above strain.

Still another object of the present invention is to provide a method for separating and purifying the thrombolytic enzyme described above in large quantities by a simple process while maintaining high thrombolytic activity.

In order to achieve the above object, the present invention provides a Bacillus sp. HLN-21 strain (KFCC-11306) having thrombolytic activity.

The present invention also provides a novel thrombolytic enzyme derived from the strain and having a molecular weight of about 17 kDa.

In addition, the present invention is thrombolytic from the culture of Bacillus HLN-21 strain (KFCC-11306) by a combination of Toyopearl butyl 650M column chromatography, dialysis and Sephacryl S-200 column chromatography. Provided are methods for separating and purifying enzymes.

The present invention will be described in more detail as follows.

The present invention provides a novel Bacillus sp. HLN-21 strain having thrombolytic activity.

The strain of the present invention is mixed with each of the straw and soybean collected from all over the country, the culture produced by spreading the bacteria on a suitable medium, and cultured to select a strain forming a monocolon (monocolony), then fibrin plate By confirming the thrombolytic activity of each strain by the method, the best strains were selected.

In order to identify the selected strains of the present invention, after culturing the selected strains, the collected strains were collected and observed using a scanning electron microscope to observe their morphological characteristics, and Gram staining resulted in Gram positive bacilli having a size of 4 μm. (See FIG. 1). In addition, in order to investigate the physiological activity of the strain, the presence or absence of activity for various carbon sources was also measured according to the Buggy's Manual (see Energy 2). As a result, the strain of the present invention was found to be a strain of the genus Bascillus sp .

However, as a result of comparing the Bacillus sp. Strains isolated from commercial Natto and the activity of various carbon sources (see Table 2), the Bacillus sp. Strain of the present invention is an enzyme different from the strains of the Bacillus sp. It was confirmed that it has activity.

Therefore, the present inventor named this strain HLN-21 of the genus Bacillus, and deposited on July 2, 2002 with the Korea Microorganism Conservation Center and was given accession number KFCC-11306.

The present invention also provides a thrombolytic enzyme produced by the strain. The thrombolytic enzyme of the present invention is an enzyme having a molecular weight of about 17 KDa, and is stable in a wide pH range of 3.5 to 11 and a temperature range of 40 ° C or less.

The thrombolytic enzyme of the present invention can be isolated and purified from the culture of Bacillus HLN-21 strain (KFCC-11306) using methods commonly known in the art. However, since these processes are very complicated and have to go through several steps, the yield is also not easy to purify in large quantities. In the present invention, after culturing the strain in an optimal liquid medium, a culture medium having high thrombolytic activity is prepared. , Toyopearl butyl 650M column chromatography, dialysis and Sephacryl S-200 column chromatography are combined and purified.

A method for isolating and purifying thrombolytic enzymes from the Bacillus HLN-21 (KFCC-11306) strain of the present invention shown in FIG. 2 will now be described in detail.

Bacillus HLN-21 (KFCC-11306) strain is incubated in a liquid medium for 24 to 48 hours, followed by centrifugation and filtration to obtain a supernatant from which the cells are removed. The supernatant is treated with 30% ammonium sulfate and then centrifuged and filtered again to obtain the supernatant.

At this time, the medium contains 3 to 7% and 1 to 5% of sucrose and potato as the carbon source, and 1 to 5% of soy peptone as the nitrogen source, based on the total weight of the medium composition. It is preferable to use a thing whose pH is 5-11 in order to acquire high thrombolytic activity. More preferably, a medium containing sucrose, potato and soybean peptone in amounts of 5% each is used. Other components of the medium may be appropriately determined depending on the characteristics of the strain and the genus to which the strain belongs. Select and blend.

The obtained supernatant was loaded on an anion exchange column, preferably a Toyopearl butyl 650M column, and then using a concentration gradient of 30% ammonium sulfate solution and 20 mM Tris-HCl buffer (pH 7.2), Fractions with thrombolytic activity are eluted. At this time, the activity of the enzyme is measured by the fibrin plate method.

Fractions having this activity are collected and 90% ammonium sulphate is added to give a precipitate, which is then dialyzed with 20 mM Tris-HCl buffer (pH 7.2).

The dialysis solution is loaded onto a gel filtration column, preferably a Sephacryl S-200 column, and chromatographed to purify enzymes with thrombolytic activity.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited only to these examples.

Example 1 Isolation of Strain with Thrombolytic Activity

(1) Isolation of Strains

Crests collected from 28 regions across the country, including Hampyeong, Jeonnam, were treated in a boiling water at 95-100 ° C, and then each 10g was placed in a sterilized container containing 10g of soybean sterilized at 121 ° C for 30 minutes, mixed, and then 35 Incubated for 24 hours at ℃. The bacteria produced by the culture are suspended in physiological saline, and the suspension is plated in agar plate medium (composition: glucose 0.1%, peptone 1%, beef extract 0.05%, sodium chloride 0.05%, agar 1.5%, pH 7.0), Colonies were formed by incubation at 35 ° C. for 24 hours. The colonies formed were screened and then shaken in a liquid medium (medium composition: 1% peptone, 0.3% beef extract, 0.05% sodium chloride, pH 7.0) under a temperature of 35 ° C for 24 hours. The number of viable cells of each culture was measured and inoculated with 1 × 10 ⁵ bacteria / g of the same soybeans and cultured at 35 ° C. for 24 hours. The bacteria thus produced were suspended in physiological saline, and the suspension was plated on agar plate medium and incubated at 35 ° C. for 24 hours, and then a single colony was formed to select each strain.

 (2) measurement of thrombolytic activity

The thrombolytic activity of the isolated strains was determined by fibrin plate method (Astup, T., et all., The National Danish Society against Rheumatic Diseases 346, 1952).

That is, 200 µl of 50 units / ml of thrombin solution was dipped into a petri dish having a diameter of 90 mm, and then 10 ml of 0.5% fibrinogen solution was added without foaming, and mixed with thrombin to form a flat plate. After 10 to 30 minutes, the fibrin plate is formed when the gel becomes. 5 μl of the culture medium of the above-dispersed strains was added to the fibrin plate, and then, a clear zone generated by reacting at 37 ° C. for 3 hours was checked, and the diameter of the clear ring was measured to determine the thrombolytic activity ( Casein unit: Cu / g or Cu / ml). At this time, as a control, the activity of plasmin (Sigma) was measured in the same manner as above and used for unit conversion. The results are shown in Table 1 below. On the other hand, in Table 1 below, J strain is a strain of genus Bacillus isolated from Natto purchased from T company in Japan in the same manner as in (1), and K strain is (1) and Natto purchased from S company in Korea. Bacillus sp. Strain isolated in the same way.

Strain Thrombolytic activity (Cu / g) Strain Thrombolytic activity (Cu / g) One 750 16 1,500 2 350 17 0 3 80 18 250 4 0 19 450 5 0 20 380 6 250 21 2,000 7 400 22 450 8 380 23 250 9 200 24 0 10 0 25 0 11 50 26 450 12 100 27 450 13 500 28 800 14 0 Control 1 (J strain) 1,125 15 1,600 Control 2 (K strain) 940

The 21st strain having the best thrombolytic activity among the above strains was named HLN-21.

Example 2 Identification of HLN-21 Strains

(1) morphological observation

In order to observe the form of the HLN-21 strain obtained in Example 1, 35 HLN-21 strain in a liquid batch (medium composition: beef extract 0.5%, peptone 1%, glucose 0.1%, sodium chloride 0.5%, pH 7.0) 35 After culturing for 24 hours at a temperature of ℃, the cells were recovered by centrifugation and fixed. This was observed at a magnification of 1,000 times using a scanning microscope, and when Gram stained, it was found to be Gram-positive bacilli having a size of 4 μm (see FIG. 1).

(2) Activity against the carbon source of the strain

The HLN-21 strain was tested for activity on various carbon sources using AP150 / CHB according to the Buggy's Manual. On the other hand, J and K strains of Example 1 were also tested for activity in the same manner. The results are shown in Table 2 below.

Carbon source activation Carbon source activation HLN-21 J strain K strain HLN-21 J strain K strain Glycerol + + + Salicine + - - Erythritol - - - Cellobiose + - - D-Arabinose - - - Maltose + + + L-Arabinose + + + Lactose + - - Ribose + + + Melibiose + - - D-Xylose + + + Saccharose + + + L-Xylose - - - Trehalose + + + Adonitol - - - Inuline - + + β-Methyl-Xyloside - - - Melezitose - - - Galactose - - - D-Raffinose + - - D-Glucose + + + Amidon + - - D-Fructose + + + Glycogene + - - D-Mannose + + + Xylitol - - - L-Sorbose - - - β-Gentiobiose - - - Rhamnose - - - D-Turanose + - - Dulcitol - - - D-Lyxose - - - Inositol + + + D-Tagatose - - - Mannitol + + + D-Fucose - - - Sorbitol + + + L-Fucose - - - α-Methyl-D-mannoside - - - D-Arabitol - - - α-Methyl-D-glucoside + - + L-Arabitol - - - N-Acetylglucosamine - - - Gluconate - - - Amygdaline + - - 2-Keto-gluconate - - - Arbutine + - - 5-Keto-gluconate - - - Esculine + + + Control - - -

From the above results, the HNL-21 strain of the present invention was a strain belonging to the genus Bacillus ( Bacillus sp. ), It was confirmed that the strain for the carbon source is different from known strains (J strain, K strain).

Therefore, the present inventors named this strain Bacillus HLN-21 strain and deposited it with the Korea Microorganism Conservation Center on July 2, 2002 and received Accession No. KFCC-11306.

Example 3 Liquid Culture Conditions of Strains for Isolation and Purification of Thrombolytic Enzymes

(1) Review of badge formation

① Comparative experiment on sugar as carbon source

A 100 ml flask containing 20 ml of medium (composition: 0.5% beef extract, 1% peptone, 0.1% glucose, 0.5% sodium chloride, pH 7.0) was inoculated with Bacillus HLN-21 strain (KFCC-11306) and then 35 ° C. Species were cultured for 18 hours at. In this culture, in order to determine the appropriate amount of sugars and sugars when other nutrients are added to the soybean, medium (common medium composition: soybean 2%, potassium dihydrogen phosphate (KH ₂ PO ₄ ) 0.15%, potassium dihydrogen phosphate ( To a 500 ml flask containing 100 ml of K ₂ HPO ₄ ) 0.3%, calcium chloride 0.05%, pH 7.0), the sugars shown in Table 3 were added by concentration, sterilized, and then inoculated with 2% of the seed culture at 35 ° C. Shake culture for 48 hours. The thrombolytic activity was measured in the same manner as in Example 1 using the supernatant obtained by centrifugation of the culture, and the results are shown in Table 3.

(Unit: Cu / ml) 0.5% 1.0% 2.0% 3.0% 5.0% Maltose 1,058 1,564 2,070 2,530 3,542 Sucrose 1,564 2,070 2,070 2,990 4,048 Fructose 2,530 2,070 2,070 2,530 2,990 Glucose 2,760 2,990 2,070 2,070 2,530 Starch 2,070 2,070 2,300 2,530 2,530

As can be seen from Table 3, the sucrose is preferably used for the liquid culture of the strain of the present invention, the amount of use is preferably 3.0 to 7.0%, particularly preferably 5% relative to the total weight of the medium composition. Do.

② Comparative experiment on carbon source other than sugar

To determine the availability of other carbon sources in addition to soybeans, 500 containing 100 ml of culture (common medium composition: soybean 2%, KH ₂ PO ₄ 0.15%, K ₂ HPO ₄ 0.3%, CaCl ₂ 0.05%, pH 7.0) Corn and potatoes were added to the ml flask as a carbon source other than soybean in the amounts shown in Table 4 below, sterilized, and incubated at 35 ° C. for 48 hours by inoculating 2% of the seed culture. The thrombolytic activity was measured in the same manner as in Example 1 using the supernatant obtained by centrifugation of the culture, and the results are shown in Table 4 below.

corn One% 3% 5% 10% - - - - potato - - - - One% 3% 5% 10% Thrombolytic activity (Cu / mL) 2,990 1,564 2,530 2,530 5,060 5,060 5,520 2,990

As can be seen from Table 4, it is preferable for the thrombolytic activity to use potatoes in an amount of 1 to 5%, particularly in an amount of 5% based on the total weight of the medium composition.

③ Comparative experiment on nitrogen source

To determine the availability of the nitrogen source, in a 500 ml flask containing 100 ml of culture (common medium composition: soybean 2%, KH ₂ PO ₄ 0.15%, K ₂ HPO ₄ 0.3%, CaCl ₂ 0.05%, pH 7.0) The nitrogen source of Table 5 was added in the amounts described in Table 5, sterilized, and then inoculated with 2% of the seed culture and incubated for 48 hours at 35 ° C. The thrombolytic activity was measured in the same manner as in Example 1 using the supernatant obtained by centrifugation of the culture, and the results are shown in Table 5 below.

Unit (Cu / mL) 0.25% 0.5% 1.0% 2.0% 5.0% Bactopeptone 2,300 2,070 1,840 1,564 2,530 Soybean Peptone 2,530 2,530 3,818 4,048 4,508 Glutamic acid 2,530 2,530 2,530 2,300 2,530

As can be seen from Table 5, it is preferable to use soy peptone in an amount of 1 to 5%, in particular in an amount of 5% with respect to the total weight of the medium composition, for thrombolytic activity.

(2) Review of culture conditions

① temperature

To determine the appropriate incubation temperature, sterilize a 500 ml flask containing 100 ml of culture (common medium composition: soybean 5%, KH ₂ PO ₄ 0.15%, K ₂ HPO ₄ 0.3%, CaCl ₂ 0.05%, pH 7.0) Then, 2% of the seed culture was inoculated and shaken for 48 hours at a temperature in the range of 30-42 ° C. At this time, in order to minimize the influence of other factors, a medium consisting only of soybeans and minerals was used. Then, using the supernatant obtained by centrifugation of the culture solution, the thrombolytic activity was measured in the same manner as in Example 1, and the results are shown in Table 6 below.

(Unit: Cu / ml) Temperature 30 ℃ 35 ℃ 37 ℃ 39 ℃ 42 ℃ 24-hour incubation 590 4,048 4,048 4,048 3,542 48 hours incubation 2,530 5,980 5,060 4,508 2,070

As can be seen from Table 6, the strain of the present invention is preferably incubated for 24 to 48 hours at a temperature of 35 to 39 ℃, especially for 48 hours at a temperature of 35 ℃ for thrombolytic activity.

 ② pH

To determine if the acidity of the medium affected the thrombolytic activity, 100 ml of each medium with different pH (medium composition: 5% soybean, KH ₂ PO ₄ 0.15%, K ₂ HPO ₄ 0.3%, CaCl ₂ 0.05%) The containing 500 ml flask was sterilized and then inoculated with 2% of the seed culture and shaken for 48 hours at a temperature of 35 ° C. The thrombolytic activity was measured in the same manner as in Example 1 using the supernatant obtained by centrifugation of the culture, and the results are shown in Table 7 below.

pH 3.0 5.0 7.0 9.0 11.0 Thrombolytic activity (Cu / mL) 0 2,990 3,496 2,990 3,496

As can be seen from Table 7, it was shown that pH 5 to 11 are not significantly affected by thrombolytic activity except in case of strong acidity.

Example 4 Isolation and Purification of Enzymes Having Thrombolytic Activity

A 500 ml flask containing 150 ml of medium (composition: 0.5% beef extract, 1% peptone, 0.1% glucose, pH 7.0) was inoculated with a Bacillus HLN-21 strain (KFCC-11306) and shaken at 35 ° C in an incubator. , And incubated for 24 hours while stirring at 200 rpm. Subsequently, 5% of the above cultures were added to 3 l of the culture medium (composition: soybean 5%, potato 1%, sucrose 5%, KH ₂ PO ₄ 0.15%, K ₂ HPO ₄ 0.3%, CaCl ₂ 0.05%, pH 7.0). Inoculation, while maintaining a temperature of 35 ℃ was adjusted to a stirring speed according to pH 6 ~ 8, dissolved oxygen (DO) concentration to 300 ~ 1,000rpm and incubated for 24 hours while stirring. At this time, the incubator (B. Braun Biotech) is a 5 L top fermenter equipped with a thermostat, a stirring speed controller, a dissolved oxygen concentration sensor and a pH sensor, and is automatically controlled in connection with a control indicator. On the other hand, the oxygen supply into the incubator was introduced into the incubator through a filtration filter using an air compressor.

The cultured medium was centrifuged at 8,000 rpm (4 ° C) and filtered to obtain a supernatant from which the cells were removed. The supernatant obtained was treated with 30% ammonium sulfate and centrifuged at 8,000 rpm (4 ° C.) for 20 minutes to separate the supernatant. The separated supernatant was loaded on a Toyopearl butyl-650M ion exchange column, and then a concentration gradient was formed with 30% ammonium sulfate solution and 20 mM Tris-HCl buffer (pH 7.2) to elute the thrombolytic active ingredient. The eluted active fractions were collected, then 90% ammonium sulfate was added to recover the precipitate and dialyzed with 20 mM Tris-HCl buffer (pH 7.2). The dialysis solution was loaded on a Sephacryl S-200 gel filtration column to purify thrombolytic enzymes. The amount, total activity, specific activity, yield and purity of the protein in each purification step were measured, and the results are shown in Table 8. At this time, the protein amount was determined by the method of Bradford (Bredford), the total activity was measured by the fibrin plate method described in Example 1, the specific activity was calculated by dividing the total activity by the total protein amount.

Purification stage Protein amount (mg) Total activity (Cu) Specific activity (Cu / mg) yield(%) Purification degree Culture filtrate 6,470 5,583,700 909 100 1.0 30% Ammonium Sulfate Supernatant 3,370 5,455,400 1,618 93 1.8 Saturday Pearl Butyl-650M Column 961 3,974,800 4,136 68 4.6 Sephacryl S-200 50 2,183,200 43,664 37 48

As can be seen from Table 8, an enzyme having high thrombolytic activity can be easily obtained by a two step process.

Example 5 Identification of Enzymes Having Thrombolytic Activity

The activity of the enzyme isolated in Example 4 was measured by the fibrin plate method of Example 1, and the results are shown in FIG. 3. At this time, a well known plasmin (unit / ml or unit / ml) was used as a thrombolytic agent.

3, the thrombolytic activity of the enzyme is 5,080 Cu / ml, the plasmin is 500 Cu / ml, and it can be seen that the enzyme of the present invention has excellent thrombolytic activity.

In addition, the enzyme of Example 4 was loaded into lane 1 and the molecular weight standard was loaded into lane 2, followed by electrophoresis with 12% SDS-PAGE. In this case, as the molecular weight standard material, cytochrome C monomer (12.4 KDa), cytochrome C dimer (24.8 KDa), cytochrome C trimer (37.2KDa), and cytochrome C hexamer (74.4 KDa) were used.

As a result, it can be seen that the enzyme isolated and purified in Example 4 is an enzyme having a molecular weight of about 17 KDa having thrombolytic activity (see FIG. 4).

Example 6 Stability of Temperature and pH of Enzyme

(1) stability to temperature

After reacting the thrombolytic enzyme purified in Example 4 in each water bath adjusted to the temperature shown in Table 9 for 3 hours, the activity of the enzyme was measured in the same manner as in Example 1, The results are shown in Table 9.

Temperature 4 ℃ 10 ℃ 20 ℃ 30 ℃ 35 ℃ 40 ℃ 50 ℃ 60 ℃ Activity (Cu / mL) 1,564 1,564 1,564 1,564 1,564 1,564 828 0

As can be seen from Table 9, the enzyme showed little change in activity in the temperature range of 4 ~ 40 ℃, the activity began to decrease little by little above 50 ℃ and completely lost the activity of the enzyme at 60 ℃ .

(2) stability to pH

After preparing buffers corresponding to the respective pHs shown in Table 10, 5 μl of the buffer solution and 45 μl of the enzyme sample of Example 4 were left in a low temperature room (4 ° C.) for 3 hours. It measured and the result is shown in Table 10.

pH 3.6 4.0 5.0 5.7 6.0 7.0 8.0 9.0 10.0 Enzyme Activity (Cu / mL) 828 828 828 828 828 828 828 828 828

As can be seen from Table 10, the enzyme of the present invention was not significantly affected by the pH.

Experimental Example 1 Comparison of Thrombolytic Activity According to Culture Methods

As in the present invention, liquid culture of the Bacillus HLN-21 (KFCC-11306) strain is preferable for the thrombolytic activity of the finally obtained thrombolytic enzyme. To confirm this, after culturing Bacillus genus HLN-21 (KFCC-11306) strain in a solid medium, the thrombolytic activity was measured.

That is, 5% of the seed culture solution of Example 4 was inoculated into a solid medium having the composition shown in Table 11, and then cultured at 39 ° C. for 24 hours, and left at 4 ° C. for 24 hours, followed by adding 200 ml of distilled water to 4 ° C. Extraction was performed with shaking at 1 hour. 1 ml of the extract was centrifuged, 5 µl of the supernatant was taken, and thrombolytic enzyme activity was measured by the fibrin plate method of Example 1, and the results are shown in Table 11.

No Badge composition Thrombolytic enzyme activity (Cu / g) One       40g soybean             598 2       40 g of corn               0 3       Soybean 28g + Carrot 12g           1,058 4       Soybean 28g + Corn 12g           2,530 5       Soybean 28g + Potato 12g           1,058 6       Soybean 28g + Carrot 4g + Corn 4g + Potato 4g           1,564 7       Soybean 40g + Sucrose 5%           1,564

As can be seen in Table 11, the thrombolytic activity was highest in the solid medium containing corn, which was significantly higher than the activity (5,080 Cu / mL) of the thrombolytic enzyme obtained by liquid culture according to the method of the present invention. Low (see FIG. 3).

Experimental Example 2 Comparison of Thrombolytic Activity According to Medium Composition

[2-1] The same conditions as in Example 4 except that the composition of the culture liquid medium (pH 7.0) was 5% soybean, 0.15% KH ₂ PO ₄ , 0.3% K ₂ HPO ₄ , and 0.05% CaCl _2. After culturing under the following conditions, enzymes were isolated and purified in the same manner, and the thrombolytic activity was measured by the fibrin plate method of Example 1. As a result, the thrombolytic activity was 3,820 Cu / ml.

[2-2] The composition of this culture liquid medium (pH 7.0) is 5% busy (domestic), 1% potato, 5% sucrose, 0.15% KH ₂ PO ₄ , 0.3% K ₂ HPO ₄ , and CaCl ₂ 0.05 After culturing under the same conditions as in Example 4 except for%, the enzyme was isolated and purified in the same manner, and the thrombolytic activity was measured by the fibrin plate method of Example 1. As a result, the thrombolytic activity was 2,450 Cu / ml.

Bacillus sp. HLN-21 strain (KFCC 11306) of the present invention is a novel strain having thrombolytic activity, and the thrombolytic component can be easily obtained by culturing it.

In addition, the thrombolytic enzyme derived from Bacillus HLN-21 strain (KFCC 11306) of the present invention is a novel enzyme having a molecular weight of about 17 KDa, and has a thrombolytic activity of about 10 times or more than plasmin, which is widely known as a thrombolytic agent. Since there is about 1.7 times more thrombolytic activity than enzymes derived from Bacillus sp. Isolated from natto, the pharmaceutical composition containing it as an active ingredient can be effectively used for the treatment of thrombosis-related diseases such as thrombosis and arteriosclerosis.

In addition, in the case of liquid culture of the strain by the method of the present invention, and the separation and purification of thrombolytic enzymes from the culture, enzymes with high thrombolytic activity can be obtained simply without going through several steps as in the conventional method. Therefore, mass production is possible.

1 is a micrograph by Gram staining of the strain of the present invention (× 1,000).

Figure 2 is a flow chart showing a process for separating and purifying the thrombolytic enzyme of the present invention.

Figure 3 is a photograph confirming the thrombolytic activity of the enzyme purified from the culture medium of the strain of the present invention.

Figure 4 is a photograph showing the SDS-PAGE results of the thrombolytic enzyme of the present invention.

<Description of Drawing>

A: plasmin

B: Thrombolytic Enzyme of Example 4

C: solid culture extract in medium 4 of Experimental Example 1

Claims (10)

Bacillus sp. HLN-21 strain having thrombolytic activity (KFCC-11306). delete The thrombolytic enzymes were removed from the culture of Bacillus HLN-21 strain (KFCC-11306) of claim 1 by a combination of Toyopearl butyl 650M column chromatography, dialysis and Sephacryl S-200 column chromatography. Separation and Purification Method The method of claim 3, wherein (a) After loading the culture solution of Bacillus HLN-21 strain (KFCC-11306) on a Toyopearl Butyl 650M column, the concentration gradient of 30% ammonium sulfate solution and 20 mM Tris-HCl buffer (pH 7.2) Eluting the fraction having thrombolytic activity using; (b) adding 90% ammonium sulfate to the eluate to obtain a precipitate, followed by dialysis of the precipitate with 20 mM Tris-HCl buffer (pH 7.2); And (C) loading the dialysed solution on a Sephacryl S-200 column to obtain an enzyme having thrombolytic activity. The culture solution according to any one of claims 3 to 4, wherein the culture solution of the genus Bacillus HLN-21 strain (KFCC 11306) is centrifuged and filtered to obtain a supernatant from which the cells are removed by centrifugation and filtration. To 30% ammonium sulfate, followed by centrifugation and filtration to obtain a supernatant containing the thrombolytic active ingredient. The method of claim 5, wherein the liquid culture is based on the total weight of the medium composition in the amount of sucrose in the amount of 3-7%, the potato in the amount of 1-5%, the soy peptone (soypeptone) in the amount of 1-5% And is carried out in a medium having a pH of 5-11. 7. The liquid culture method according to claim 6, wherein the liquid culture contains 5% sucrose, 5% potato, 5% soybean peptone, and a pH of 5-11 based on the total weight of the medium composition. Characterized in that it is carried out in the medium. The method of claim 5, wherein the liquid culture is characterized in that carried out for 24 to 48 hours at a temperature of 35 ~ 39 ℃. 9. The method of claim 8, wherein the liquid culture is carried out for 48 hours at a temperature of 35 ℃. The method of claim 5, wherein the liquid culture is performed at pH 5 to pH 11.