KR20030084073A - Novel strain bacillus sp. brd-54(kctc 10183bp) and method for preparing thrombolytic enzyme protein from the microorgarnism - Google Patents

Abstract

PURPOSE: A microorganism Bacillus sp. BRD-54 (KCTC 10183BP) and a method for preparing thrombolytic enzyme protein therefrom are provided. The thrombolytic enzyme protein has higher thrombolysis activity. CONSTITUTION: A microorganism Bacillus sp. BRD-54 (KCTC 10183BP) capable of producing thrombolytic enzyme protein is isolated from Korean traditional fermented bean paste, chungkukjang, wherein the thrombolytic enzyme protein has the molecular weight of 29,000 dalton. A method for preparing the thrombolytic enzyme protein comprises culturing the microorganism Bacillus sp. BRD-54 (KCTC 10183BP) in medium, and isolating the thrombolytic enzyme protein from the cultured medium and cells.

Description

Bacillus sp. Mycobacterium strain VR-5-4 (BTCC 129８3XP) and the production method of enzyme protein with thrombolytic ability produced by the strain {NOVEL STRAIN BACILLUS SP. BRD-54 (KCTC 10183BP) AND METHOD FOR PREPARING THROMBOLYTIC ENZYME PROTEIN FROM THE MICROORGARNISM}

The present invention relates to a new strain BRD-54 of Bacillus (KCTC 10183BP) and an enzyme protein having a thrombolytic ability produced by the strain. More specifically, Bacillus sp. BRD-54 (Accession No .: KCTC 10183BP), which produces thrombolytic enzyme protein isolated from Cheonggukjang, a traditional fermented food, and an enzyme protein having thrombolytic ability obtained from the present strain It is about.

Thrombus is the product of metabolic processes that naturally produce and degrade in reactions in vivo and is associated with blood coagulation. Blood coagulation can occur when changes in blood composition occur, such as damage to the blood vessel wall, an increase in the number of platelets in the blood due to a change in blood flow rate after surgery, or an increase in fibrinogen. The production of thrombi begins when platelets adhere to damaged vascular endothelial cells to form fibrin. Drugs for the treatment of blood clots inhibit the formation of blood clots or dissolve the generated blood clots. Drugs that inhibit the formation of blood clots include heparin, ankrod, and warfarin. Plasma dissolving drugs include plasmin. Urokinase, which activates or indirectly dissolves blood clots.

Thrombosis is a substance that occurs as a part of hemostasis, and hemostasis occurs when blood vessel walls are damaged and blood leaks to prevent blood vessels from contracting, narrowing the inner diameter, and coagulating blood by coagulation factors. Hemostasis can be divided into four stages: 1) contraction of blood vessels, 2) thrombus formation due to platelet aggregation, 3) blood coagulation, and 4) proliferation of coagulated fibrous tissue in the blood. In other words, platelets and tissues are collected in collagen exposed from the wound of blood vessels and blood coagulation is started, and fibrous tissue proliferates in this region to block blood vessels. Sometimes. Once the platelets gather around the collagen exposed by the wound, the coagulation of platelets occurs and cohesion of the platelets occurs, freeing and oxidizing the arachidonic acid and reorganizing the platelets to form a blood clot. Many factors are involved in the process of blood clotting and blood clotting.

The product of this blood coagulation process is fibrin, and also in the blood vessels coagulation or blood clot production in vivo, the mechanism of self-degrading it, the plasmin (serine protease) called (serine protease) is involved. Plasmin (plasmin) dissolves fibrinogen or fibrin (fibrin) to inhibit blood coagulation, heparin (heparin) released from the liver exhibits anticoagulant action by inhibiting the action of thrombin.

The cause of thrombus has not yet been accurately identified, but congenital hereditary diseases such as disseminated intravascular coagulation (DIC), changes in physiological function according to aging of the body, and stress are pointed to the cause. Stress caused by overworking in modern people is one of the causes of abnormal thrombus by changing the amount of factors involved in blood clotting. When blood clots are generated in the cerebrovascular system due to bioreaction mechanisms, thrombosis occurs and the body becomes indeterminate. In addition, clogged heart vessels due to blood clots will lead to heart failure or heart failure. Therefore, many studies have been conducted to remove the blood clots, which is an important cause of death.

Many studies have been conducted to remove the blood clots, which are important causes of death, and as a result, plasminogen activators such as streptokinase and urokinase have been found to be useful. ) Has been used for a long time. Clinical trials have demonstrated that these two drugs have a significant effect on thrombolysis when actually used, but side effects such as systemic bleeding have also been reported. Recently, many efforts have been made to develop new thrombolytics that overcome the disadvantages of these existing thrombolytics.

One method for developing new thrombolytics has been an attempt to develop new thrombolytics derived from animals, plants or microorganisms with superior thrombolytic activity from linkages, and the components extracted from ginkgo biloba have been found to facilitate blood circulation. The formulations used were developed and commercialized. Many substances have been reported in animals that are known to dissolve blood clots.A novel plasminogen activator (Gardell, SJ et al., (1991), hemetin isolated from leeches isolated from salivary glands of vampire bats). (hemetin), fibrinolytic enzymes isolated from centipedes. In addition, various thrombolytic substances have been identified in microorganisms, such as Streptokinase isolated from Streptococcus sp. , Staphylokinase isolated from Staphylococcus sp. Fibrinase isolated from brinolase isolated from Aspergillus sp. , Fibrinolysin isolated from Yersinia sp. , And Bacillus sp. fibrinolytic enzymes have been reported as thrombolytics.

It is known that natto, a traditional Japanese food, contains thrombolytic enzymes, and the enzyme was directly isolated and named as nattokinase, and this enzyme is reported to increase the thrombolytic ability in vivo even when administered orally. For that reason, natto is known to be a thrombolytic product in Japan and the sales volume is increasing rapidly as a health food (Sumi, H. et al., 1987).

Therefore, the present inventors have tried to isolate strains and thrombolytic enzymes excellent in thrombolytic activity in Cheonggukjang, a traditional fermented food of our country, isolate strains producing thrombolytic enzymes with excellent thrombolytic activity, and produce new strains from the isolated strains. The present invention was completed by identifying an enzyme protein having thrombolytic ability.

An object of the present invention is to provide a strain that produces an enzyme protein with excellent thrombolytic ability isolated from the traditional fermented food of Korea.

Another object of the present invention is to provide an enzyme protein having a thrombolytic ability of about 29,000 Daltons in molecular weight obtained by culturing the strain and the culture medium and the cells.

Another object of the present invention is to provide a thrombolytic agent using the thrombolytic enzyme protein produced by the strain as an active ingredient.

Another object of the present invention is to provide a dietary supplement that helps thrombolysis, including the thrombolytic enzyme protein produced by the strain.

Figure 1 is a photograph of the degradation results of artificial blood clots by the strain BRD-54 of the genus Bacillus of the present invention.

Figure 2 is an electron micrograph of a strain of genus BRD-54 of the present invention.

Figure 3 is a photograph of the molecular weight of the thrombolytic enzyme protein produced by the strain BRD-54 of the genus Bacillus of the present invention.

According to the first aspect of the present invention, Bacillus sp. BRD-54 strain (Accession No .: KCTC 10183BP), which produces thrombolytic enzyme protein isolated from Chungkookjang, a traditional fermented food, is provided.

According to the second aspect of the present invention, there is provided an enzyme protein having a thrombolytic ability of about 29,000 Daltons in molecular weight obtained by culturing the strain and the culture medium and the cells.

According to the third aspect of the present invention, there is provided a thrombolytic agent using an enzyme protein having thrombolytic ability produced by the strain as an active ingredient.

According to the fourth aspect of the present invention, there is provided a dietary supplement for helping thrombolysis, which comprises an enzyme protein having a thrombolytic ability produced by the strain.

Hereinafter, the present invention will be described in more detail.

The present inventors isolated strains producing enzyme proteins with excellent thrombolytic activity from Cheonggukjang and Traditional Doenjang collected from Anseong, Daejeon, Jeonju, Suncheon, Masan, Chuncheon, etc. in Korea. Isolated strains of the present invention can be found in the Bugis Manual (Peter, HAS et al., Bergey's manual of systemic bacteriology, vol. 2. Baltimore, London) and the Cowan, NR and KJ Steel, Manual of the identification According to of medical bacteria, Cambridge Univ. Press, London, it was determined to be a strain of Bacillus sp. , and the isolated strain was named BRD-54. This strain was deposited with the accession number KCTC 10138BP dated February 22, 2002 at the Korea Research Institute of Biotechnology.

Morphological, culture, physiological and biochemical characteristics of the Bacillus sp. BRD-54 (KCTC 10138BP) as described above are shown in Table 1 below.

As a result of measuring the thrombolytic activity using the strain of the present invention having the above characteristics, the thrombolytic activity of the enzyme protein produced in the strain of the present invention was 2.4 times higher than that of the strain isolated from Japanese natto. Activity was shown (see Examples).

Since the enzyme protein produced by the strain of the present invention has excellent thrombolytic ability, therefore, a pharmaceutical composition such as thrombolytic agent having them as an active ingredient may be provided. For example, such pharmaceutical compositions may be used for the treatment of thrombotic diseases such as coronary artery disease, thrombosis, cerebrovascular disease, and the like.

In addition, according to the present invention may be provided a dietary supplement to help the thrombolysis including the enzyme protein.

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

Example 1 Isolation of Microbial Mycobacteria

The present inventors collected a small amount of samples from Cheonggukjang and traditional doenjang collected from Anseong, Daejeon, Jeonju, Suncheon, Masan, Chuncheon, etc. in Korea, put them in sterile saline solution, shake them, and dilute them by plate dilution. Was smeared on LB agar medium containing 1% skim milk (1.0% by weight of tryptone, 1.0% by weight of sodium chloride, 0.5% of yeast extract, 1-2% by weight of agar, pH 7.0) and After time incubation, dozens of single colonies were first selected. In order to reconfirm the thrombolytic ability of the primary screened strains and to compare their relative thrombolytic enzyme production, each strain was screened for LB culture (1-5 wt% tryptone, 1-5 wt% sodium chloride, yeast). The extract was incubated for 24 hours at 37 ℃ in 0.1 ~ 1.0%, agar 1-2 wt%, pH 7.0) and centrifuged for 10 minutes at 12,000 rpm. 50 μl of the culture supernatant was added to fibrin plates (0.3-0.9 wt% fibrinogen, 100 NIU thrombin, 0.7-1.0 wt% agarose) and reacted for 5 hours at 37 ° C. More than 10 larger strains were secondarily screened (see FIG. 1).

In order to quantify the activity of thrombolytic enzyme protein produced by microorganisms, each strain was 1-5% by weight of soy flour, 1-4% by weight of glucose, 0.1-0.5% by weight of yeast extract, and 0.1-0.5 of potassium phosphate. After 48 hours incubation in a liquid medium consisting of weight%, 0.01 to 0.05% by weight of potassium diphosphate, 0.01 to 0.05% by weight of magnesium sulfate (pH 7.0), centrifuged to remove the cells and obtain a supernatant. Thrombolysis was measured by the following method using a spectrophotometer. That is, 2 ml of 4% alpha-casein solution and 1.6 ml Tris-lysine-sodium chloride-IDT buffer were mixed and 200 µl of the culture supernatant was added. After reaction at 37 ° C. for 30 minutes, the reaction was terminated by adding 15% TCA (Trichloroacric acid) solution, and left at 4 ° C. for 18 hours, and then absorbance was measured at 280 nm. The amount of tyrosine, an amino acid, was measured and thrombolytic activity was calculated by the following equation (Robbin et al, 1966). At this time, the plasmin casein unit (PCU) was defined as 1 unit of the amount of enzyme that releases 450 μg of TCA-soluble tyrosine for 1 hour. Among the isolated strains, one strain producing the enzyme protein having the highest fibrin resolution was selected, named BRD-54, and the strains were identified.

Example 2 Identification of Isolated Microorganisms

Morphological characteristics of the isolate strain are rod-shaped, 0.5-0.7 μm × 2.0-2.5 μm in size, as shown in the electron micrograph of FIG. 1, and are motile, gram-positive, and form endogenous spores. The culture characteristics were aerobic, and catalase production test results were positive, and oxidase production test results were negative (see FIG. 2).

Physiological and biochemical properties of the isolates were tested primarily for the availability of sugars, pH, temperature and stability to salts, and the results are as mentioned in Table 1 above.

As a result of analyzing the morphological, culture, physiological and biochemical characteristics of the microbial microorganisms, the new strain isolated from the present invention (Peter, HAS et al., Bergey's manual of systemic bacteriology, vol. 2. According to Baltimore, London) and Cowan, NR and KJ Steel, Manual of the identification of medical bacteria, Cambridge Univ. Press, London, the strain is considered to be a strain of the genus Bacillus. It was named 54, and it was deposited with the Korea Institute of Bioscience and Technology Genetic Resources Center, a depositary institution for Korean patent strains, and received accession number KCTC 10138BP (February 22, 2002).

Example 3 Isolation and Purification of Thrombolytic Enzyme Protein

BRD-54 in Bacillus, soy flour 1-5%, glucose 1-4%, yeast extract 0.1-0.5%, potassium monophosphate 0.1-0.5%, potassium diphosphate 0.01-0.05%, magnesium sulfate 0.01 The supernatant was taken by incubating for 24 hours at 37 ℃ in a liquid medium consisting of ~ 0.05% by weight (pH 7.0). The separated supernatant was treated with ammonium sulfate corresponding to 50 to 75% by weight to precipitate enzyme protein, and then centrifuged at 9,000 rpm for 30 minutes to discard the supernatant and recover only the precipitate. The obtained precipitate was redissolved in 10 mM phosphate buffer solution whose pH was adjusted to 7.2 and then desalted by dialysis at 4 ° C. for 18 hours. After desalination, an enzyme protein having thrombolytic activity was purified through an alkyl superose column by hydrophobic action and its properties were investigated.

SDS-PAGE electrophoresis was performed at the same time as the standard protein and its activity was confirmed to be a protein having a molecular weight of about 29,000 Daltons (see FIG. 3). (As a marker, lysozyme and carbo as a standard protein (BIO-RAD, USA) Nic anhydrase (carbonic anhydrase), phosphorylase b (phosphorylase b) was used, in Figure 3 lane 1 is the result of electrophoresis in the step of treating ammonium sulfate, lane 2 is an alkyl superrose column (alkyl results of electrophoresis at the stage of purification using a superose column).

Example 4 Thrombolytic Activity of Enzyme Protein Produced by BRD-54 in Bacillus

The thrombolytic enzyme protein produced by the microorganism isolated from natto, a traditional Japanese fermented food, produced by the three microorganisms including BRD-54 of the Bacillus genus. To compare the results of the comparative analysis according to the method of Example 1 shown in Table 2 below.

The microbial strains producing the thrombolytic enzyme protein isolated in the present invention showed 1.7 to 2.4 times higher enzyme activity than the strain isolated from Natto in Japan. Particularly, thrombi of the enzyme protein produced by BRD-54 in Bacillus spp. Solubility was 2.4 times the highest activity.

Comparison of Thrombolytic Activity of Enzyme Produced by Bacillus BRD-54 Test strain Thrombolytic activity (PCU / ml) Bacillus in BRD-54 34,479 Bacillus genus BRD-73 24,331 Bacillus genus BRD-102 25,405 Microbial Strains Isolated from Natto 14,239

Example 5 Measurement of Hemolytic Activity

In order to confirm the usefulness of the new enzyme protein produced by the strain Bacillus genus BRD-54 of the present invention in the pharmaceutical or food industry, an experiment was performed to determine whether hemolytic activity is present in the new strain of the present invention. The strain of BRD-54 in Bacillus was placed on a flake and added to human blood, followed by cultivation and observation under a microscope. As a result, the strain of the present invention had no hemolytic activity. there was.

The thrombolytic enzyme protein produced by the Bacillus genus BRD-54 of the present invention has a thrombolytic ability of 2.5 times or more than the conventional thrombolytic plasmin, so that the pharmaceutical composition having the active ingredient is coronary artery disease, thrombosis It can be useful for diagnosing and treating cerebrovascular diseases.

In addition, the thrombolytic enzyme protein of the present invention can be used orally as well as vascular injection as compared with the conventional thrombolytic agent is administered mainly through the vein has an excellent advantage that can be used in combination with the vascular injection. Therefore, it can be developed as a dietary supplement as well as clinically orally administered drugs. In addition, the Bacillus genus BRD-54 of the present invention does not have a hemolytic activity and is easy to use clinically.

Claims (4)

Bacillus sp. BRD-54 strain (Accession No .: KCTC 10183BP) which produces Bacillus sp. BRD-54, which produces thrombolytic enzyme protein isolated from Chungkookjang, a traditional fermented food. Enzyme protein having a thrombolytic ability of about 29,000 Daltons in molecular weight obtained from the culture medium and the bacterium BRD-54 strain (KCTC 10183BP). Thrombolysis using the enzyme protein of claim 2 produced by Bacillus strain BRD-54 (KCTC 10183BP) as an active ingredient. A dietary supplement for thrombolysis, comprising the enzyme protein of claim 3 produced by a strain of BRD-54 of the genus Bacillus (KCTC 10183BP).