KR100229282B1 - Novel bacillus licheniformis and fibrinolytic enzyme - Google Patents

Abstract

The present invention relates to a new Bacillus licheniformis DJ-3 strain having a thrombus dissolving ability isolated from doenjang, a fermented food of Korean tradition, and a thrombin soluble enzyme protein produced by the strain. And the pharmaceutical composition comprising the enzyme protein as an active ingredient can be administered orally can be useful as a therapeutic agent and dietary supplement for thrombosis-related diseases.

Description

New Bacillus licheniformis Strain and Thrombolytic Enzyme Protein Produced by the Strain

One of the biggest causes of death in modern people is reported to be cardiovascular disease. Specifically, cardiovascular diseases include cerebrovascular diseases that cause seizures, such as coronary artery thrombosis and stroke, which cause myocardial infarction ( cerebrovascular thrombosis, and venous thrombosis that causes pulmonary embolism. Currently, the incidence of cardiovascular diseases is increasing every year. In Korea, as the national income is improved and the diet is westernized, the rate of death from cardiac diseases is increasing.

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 these blood vessels is surrounded by endothelial cells, and these endothelial cells act to inhibit the coagulation of blood when normal, and to act quickly to hemostatic in case of injury. The reason why vascular endothelial cells have antithrombosis is that the surface is negatively charged like the surface of platelets, and in these cells strong anticoagulant factors such as heparan sulfate or thrombomodulin, etc. Is secreted. At this time, heparan sulfate promotes the activity of antithrombin III, and thrombomoduline inhibits the enzymatic activity of thrombin and at the same time activates protein C to prevent blood coagulation.

In addition, when blood vessels are damaged by any factor and bleeding occurs, a physiological defense mechanism triggers a hemostatic response in the blood vessels, thereby inhibiting blood loss. Done. When platelets adhere to agglomerate, a plug is formed at the site of damage, and thrombin is exploded through a blood coagulation pathway by the action of endothelial cells and platelets around the damaged blood vessel. 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 activating plasminogen in the thrombolytic system. As such, plasmin acts as an enzyme that dissolves fibrin, but also has an activity of degrading fibrinogen, which is important for blood coagulation. When excessive amounts of free plasmin are present, blood coagulation is suppressed as a whole and thus a great risk exists to the living body. To overcome this problem, there is an inhibitor of plasmin in the blood and plasmin so that plasminogen activation by plasminogen activator occurs only in the place where fibrin clot is formed. Genome inhibitors (plasminogen activator inhibitors, PAI) are finely regulated.

When blood clots are generated in the cerebrovascular system due to bioreaction mechanisms, thrombosis occurs. If the blood vessels are blocked by blood clots, heart failure or heart failure will occur, and many studies have been conducted to treat thrombus. Specifically, plasminogen activators such as streptokinase and urokinase are known to be useful for removing blood clots, and intravenous injections of these plasminogen activators have been used for the past 30 years to activate the fibrinolytic system. The two drugs have already been demonstrated in many clinical cases that actually have a significant effect on thrombolytic dissolution, but on the other hand, side effects such as systemic haemorrhage occur during treatment due to lack of 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 because it can bind directly to thrombi and has high selectivity for thrombi. However, as a result of the clinical use, the half-life of the blood is very short, about 6 minutes, which is inconvenient to use, and there are still problems such as the side effects of streptokinase or urokinase administration.

Thus, in recent years, attempts have been actively made to provide better thrombolytic agents that overcome the problems 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 new plasminogen activator isolated from the vampire tissue of vampire bats at the Merck Laboratories 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 CA-7 found in Aspergillus oryzae, fibrinolysin, Bacillus sp. Found in Yersinia pestis Fibrinolytic enzymes found in have been reported as thrombolytic agents.

To date, the drugs widely used in the treatment of thrombosis, such as streptokinase, urokinase and tPA (tissue-type plasminogen activator), have some problems in clinical use. Specifically, the drug is not only high risk of side effects, very expensive, and is not oral administration except for eurokinase. Therefore, in recent years, attention has been focused on preparations that can increase the thrombolytic ability in blood by direct oral administration or in combination with vascular injection in addition to thrombolytic agents which can be administered only by vascular injection. Specifically, six thrombolytic enzymes were isolated from the worm (Lumbricus lubellus) as orally administered thrombolytic agents [Nakajima, N. et al., (1993) Biosci. Biotech. Biochem., 57, 1730; Mihara, H. et al., (1991) Jpn. J. physiol., 41, 461].

In addition, Sumi et al. Reported that when urokinase was introduced into the intestine and absorbed, the thrombolytic ability in the blood was significantly increased. In other words, this indicates that the urokinase absorbed by the intestine is transported to the liver, thereby promoting the synthesis of an enzyme that dissolves blood clots, thereby increasing the blood clot dissolving ability. Confirms [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, 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 if it is used, and natto is commercialized as a health food with thrombolytic ability in Japan, and its sales are rapidly increasing [Sumi, H. et al., ( 1987) Experimentia, 43, 1110;須 見 洋行 (1990) Bioindustry, 7, 725].

In addition, the present inventors also purified various thrombolytic enzyme proteins using Bacillus sp. Isolated from Korean fermented soybean paste, which is a traditional Korean fermented food, and a therapeutic agent for thrombosis-related diseases in which the pharmaceutical composition containing the same can be administered orally. As already filed (Korean Patent Application No. 96-10045).

In addition, the present inventors have continued to find strains and thrombolytic enzymes with excellent thrombolytic ability in Korean traditional fermented foods such as doenjang, cheonggukjang, soy sauce, and kimchi. Bacillus licheniformis DJ-3) strain was isolated, and from this, the enzyme protein excellent in thrombus solubility was identified to complete the present invention.

It is an object of the present invention to provide a new Bacillus licheniformis strain having a thrombus lytic ability. The present invention provides a Bacillus licheniformis strain isolated from Korean traditional miso which is a fermented food.

Specifically, the present invention screened the new Bacillus Richenomyces DJ-3 strain with the best thrombolytic ability and deposited it on Gene Bank of Korea Institute of Science and Technology, Biotechnology Research Institute, on July 20, 1996 (Accession Number: KCTC 0266 BP). ).

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

Specifically, the present invention provides an enzyme protein having a molecular weight of 25,000 Daltons and 64,000 Daltons as a thrombolytic enzyme protein produced by Bacillus Richenomyces DJ-3 strain.

In addition, an object of the present invention is to provide a pharmaceutical composition for a thrombus-related therapeutic agent comprising the thrombi soluble enzyme protein produced in Bacillus licheniformis DJ-3 strain having a thrombus dissolving ability as an active ingredient.

Specifically, the pharmaceutical composition of the present invention may be usefully used for diagnosing and treating coronary artery disease, thrombosis, cerebrovascular disease, and the like, which may be administered orally as well as vascular injection.

In addition, an object of the present invention is to provide a dietary supplement comprising the thrombi soluble enzyme protein produced in the Bacillus licheniformis strain as an active ingredient.

Figure 1 shows the thrombolytic ability of the strain culture solution (A) of the present invention compared with the thrombolytic ability of plasmin (B, 1 U / ml) by a fibrin plate assay method.

Figure 2 shows the thrombolytic enzyme protein contained in the strain culture of the present invention by SDS-polyacrylamide gel electrophoresis.

A: standard protein, B: enzyme protein

Figure 3 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 provides a novel Bacillus licheniformis strain having a thrombus lytic ability.

In order to isolate strains with thrombolytic ability, the present invention collects doenjang, a traditional Korean fermented food, spreads them on a suitable medium, selects strains that form a single colony, and then measures each strain by fibrin plate measurement. Check thrombolytic ability (see FIG. 1).

In order to identify the strain of the present invention isolated by the above process, after culturing the strain and then observed its morphological characteristics using a scanning electron microscope and a phase contrast microscope, the gram staining or fatty acid again Analyze its composition and examine its culture and physiological characteristics.

As a result, the strain was in the form of bacilli and has elliptical spores and confirmed the following characteristics. In general, 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 system of each bacteria. Since the long chain fatty acid composition of bacteria is very diverse in each strain, the fatty acid composition is a taxonomic unit that can reliably classify strains at the species level. Accordingly, the present invention analyzes the fatty acid composition of the strain by gas-liquid chromatogram (see Table 1 and FIG. 3). As a result, the side chains present in this strain are mostly iso carbon (iso-Cn) and anthiso carbon (anteiso-Cn), and this strain was identified as 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].

In addition, all other experiments required to identify strains of the present invention are based on 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 strain of the present invention shows the general characteristics of the microorganisms of the genus Bacillus, and confirmed as a Gram-positive bacteria as a result of gram staining in the optimum medium. As a result of analyzing the above characteristics and fatty acid composition of the cell wall, it was confirmed that the strain of the present invention has a unique and useful characteristic of producing an enzyme protein that strongly decomposes fibrin involved in thrombus formation, unlike the general Bacillus strain. do.

Therefore, the strain of the present invention was identified as a new strain of Bacillus licheniformis and named as Bacillus licheniformis DJ-3, and on July 20, 1996, the Gene Science Bank of Korea Institute of Science and Technology Was deposited (Accession Number: KCTC 0266 BP).

The present invention also provides a thrombolytic enzyme protein produced by the strain. Specifically, the Bacillus licheniformis DJ-3 strain of the present invention is cultured on a trytic soy broth medium and the like and centrifuged to obtain the culture supernatant. The culture supernatant is used to measure the activity of thrombolytic enzymes from which the enzyme protein secreted is identified (see FIG. 2). In addition, by measuring the fibrin plate lysis area of each strain to compare the superiority of thrombolysis, at this time, 1 U / ml plasmin, a thrombolytic agent well known as a control group is used. Enzyme protein of the present invention exhibits 1.5 times higher thrombus solubility than the comparative plasmin (see FIG. 1).

In order to investigate the characteristics of the enzyme protein having the thrombolytic ability, the culture supernatant is treated with ammonium sulfate to obtain a precipitate, which is subjected to DEAE-Sephasil column chromatography to isolate and purify the new thrombolytic enzyme protein. As a result, a thrombolytic enzyme protein having a molecular weight of 25,000 Daltons and 64,000 Daltons having excellent thrombolytic ability is identified.

To determine if the enzyme protein of the present invention can be used clinically, its hemolytic activity is examined. As a result, it was confirmed that the Bacillus licheniformis strain producing the enzyme protein does not show any hemolytic activity.

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

The following examples illustrate the invention and do not limit the scope of the invention.

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. Tryptic soy agar medium (17 g / l bacto tryptone, 3 g / l bactosodium, 2.5 g / l bactodextrose, 5 g / l sodium chloride, 2.5 g / l potassium phosphate, 20 g / l bacto agar) and incubated at 37 ° C. for 24 hours to select a single colony It was. Fibrin plate (fibrin plate) measuring method using each of the selected strains in the following process to confirm the thrombolytic ability exhibited by the strain of the present invention was selected strains having excellent thrombolytic ability.

In the present invention, in order to select strains by the fibrin plate measuring method, first prepare a 5ml solution completely dissolved so that the final concentration of fibrinogen (fibrinogen) 1% with 50mM phosphate buffer (pH 7.4), and tryptic soy agar medium 5 ml were mixed. 0.1 ml of 100 NIH U / ml thrombin solution was added to the solution, mixed, and then 15 ml of tryptic soy agar medium was added to the solidified petri dish and left to stand at room temperature for 5 to 10 minutes to solidify. I was. Herein was inoculated strain isolated from the above and incubated for 24 hours at 37 ℃ and 15% trichloroacetic acid (trichloroacetic acid) was added to confirm the clear ring (clear zone) generated.

The strain having a broad transparent ring isolated in the above process was named Bacillus licheniformis DJ-3, and the strain was named July 20, 1996 by the Korea Institute of Science and Technology. Was deposited (Accession Number: KCTC 0266 BP).

Example 2 Electron Microscopy

In order to observe the form of the strain isolated in the present invention, the strain obtained in Example 1 was tryptic soy broth medium (17 g / l Bakto tryptone, 3 g / l Bakto Soyton, 2.5 g / l bactodextrose, 5 g / l sodium chloride, 2.5 g / l potassium phosphate) was incubated at 37 ° C. for 24 hours and then centrifuged to recover and fix the cells. This was photographed at 8,400 magnification using a scanning electron microscope to observe the shape of the strain.

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, 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 anisoisocarbon (anteiso-Cn), which were characteristics found in Gram-positive bacteria. In particular, the total composition of the side chain chain is 0.771, it could be confirmed that the strain of the present invention is Bacillus licheniformis strain of the genus Bacillus (Bacillus sp.).

Further, according to the theory of Kaneda not significantly unsaturated fatty acids or a very few (3%), Arndt iso -C ₁₅ acid (anteiso-C ₁₅ acid) and isophthalic acid -C ₁₅ (iso-C ₁₅ acid), respectively. 26 to Group A (B. alvei, B. brevis, B. circulans, B. licheniformis, B. marcerans, B. megaterium, B. pumilus, B. subtilis, 60-60, 13-30%) It is said to belong. 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, 30.21% and 34.34% Although C ₁₂ and C ₁₈ are present in small amounts of 0.8% and 9.85%, respectively, there are differences in the length of fatty acids. Factor and solved it [Sehgal, SN et al., (1962) Can. J. Biochem. Physiol., 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 to F) of Kaneda.

Example 4 Identification of Enzyme Proteins with Thrombus 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 activity measurement method of thrombolytic enzyme.

Fibrin plates were prepared in the same manner as the fibrin plate measuring method described in Example 1, and then a paper disc was placed on the bone plate. 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, well known as thrombolytic enzyme, was used. As a result, it is confirmed that the enzyme protein of the present invention exhibits 1.5 times higher thrombus solubility than the plasmin used in the comparative group.

Example 5 Isolation and Purification of Enzyme Proteins Having Thrombus Solubility

In order to isolate the enzyme protein having thrombolytic ability of the present invention, the culture solution of the strain obtained in Example 1 was centrifuged at 10,000xg for 20 minutes to obtain a supernatant and treated with 30-80% of ammonium sulfate. The protein precipitate thus obtained was desalted and then passed through a DEAE-Sephacel column (Sigma, USA) to isolate and purify new proteins with thrombolytic ability and to investigate the properties of the isolated enzyme.

SDS-polyacrylamide gel electrophoresis was performed at the same time as the standard protein and its activity was measured. As a result, proteins having molecular weights of 25,000 and 64,000 daltons were isolated and purified (see FIG. 2).

Example 6 Measurement of Hemolytic Activity

In order to confirm the usefulness of applying the new enzyme protein produced by the strain of the present invention to the pharmaceutical or food industry, it was examined whether the strain has hemolytic activity. The Bacillus licheniformis DJ-3 strain selected in Example 1 was placed on a plate, cultured by adding human blood thereto, and observed under a microscope. It can be seen that the strain of the present invention shows no hemolytic activity at all.

The thrombolytic enzyme protein produced by the Bacillus licheniformis DJ-3 strain of the present invention has a thrombolytic ability of 1.5 times or more than the conventional thrombolytic plasmin, so that the pharmaceutical composition comprising the active ingredient is a thrombosis-associated coronary artery disease. In addition, the present invention may be useful for diagnosing and treating thrombosis and cerebrovascular disease.

In addition, the thrombolytic enzyme protein of the present invention can be administered orally as well as vascular injection as compared with 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, it can be developed as a dietary supplement as well as clinically orally administered drugs. In addition, the Bacillus licheniformis strain of the present invention does not show any hemolytic activity, it is easy to use clinically.

The present invention relates to a novel Bacillus licheniformis strain and an enzyme protein having thrombus lytic ability produced by the strain.

More specifically, the present invention relates to a novel Bacillus licheniformis DJ-3 strain having excellent thrombolytic ability isolated from doenjang, a fermented food of Korean tradition, and a thrombolytic enzyme protein produced by the strain. The pharmaceutical composition comprising the strain and the enzyme protein as an active ingredient may be administered orally, and thus may be usefully used as a therapeutic agent and dietary supplement for thrombosis-related diseases.

Claims (2)

New Bacillus licheniformis DJ-3 strain with accessibility to thrombosis isolated from fermented soybean paste (Accession No .: KCTC 0266BP). An enzyme protein having a thrombus soluble capacity of 64,000 Daltons produced by the Bacillus licheniformis DJ-3 strain (Accession No .: KCTC 0266BP) of claim 1.