KR100246123B1 - Novel bacillus sp. and fibrinolytic enzyme - Google Patents

Abstract

The present invention relates to a strain of the genus Bacillus having a blood clot resolution isolated from doenjang.

The present invention also relates to an enzyme protein having a thrombus resolution produced by a strain of genus Bacillus.

More specifically, the present invention relates to a Bacillus strain having excellent thrombolytic activity in Korean doenjang, a traditional Korean food, and an enzyme protein having thrombolytic activity produced by the present strain, and a thrombus comprising the present strain and the present enzyme protein as an active ingredient. It is intended to be used as a therapeutic and health supplement for related diseases.

Description

Bacillus spp. Strain and thrombolytic enzyme protein produced by the strain

Figure 1 shows the screening results of strains with thrombus resolution.

Figure 2 compares the activity of the fibrinolytic enzyme (plasmin) and plasmin of the present invention by the fibrin plate assay (fibrin plate assay).

A: plasmin (1 U / ml) B: strain culture

Figure 3 shows the form of the strain of the present invention observed by scanning electron microscopy (scanning electron microscopic photogram, 8,400 magnification).

Figure 4 shows the analysis of the fatty acid composition of the strain of the present invention by gas-liquid chromatogram (gas-liquid chromatogram).

The present invention relates to a strain of the genus Bacillus sp. Having a blood clot resolution isolated from doenjang.

The present invention also relates to an enzyme protein having a thrombus resolution produced by Bacillus sp.

More specifically, the present invention relates to a Bacillus sp. Strain having excellent thrombolytic activity in Korean doenjang, a Korean traditional food, and an enzyme protein having thrombolytic activity produced by the present strain. It is to provide a therapeutic agent and a health supplement for a blood clot-related disease as an active ingredient.

[Background of invention]

One of the biggest causes of death in modern people is reported to be cardiovascular disease. Cardiovascular diseases include cerebrovascular thrombosis that causes seizures, such as cocronary artery thrombosis and stroke, which specifically cause myocardial infarction. And venous thrombosis that causes pulmonary embolism. This disease is increasing every year, and recently, in Korea, death rate due to heart disease is the number one cause of death due to the improvement of national income and westernization of diet.

In general, blood circulates in the body with fluidity within blood vessels, but once exposed outside the blood vessels, they coagulate rapidly. When the blood vessels are damaged, bleeding occurs. Usually, when the damaged blood vessels are large blood vessels such as arteries, surgery should prevent bleeding, but small injuries naturally hemostasis in the blood vessels.

The inner surface of blood vessels is surrounded by endothelial cells, and these endothelial cells act to inhibit the coagulation of blood in normal cases, and to hemostatically in case of injury.

The antithrombosis activity of vascular endothelial cells is negatively charged on the surface of the vascular endothelial cells and is maintained by secreting strong blood coagulation factors such as heparan sulfate or thrombomodulin. do. At this time, heparan sulfate promotes the activity of antithrombin III, and thrombomoduline inhibits the enzymatic activity of thrombin and activates protein C, thereby inhibiting blood coagulation. prevent.

However, if a blood vessel is damaged by any factor and bleeding occurs, a hemostatic reaction occurs as a physiological defense mechanism in the blood vessel to prevent blood loss. In other words, when blood vessels are damaged, blood flow and outflow are first inhibited, and then platelets circulating in the blood gather and adhere to each other. Subsequently, as a result of platelet adhesion and aggregation, a plug is formed at the injury site, and finally, explosive thrombin generation is caused by the action of endothelial cells and platelets around the damaged blood vessel through the blood coagulation pathway. In vivo physiological conditions, hemostasis necessarily occurs only around damaged blood vessels and do not affect other areas.

When the hemostasis process is completed and regeneration of vascular tissue is performed, the fibrin polymer formed through this process must be dissolved. Fibrin polymers are dissolved by a substance called plasmin, which is produced by the activation of plasminogen in the thrombolytic system.

As such, plasmin acts as a major enzyme to dissolve fibrin, but has high degradation activity against fibrinogen, which is important for blood coagulation. Therefore, excessively large amounts of free plasmin inhibit blood coagulation so that there is a high risk of bleeding. In order to overcome this problem, an inhibitor of plasmin exists in the blood in vivo. In addition, plasminogen activator inhibitor (PAI) is precisely regulated so that plasminogen activation by plasminogen activator occurs only in the place where fibrin clot is formed. .

[Prior art]

When blood clots are generated in the cerebrovascular system due to bioreaction mechanisms, thrombosis occurs and the body becomes incapacitated, and when blood vessels are ruptured due to rupture of the blood vessels of the brain, a hemorrhage occurs in the space between the brain and the head bone. In addition, clogging of blood vessels by blood clots can lead to heart failure or heart failure. Therefore, many studies have been conducted to remove the thrombus, which is an important cause of death. As a result, plasminogen activators such as streptokinase and urokinase are known to be very useful, and they are injected intravenously. Therapies to activate the fibrinolytic system have been in common use for over 30 years. Many clinical cases have already demonstrated a significant effect on thrombolytic dissolution when these two drugs are actually used. On the other hand, there is no side effect such as systemic haemorrhage during treatment because there is no specificity for thrombosis.

In addition, in 1987, a tissue-type plasminogen activator (tPA) was developed by genetic engineering method, which was first recognized as an ideal thrombolytic agent due to its high selectivity by binding directly to thrombi. However, in actual clinical results, the same side effects as when administration of streptokinase or urokinase were still observed, and the half-life of the blood was very short as about 6 minutes.

Therefore, in recent years, attempts have been made to actively provide better thrombolytic agents that overcome the disadvantages of these conventional thrombolytic agents. For example, the first is to develop new mutant activators with strong thrombolytic activity, and the second is to develop new thrombolytic agents derived from animals, plants, or microorganisms having better thrombolytic ability from natural substances.

Numerous attempts have been made for this purpose, since substances capable of dissolving thrombi existing in various organisms in nature can be developed as new thrombolytic agents using a wide range of screening techniques.

In particular, studies using plants are very active, and many studies on tissue factor pathway inhibitors (TFPI) have already been reported. Specifically, ingredients extracted from ginkgo biloba are known to facilitate blood circulation, and formulations using the same have been developed and commercialized.

On the other hand, it is reported that there are many substances known to dissolve blood clots in animals. For example, a novel plasminogen activator isolated from the vampire tissue of vampire bats at the Merck laboratory in the United States in 1989 [Gardell, SJ et al., (1991) Circulation 84, 244-253]. , Salmonase isolated from snake venom [Chung, KH and Kim, DS, (1991) Thromb. Haemost., 65, 953], hementin isolated from leeches, and fibrinolytic enzymes isolated from centipedes [Kim, K. Y. et al., (1993) Thromb. Haemost., 69, 839] have been reported as new thrombolytic agents.

In addition, various thrombolytic substances have been identified in microorganisms. Representative of this is streptokinase isolated from β-haemolytic streptococci, as well as staphylokinase and asphysis present in Staphylococcus, reported in 1948. Strains of CA-7 found in Aspergillus oryzae, fibrinolysin found in Bacillus sp., Brinolase, Yersinia pestis, and Bacillus sp. Fibrinolytic enzymes found in have been reported as thrombolytic agents.

Drugs such as streptokinase, urokinase, and tissue-type plasminogen activator (tPA), which have been widely used to treat thrombosis, have some disadvantages for clinical use. . Specifically, the present drugs are not only high risk of side effects, very high price, but also cannot be orally administered except urokinase.

Therefore, in recent years, there has been an interest in preparations that can increase the thrombolytic ability in the blood by directly oral administration or in combination with vascular injection in addition to the thrombolytic agent that can be administered only by vascular injection. So far known orally administrable thrombolytic agents include six thrombolytic enzymes isolated from worms (Lumbricus lubellus), which are restricted in Korea (Nakajima, N. et al., (1993) Biosci. Biotech.Biochem., 57, 1730; Mihara, H. et al., (1991) Jpn. J. physiol., 41, 461).

In addition, according to a report by Sumi et al., When urokinase is injected into the intestine and absorbed, the thrombolytic ability in the blood is significantly increased. In other words, this indicates that the urokinase absorbed by the intestine is transported to the liver, thereby promoting the synthesis of the enzyme that dissolves the blood clot, thereby increasing the blood clot dissolving ability. Suggests an applicability that can be used to increase [Sumi, H. et al., (1985) Enzyme, 33, 121; Sumi, H. et al., (1983) Acta Haemat., 70, 289; Sumi, H. et al., (1980) Thromb. Res., 20, 711].

In addition, it is known that thrombolytic enzymes exist in shiokara and natto, which are traditional Japanese fermented foods, and the enzyme was directly isolated. Among them, an enzyme called nattokinase isolated from NATO was orally administered. It is reported that the thrombolytic ability in vivo can be improved even in the case of the above. Therefore, natto is known as a product having thrombolytic ability in Japan and the sales volume is rapidly increasing as a health food [Sumi, H. et al., (1987) Experimentia, 43, 1110; Inhabitants (1990) Bioindustry, 7, 725].

Therefore, the present inventors have tried to find strains and thrombolytic enzymes excellent in thrombolytic ability in fermented foods such as doenjang, cheonggukjang, soy sauce, and kimchi, which are traditional fermented foods of Korea, and have excellent thrombolytic ability in thrombosis from doenjang, a traditional fermented food in Korea The strain producing the enzyme is isolated, and among them, a strain having excellent efficacy is selected among them, and a new thrombus soluble enzyme protein produced by the strain is identified to complete the present invention.

[Summary of invention]

An object of the present invention is to provide a new Bacillus sp. (Bacillus sp.) Strain having a specific and excellent function in thrombolysis isolated from doenjang, a fermented food of Korean tradition.

It is also an object of the present invention to provide a novel Bacillus sp. JK961 (Bacillus sp. JK961) strain (Accession No .: KCTC 8731P) with excellent thrombolytic effect.

It is also an object of the present invention to provide a thrombolytic enzyme protein produced in a strain of Bacillus sp.

It is also an object of the present invention to provide a thrombolytic enzyme protein capable of oral administration as well as vascular injection.

It is also an object of the present invention to provide a pharmaceutical composition comprising the Bacillus sp. Strain of the present invention having a thrombolytic activity and a thrombolytic enzyme protein produced by the strain, as an active ingredient, and the pharmaceutical composition is a coronary artery which is a blood clot-related disease. It is provided as a therapeutic agent for diseases, thrombosis, cerebrovascular disease and the like.

It is also an object of the present invention to provide a dietary supplement comprising the Bacillus sp. Strain of the present invention having a thrombolytic activity and a thrombolytic enzyme protein produced by the strain as an active ingredient.

Detailed description of the invention

Hereinafter, the present invention will be described in detail.

In order to isolate the strain of the present invention, traditional soybeans are collected from various parts of Korea including Yuseong, Gwangju, Nonsan, and Seoul, and this sample is collected by platinum and tryptic soy agar medium by plate dilution culture method. medium) and the like, and cultured under appropriate conditions to select a single colony (colony) and to determine the thrombolytic ability by fibrin plate measurement method and the like from which strains having high thrombolytic ability were isolated.

In order to observe the morphology of the isolates, the strains were cultivated and recovered and observed using a scanning electron microscope, which showed the form of bacillus. 3).

The fatty acid composition was analyzed to investigate the physiological characteristics of the strain. Lipids contain long-chain fatty acids as major constituents of bacterial cell membranes and cell walls, and their composition is characterized by the fatty acid biosynthesis of each organism. Especially in bacteria, the long-chain fatty acid composition is very diverse in each strain. In addition, fatty acid composition can be a taxonomic group, for example, a taxonomic group having certain characteristics at the species level, so that the taxonomic position can be confirmed by examining the fatty acid composition of the cells.

Therefore, in the present invention, the fatty acid composition of the strain was analyzed by gas-liquid chromatogram (see FIG. 4), and the side chains present in the strain were iso-Cn and anisoisocarbon. (anteiso-Cn) was mostly. In view of the overall composition of the side chains, the strains were characterized by the strain Bacillus sp. [Kaneda, T., (1967) J. Bacterio., 93, 894-903; Kaneda, T., (1977) B. Review, 41, 391-428; Rasoamananjaka, D. et al., (1985) J. Gen. Microbiol, 132, 2723-2732].

All experiments required to identify the strains are according to Berger's method of histologic cell sorting [Peter H. A. Sneath, Endosporeforming Gram-positive Rods and Cocci Bergey's Manual of systematic Bacteriology, Vol. 2, 1104-1137 (1986).

Therefore, the strains identified and selected as having excellent thrombolytic ability in the present invention were identified as follows.

The strain showed the general characteristics of the microorganisms of the genus Bacillus, and the strain was cultured under optimal media conditions and then stained with Gram, and identified as Gram-positive bacteria. Scanning electron microscopy showed bacilli, and phase microscopy showed elliptical spores.

Analysis of the morphological, culture and physiological characteristics and cell wall fatty acid composition revealed that the microorganism of the present invention, unlike the general Bacillus, has a unique and useful property of producing an enzyme protein that strongly degrades fibrin involved in blood clots. Confirmed.

Therefore, the present inventors judged this as a strain of the genus Bacillus sp., And named it Bacillus sp. JK 961 (Bacillus sp. JK 961).

In addition, in order to identify the enzyme protein produced by the strain selected as having excellent thrombolytic ability, each strain is incubated in trytic soy broth medium and centrifuged to remove the cells and obtain the supernatant. . The liquid medium is used to measure the thrombolytic activity according to the method for measuring the activity of the thrombolytic enzyme, and the enzyme protein secreted from the strain of the present invention is identified. The fibrin lysis superiority is compared by measuring the fibrin plate lysis area of each strain. At this time, a well-known thrombolytic agent 1 U / ml plasmin is used as a control. The enzyme protein of the present invention exhibits a thrombolytic ability of 2.25 times higher than that of the comparative plasmin.

In order to isolate and purify the enzyme protein having the thrombus lytic ability as described above, and to investigate its characteristics, the enzyme supernatant is isolated and purified by treating the culture supernatant with ammonium sulfate and carrying out DEAE-Sephasil column. . This identified thrombolytic enzyme proteins having molecular weights of 14,000, 21,000, 36,000, 55,000 and 66,000 Daltons.

In order to know whether the enzyme protein is suitable for clinical use, it is confirmed that the hemolytic activity (hemolytic activity), the microorganism of the present invention shows no hemolytic activity at all.

Thus, the enzyme protein of the present invention may be provided as a pharmaceutical composition comprising a pharmaceutically acceptable salt or excipient.

Such pharmaceutical compositions may be administered orally or parenterally, and the effective dosage of the enzyme protein of the present invention is preferably 10,000 to 1,000,000 U per day.

In the pharmaceutical use of the enzyme protein of the present invention can be prepared by a method known in the pharmaceutical field, it can be used in the form of tablets, capsules, powders, solutions, suspensions, injections, etc. using an appropriate excipient. .

Hereinafter, the present invention will be described in detail by the following examples.

The following examples illustrate the invention but do not limit the scope thereof.

Example 1

[Isolation of Strains with Thrombolytic Solubility]

A sample of platinum is taken from traditional soybeans collected from Yuseong, Gwangju, Nonsan and Seoul in Korea and tryptic soy agar medium (17g / l bacto trypton) , 3g / l bactosodium, 2.5g / l bactodextrose, 5g / l sodium chloride, 2.5g / l potassium phosphate, 20g / l bacto agar), incubated at 37 ° C for 24 hours, and then select single colonies. Using this, the blood clot dissolution ability was confirmed by the following fibrin plate measurement method.

Fibrin plate measurement method, which is a method for measuring thrombolytic activity used to isolate strains in the present invention, was performed as follows.

First, 5 ml of a solution completely dissolved in 50 mM phosphate buffer (pH 7.4) so that the final concentration of fibrinogen was 1% was prepared, and 5 ml of tryptic soy agar was added thereto and mixed. 0.1 ml of 100 NIH U / ml thrombin solution was added to the mixed solution, mixed, and then 15 ml of Tryptic Soy Aga was added to the solidified petri dish, which was allowed to stand at room temperature for 5 to 10 minutes to solidify. I was. Next, the isolated strain was inoculated and incubated at 37 ° C. for 24 hours, and then 15% trichloroacetic acid was added to confirm the clear zone generated at this time (see FIG. 1).

Through this process, a strain having a broad transparent ring was isolated from doenjang and named JK961 in Bacillus, and the strain was deposited on March 20, 1996 with the Gene Bank of Korea Institute of Science and Technology. KCTC 8731P).

Example 2

[Electron microscopic observation]

In order to observe the form of the present strain, the strain obtained in Example 1 was tryptic soy broth medium, 17g / l Bakto tryptone, 3g / l Bakto Soyton, 2.5g / l Bakto dextrose, 5g / l sodium chloride, 2.5g / l potassium phosphate) was incubated for 24 hours at 37 ℃ and centrifuged to recover the cells and fixed. The result was taken at 8,400 magnification using a scanning electron microscope, and the cell morphology was bacterium, the size was 0.4 μm × 0.6 μm and the length was 1.8 μm × 1.9 μm (see Fig. 3). ).

Example 3

Fatty Acid Analysis of Strains

In order to identify the present strain isolated in Example 1, its fatty acid composition was analyzed by gas-liquid chromatogram (see FIG. 4), and the results are shown in Table 1.

TABLE 1

As can be seen in Table 1, the side chains present in this strain are mostly isocarbon (iso-Cn) and anthisocarbon (anteiso-Cn), which were characteristics found in Gram-positive bacteria. In particular, the total composition of the side chain chain was found to be 96.12%, it was confirmed that the new strain according to the present invention is a strain belonging to the genus Bacillus sp.

Further, according to the theory of Kaneda in unsaturated fatty acids do not significantly very low (<3%), Arndt iso -C ₁₅ acid (anteiso-C ₁₅ acid) and isophthalic acid -C ₁₅ (iso-C ₁₅ acid) 26 each From group A (B. alvei, B. brevis, B. circulns, B. licheniformis, B. marcerans, B. megaterium, B. pumilus, B. subtilis). In the content of the fatty acid composition compared to the results of the present invention, ANT iso -C ₁₅ acid (anteiso-C ₁₅ acid) and isophthalic acid -C ₁₅ (iso-C ₁₅ acid) the group A respectively, 24.49% and 37.63% Although C ₁₂ and C ₁₈ are present in small amounts of 0.3% and 0.32%, respectively, there are differences in the length of fatty acids. Factor and solved it [Sehgal, SN et al., (1962) Can. J. Biochem. Physio., 40, 69-81]. Therefore, the MIDI system prepared for the cell wall fatty acid profile [Microbio. Identification System Clin. Microbiol. Rev., 5 (3), 302-327 (1992)] and the result of the comparative analysis showed that the strain of the present invention was the closest strain to group A among the fatty acid classification groups (A-F) of Kaneda.

Example 4

[Identification of Enzyme Protein with Thrombolytic Solubility]

In order to confirm the thrombolytic ability of the thrombolytic enzyme protein produced by each strain selected in Example 1, the selected strains were incubated at 37 ° C. for 24 hours using tryptic soy broth medium. . Cells were removed by centrifugation of these cultures, and the activity was measured using the culture supernatant according to the method for measuring thrombolytic enzyme activity.

Fibrin plates were prepared in the same manner as in the fibrin plate measuring method described in Example 1, and then paper discs were placed on the bone plates. 100 μl of each sample was taken, injected onto a paper disc, reacted at 37 ° C. for 24 hours, and the dissolution area of the fibrin plate was measured. As a control, 1 U / ml of purified plasmin, which is well known as a thrombolytic enzyme, was used. As a result, the enzyme protein of the present invention showed 2.25 times higher thrombus solubility than the comparative plasmin (see FIG. 2).

Example 5

[Isolation and Purification of Enzyme Proteins with Thrombolytic Resolution]

The supernatant was taken by centrifuging the culture medium of the microorganism obtained in Example 1 at 10,000xg for 20 minutes, and treated with 30-80% of ammonium sulfate. The crude protein extract thus obtained was desalted and passed through a DEAE-Sephacel column (Sigma, USA) to isolate and purify new proteins with thrombus resolution and to investigate the properties of the isolated enzymes.

SDS-PAGE electrophoresis was performed at the same time as the standard protein. As a result of measuring their respective activities, proteins having molecular weights of 14,000, 21,000, 36,000, 55,000, and 66,000 Daltons were isolated and purified, respectively.

Example 6

[Measurement of Hemolysis Activity]

In order to confirm the usefulness when the new enzyme protein produced by the strain of the present invention is applied to the pharmaceutical or food industry, a test was conducted to determine whether hemolytic activity was present. The Bacillus sp. JK 961 (Bacillus sp. JK 961) strain produced according to the method of Example 1 was placed on a plate, and human blood was added thereto, followed by cultivation and observation. The microorganism of the invention was judged to have no hemolytic activity.

Through the above experimental results, it can be seen that the thrombolytic enzyme protein produced in the strain of the present invention has a much higher thrombolytic property than plasmin which is a conventional thrombolytic agent.

In addition, the thrombolytic enzyme protein of the present invention can be used orally as well as vascular injection as compared to the conventional thrombolytic agent is mainly administered via intravenous injection, there is an excellent advantage that can be used in combination with vascular injection. Therefore, not only can be developed as a clinical oral medication, but also as a dietary supplement.

In order to take advantage of this advantage, if the degree of hemolytic activity of the strain of the present invention is investigated, since it does not show any hemolytic activity, it can be widely used as a thrombolytic therapeutic agent and health supplement.

Claims (7)

Bacillus sp. JK 961 (Accession No .: KCTC 8731P) with thrombus resolution isolated from fermented soybean paste. An enzyme protein having thrombus resolution produced by the strain of Bacillus sp. JK 961 (Bacillus sp. JK 961). The enzyme protein of claim 2, wherein the protein is 14,000 daltons. The enzyme protein of claim 2, wherein the protein is 21,000 daltons. The enzyme protein of claim 2, wherein the protein is 36,000 daltons. The enzyme protein of claim 2, wherein the protein is 55,000 daltons. The enzyme protein of claim 2, wherein the protein is 66,000 Daltons.