KR970000593B1 - New strain bacillus sp.ck11-4 for melting of a thrombus - Google Patents

Abstract

New strain Baccilus sp. CK 11-4(KCCM-10042) having a melting capaci- ty of thrombus is separated from fermented soybeans. For example, Baccilus sp. CK 11-4(KCCM-10042) is inoculated in 10 ml of a culture media(Bacto-beef extract 3g/L, Bacto-peptone 5g/L) and fermented at 37 deg.C for 20 hr in a water bath by stirring. 0.5mL of The fermented solution is inoculated in 1L of medium(pH 7), fermented at 37 deg.C for 20 hr in a water bath by stirring, separated from a biomass, and filtrated through a filtre of 0.2 microns. Thrombus melting activity shows 1.84 U/mL.

Description

New strain Bacillus sp. CK 11-4 (KCCM 10042), which produces thrombolytic enzymes

The present invention is a genus Bacillus genus strain that produces an enzyme with excellent lytic ability isolated from Cheonggukjang ( sp.) CK 11-4 (KCCM 10042).

The thrombus converts fibrinogen into fibrin by thrombin activated by a complex blood cascade mechanism in vivo during wound repair. At this time, the generated fibrin forms a polymer with each other, thereby creating a thrombus. Small wounds, etc. occur in vivo, and the generated blood clots circulate after the wound repair. In vivo, blood coagulates and dissolves at all times, but due to various causes, the balance is broken, and blood clots accumulate in fine blood vessels, especially cerebrovascular vessels, which block blood circulation, thereby providing a smooth supply of nutrients and oxygen to each tissue. Stops and causes an increase in blood pressure.

In other words, if blood clots are generated in the blood vessels of the brain, cerebellar thrombosis occurs. If the cardiovascular is blocked, heart failure or heart attack can cause death.

According to a recent report on mortality trends of modern people, the total number of deaths due to vascular disorders ranks first among about 40% of all deaths, and active research in basic and clinical areas is in progress.

The trend of research and development for the prevention and treatment of these diseases is focused on the development of antithrombotic agents that inhibit the production of thrombi and the development of thrombolytic agents that dissolve the generated thrombi.

Antithrombicides commonly used in clinical practice include organic synthetic coumarin and warfarin. Natural substances include heparin extracted from bovine heart, swine intestine, and leech. There are hirudin preparations, and thrombolytics are streptokinase, urokinase, and tPA (tissue type plasminogen activator), which was developed by gene manufacturing technology in 1987 by Genetech of the United States. There is).

In vivo, there is a plasmin (plamin) that dissolves blood clots, which are formed from the precursor plasminogen. Most of the thrombolytics currently being studied are plasminogen activating factors that convert these plasminogens into plasmin.

However, urokinase and the like have a problem of being known to cause many side effects such as intestinal bleeding by degrading other proteins in the blood due to low substrate specificity.

In addition to such vascular injectable tissue plasminogen activating factor (TPA) has been beginning to interest in the preparation for increasing the thrombolytic ability in the blood by direct oral administration for use in parallel therapy, but the results are still insignificant. Sumi et al. (Bioindustry, 7.11.12 ~ 18, 1990; Japanese Brewery Association Magazine 85. 518 ~ 524, 1990) reported that the ingestion and absorption of urokinase in the intestine significantly increased the thrombolytic activity in the blood. Drew attention.

In other words, it is suggested that the thrombolytic enzyme is likely to increase the thrombolytic activity by reporting the urokinase absorbed by the intestine and enhancing the synthesis of enzymes that dissolve the blood clots, suggesting that the intestinal application of thrombolytic enzymes is possible.

In addition, the development of thrombolytic activity in the body is increased when ingesting the traditional Japanese fermented food, NATO, and it has been reported that the thrombolytic ability has been increased by separating the thrombolytic enzyme from the NATO and intestinal administration. By attracting attention in that it can treat and prevent various vascular diseases. To date, there are few reports except that food intake increases thrombolytic ability.

Therefore, the present inventors have focused on the fact that there is a strain with excellent thrombolytic ability other than Nato from Korea's Cheonggukjang, since Korea has a traditional food, Cheonggukjang, which has been fermented and consumed with soybeans as well as Japan's NATO. As a result of the research and efforts, the present invention was completed by separating the strain of the present invention, which produces an enzyme having much better thrombolytic ability than nattokinase and having excellent substrate specificity for blood clots.

The present invention is a genus of Bacillus sp., A strain that produces enzymes with excellent thrombolytic ability isolated from Korean traditional fermented food, Cheonggukjang. sp.) To provide CK 11-4 (KCCM 10042).

Hereinafter, the present invention will be described in detail.

The present invention is a genus of Bacillus sp. sp.) CK 11-4 (KCCM 10042).

Referring to the present invention in more detail as follows.

The present invention is a genus Bacillus genus strain that produces enzymes with excellent thrombolytic ability isolated from the traditional fermented food Cheonggukjang ( sp.) As regards CK 11-4 (KCCM 10042), the new strain of the present invention is isolated from Cheonggukjang of Mokpo, Jeollanam-do, and produces a strain with the highest thrombolytic activity. of Systermatic Bacteriologg vol. 1) Bacillus by index. The strain belonging to the genus was identified and named Bacillus CK 11-4.

This strain was deposited with KCCM on August 26, 1993 with Accession No. KCCM 10042.

Genus Bacillus sp.) Strain characteristics such as morphological characteristics for CK 11-4 (KCCM 10042) are as follows.

Morphological characteristics

-Type: Simple, 1.0 ~ 1.1μ × 5.6 ~ 6.5μ

Mobility: positive

Gram Dye: Positive

Spore: positive

Culture characteristics

-Juicy agar plate culture: growth nurturing, mucus secretion, off-white crack

-Growth pH: 5 ~ 8

-Growth temperature: 25 ~ 45 ℃

Physiological features

Starch Hydrolysis: Positive

Casein hydrolysis: positive

Gelatin hydrolysis: positive

Carboxy methyl cellulose hydrolysis: positive

Pectin hydrolysis: positive

Pullulan hydrolysis: negative

Catalase: positive

Β-galactosidase: positive

Arginine dihydrolase: positive

Lysine decarboxylase: positive

Ornithine decarboxylase: positive

Hydrogen sulfide generation: negative

Nitric acid generation: negative

Nitrogen reduction: negative

Ureaase positive

Acetoin production: positive

Oxidase: positive

Tryptophane deamidase: negative

Availability of sugars

Glucose: positive

Mannitol: positive

Inositol: negative

Sorbitol: positive

Rhamnose: Voice

Sucrose: positive

Melibiose: negative

Amygdalin: positive

Arabinoses: positive

Morphological and biochemical characteristics of the CK 11-4 strains isolated from Cheonggukjang were investigated to identify the CK 11-4 strains. The isolates were bacillus, motility, aerobic (catalase), and Gram-positive bacteria. It is observed to form spores, so the genus Bacillus ( sp.) was identified as a microorganism.

These strains of the genus Bacillus is harmless to the human body has the advantage that can be consumed by mass production of enzymes.

As described above, the genus Bacillus genus of the present invention ( sp.) CK 11-4 (KCCM 10042) produces enzymes with excellent thrombolytic ability and can be very useful for treating vascular diseases.

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

EXAMPLE

Strains were isolated from various Cheonggukjangs sold in Korea as follows. One platinum solution was taken from each sample, plated in nutrient agar medium, incubated for 24 hours, and a single colony was separated and named CK 1-1 to CK 23-4. Each bacterium was inoculated with 5 ml of nutrient medium, one platinum each, and shaken at 37 ° C. for 24 hours, followed by centrifugation. The thrombolytic activity test method is as follows.

Dissolve the fibrinogen in a 10 mM phosphate buffer solution (pH 7.8, 0.15M NaCl) to 0.3%, and 5 mL of agarose solution to 5 mL of completely dissolved fibrinogen is added to the same buffer solution. After adding 0.1 mL of thrombin (100 NIH / mL) again, the mixture was immediately poured into the saare, left to stand at room temperature for 5 to 10 minutes to solidify, and 7 holes per one saare were made into a fibrin plate (plage). 5 μL of each sample was taken, injected into the sample hole of the fibrin plate, and reacted at 37 ° C. for 18 hours. As a control, plasmin (1.0 U / mL), a purified thrombolytic enzyme, was used and thrombolytic activity was calculated by the following method.

Thrombolytic activity (%) = (sample lysis zone / plasmin lysis zone) × 100

As a result, the bacterium isolated from Cheonggukjang of Mokpo, Jeollanam-do, produced the enzyme with the highest thrombolytic activity and was determined to belong to the genus Bacillus according to the Burgess manual. The strain of the present invention was developed by depositing with the spawn association (KCCM) (KCCM 10042).

Example 1

[Preparation of Coenzyme Solution with Thrombolytic Activity]

The present invention inoculate Bacillus CK 11-4 strain (KCCM 10042) to 10 mL of nutrient medium (Bacto-Beef Extract 3g / L, Bactopeptone 5g / L) and shake in a water bath at 37 ° C. for 20 hours. After incubating with seed, inoculate 0.5 mL of seed culture solution into 1L nutrient medium (pH 7.0), incubate for 20 hours while stirring at 500rpm at 37 ℃, centrifugation to remove cells, and pass through 0.2㎛ filter. I was. The culture solution was measured by thrombolytic activity by the activity measurement method described above, and is shown in Table 1 below. As a result, when 1.0 U / mL of plasmin was used as a control, the enzyme showed 1.84 U / mL, showing the highest thrombolytic activity among the enzymes produced by the isolates.

Two times of acetone was added to the culture, and the mixture was allowed to stand at 4 ° C for 24 hours to precipitate protein in the culture. Thereafter, the supernatant was removed by centrifugation, and the remaining precipitate was frozen at -70 ° C and dried in vacuo. In this way, the coenzyme having thrombolytic ability could be easily prepared from the culture solution of CK 11-4 strain (KCCM 10042) in Bacillus.

Example 2

[Comparative experiment on specificity of substrate reaction with conventional nattokinase]

In order to investigate the thrombolytic ability of the coenzyme prepared in Experimental Example 1 in more detail, the substrate specificity of the proteolytic enzymes including the synthetic substrate of plasmin, the thrombolytic enzyme, was investigated. The results compared with the substrate specificity of the found nattokinase are shown in Table 2 below.

The reaction mixture used in the experiment was used 20mL coenzyme solution, 5 × 10 ^-4 M substrate and 0.1M acid chlorine solution. In the case of nattokinase, the reaction was carried out at 37 ° C. for 10 minutes, and the enzyme was reacted for 5 minutes, and the absorbance was measured at 450 nm for ρ-nitroaniline. Decided. The hydrolytic results were expressed as the amount of nattokinase or hydrolyzed substrate per minute per mL of this enzyme. The values in parentheses represent the activity of the plasmin as a percentage of the activity of the other substrate when the activity of the synthetic substrate is 100. Each value represents the average value of three repeated tests.

* It is recorded in the literature of Sumi [H. Sumi et al. Experientia 43. 1110 ~ 1111) (1987)

(1) Plasmin synthetic substrate

(2) trypsin synthetic substrate

(3) thrombin synthetic substrate

(4) Eurokinase Synthetic Substrate

As shown in Table 2, the coenzyme prepared from the isolate strain Bacillus CK 11-4 (KCCM 10042) was similar to the conventional nattokinase investigated by Sumi et al. In that it did not react at all to the synthetic substrate of urokinase. Synthetic substrates of soluble plasmin showed much higher substrate reaction specificities than nattokinase.

In addition, nattokinase showed high reactivity with trypsin synthetic substrate, but the enzyme secreted by the strain did not react with trypsin synthetic substrate at all.

Compared to the activity of nattokinase on various synthetic substrates, the enzyme produced by this strain showed high activity on plasmin substrate but weaker activity on nattokinase on other substrates. Therefore, the genus Bacillus isolated in the present invention ( sp.) CK 11-4 strain (KCCM 10042) was found to produce a specific synthesis of enzymes that show a very high substrate response specificity for the synthetic substrate of plasmin to dissolve blood clots in the human body.

Claims (1)

The genus Bacillus, which produces enzymes with thrombolytic activity, sp.) CK 11-4 strain (KCCM 10042).