KR100737736B1 - Novel Bacillus sp. WRD-2 isolated from soil and fibrinolytic agent composition comprising extracellular protease produced by the WRD-2 isolated from soil as an effective component - Google Patents

Abstract

The present invention relates to a novel Bacillus sp. WRD-2 ( Bacillus sp. WRD-2, Accession No. KCTC 0869BP) isolated from soil, and a thrombolytic composition comprising an extracellular protease produced by the strain as an active ingredient. Soil samples were collected from the soil, and 30 strains of at least 2 cm in size of proteolytic enzymes were selected. Among them, WRD-2 was isolated from Bacillus, the strain having the highest enzyme activity. And the enzyme activity is rapidly increased from 6 hours of culture, the best 12 to 15 hours, cell growth according to the carbon source is the best in the medium with galactose and the enzyme activity is the best in the medium with 3% maltose, Cell growth and enzyme activity according to nitrogen source were the best in the medium containing yeast extract. Especially, enzyme activity was relatively high when 4% of yeast extract was added. Best to thrombolytic agent composition as the extracellular proteinase which the Bacillus WRD-2, and the strain of the present invention as an active ingredient in the production ability is excellent thrombolysis.

Bacillus genus WRD-2, protease, enzyme activity, thrombolytic composition, thrombolytic ability.

Description

Thrombolytic composition using novel Bacillus genus QR-2 isolated from soil and extracellular protease produced by this strain as an active ingredient {Novel Bacillus sp. WRD-2 isolated from soil and fibrinolytic agent composition comprising extracellular protease produced by the WRD-2 isolated from soil as an effective component}             

1 is a photograph of a degradation ring showing the activity of WRD-2 derived thrombolytic enzymes,

2 is a graph showing cell growth and protease activity of WRD-2 according to the incubation time;

3 is a graph showing the cell growth and protease activity of WRD-2 according to the initial pH change,

4 is a graph showing the cell growth and protease activity of WRD-2 according to the carbon source,

5 is a graph showing the cell growth and protease activity of WRD-2 according to the nitrogen source,

6 is a graph showing the enzyme activity according to the pH of WRD-2 derived protease,

7 is a graph showing the enzyme activity according to the temperature of WRD-2 derived protease,

Figure 8 is a graph showing the size of the degradation ring with time for measuring the thrombolytic ability of WRD-2.

The present invention relates to a novel Bacillus sp. WRD-2 ( Bacillus sp. WRD-2) isolated from soil, and to a thrombolytic composition comprising an extracellular protease produced by the strain as an active ingredient. Specifically, WRD-2 and the strains of Bacillus sp. Isolated from the soil sample by first selecting 30 strains, culturing in an optimal medium for measuring the activity of protease, and then examining the enzyme activity. It relates to a thrombolytic composition comprising an extracellular protease which is produced as an active ingredient.

With the development of the modern industrial society, the use of various enzymes is rapidly increasing, and the proteolytic enzymes are increasing in various fields of manufacturing industry such as protein chemistry, food, medicine and environment. About 60% of the total amount of serine (-alkaline) protease is known to be a very important enzyme protein in the fields of leather and tax industry (Ward. OP, 1985, Proteolytic Enzymes, in Comprehensive Biotechnology 1st ed. by MY Murray, Pergamon press, 789-792).

The genus Bacillus is one of the microorganisms that have already been researched and has different species to grow according to alkaline and neutral conditions, and they produce different enzymes (Horikoshi, K., 1971, Production of alkaline enzymes by alkalophilic microorganism. part 1. alkaline protease produced by Bacillus No.221 Agr Biol Chem 35:....... 1407 - 1414; Manachini, PL , etc., 1988, Themostable alkaline protease produced by Bacillus thermoruber a new species of Bacillus Appl Microbiol Biotechnol. 28 : 409-413; Tsuru.D., H. et al., 1966, Studies on bacterial protease.Part 16.purification crystallization and some enzymic properites of alkaline protease of Bacillus subtilis var. Amylosacchariticus.Agr . Biol. Chem . 30 : 1261 1268) is used in a variety of industries, in particular Subtilisin Calsberg from Bacillus subtilis (Guntelber, AV, M. Ottesen., 1952, Preparation of crystals containing th e plakalbumin forming enzyme from Bacillus subtilis.Nature (London) 170: 802), Subtilisin BPN (Smith, EL et al., Markland. 1968. Subtilism Calsberg. V. The complete sequence: comparison with subtilisin BPN: evolutionary relationship. J. Biol. Chem. 243 : 2184-2191), Subtilisin Novo (Ottesen, M., A. Spector., 1960, A comparison of two proteinases from Bacillus subtilis . CR Trav. Lab. Calsberg. 32 : 63-74).

Fibrinolytic enzymes are a type of proteolytic enzyme that dissolves fibrin. Under normal conditions, fibrin production and degradation are properly regulated by thrombin and plasmin in vivo. However, when their balance is broken and blood clots adhere to and accumulate on the walls of blood vessels, blood vessels are narrowed, or further, they invade and close microvascular vessels such as cerebrovascular vessels along the bloodstream, causing various circulatory diseases. Since our body maintains metabolism by supplying nutrients and oxygen to each tissue through blood vessels, smooth flow of the circulatory system is very important for healthy living.

At present, thrombotic adult disease is increasing in Korea due to westernization of diet and excessive intake of fat. In Korea, the highest number of deaths due to circulatory diseases (hypertensive disease, cerebrovascular disease, ischemic heart disease) is 24.6%, followed by 21.7% of neoplasms (various cancers, leukemias, etc.). The death toll from accidents is reported at 14.5% (Population Analysis. 1997. Statistics of Causes of Death in 1996. National Statistical Office. Korea). In particular, the cerebrovascular disease of the circulatory disease is reported as the first cause of death in the 50s or older age bar This circulatory disease may be referred to as senile disease. Given the aging social structure and the fact that 60% of senile dementia patients are caused by blood clots, the mortality rate from these circulatory diseases is unlikely to decrease. Therefore, the countermeasures against such circulatory diseases are an important problem that cannot be overlooked, and thus the importance and necessity for the development of thrombolytics have emerged.

Recently, researches on producing thrombolytic enzymes from traditional fermented foods have been conducted in Korea. Kim et al., Bacillus sp . It was remove the fibrinolytic enzyme from strain CK 11-4 (Kim, WK, etc., 1996, Purification and Characterization of a Fibrinolytic Enzyme Produced from Bacillus sp. Strain CK 11-4 Screened from Chungkook-jang, Appl. Environ. Microbiol., 62 : 2482-2488), it is reported that this enzyme is very stable to heat and has strong thrombolytic properties. In addition, seaweed etc. were isolated from Bacillus sp . The production and characterization of new thrombolytic enzymes using KA 38 has been reported (Kim, HK et al., 1997, Purification and Charaterization of a Novel Fibrinolytic Enzyme from Bacillus sp . KA38 Originated from Fermented Fish.J. Ferment.Bioeng . , 84: 307-312). However, there have not been any reports on the superior thrombolytic strains isolated from the soil in Korea.

The inventors of the present invention have been made in view of the above points by selecting strains excellent in the activity of protease from the soil, by determining the culture conditions that can further increase the activity of the enzyme, by measuring the thrombolytic ability accordingly The present invention has been completed.

Accordingly, it is an object of the present invention to provide a novel thrombolytic composition having excellent thrombolytic activity using the novel Bacillus genus WRD-2 isolated from soil and extracellular protease produced by the strain.

The object of the present invention is to collect the soil as a sample to select 30 strains of the size of the degradation ring of the proteolytic enzyme more than 2cm and then finally select the strains with the highest enzyme activity microbiologically identified, incubation time This was achieved by measuring cell growth and enzyme activity according to pH, carbon source and nitrogen source, and enzyme activity and thrombolytic activity of crude enzyme solution according to pH and temperature.

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

Example 1 Isolation and Identification of Strains Used

Samples were collected from paddy and field soils of Gimhae-si, Gyeongnam, in order to select strains producing protease with excellent enzyme activity. After properly diluting the sample with sterile water, skim milk agar medium (skim milk agar medium: skim milk 5g / ℓ, bactotryptone 10g / ℓ, yeast extract 5g / ℓ, NaCl 5g / ℓ, bactoagar 15g / ℓ) Among the strains produced by incubation for 15 hours at 30 ° C. incubator at 30 ° C., strains with relatively large clear zones or halo formed by digestion of the skim milk by extracellular protease were selected first. After that, 30 strains of which the diameter of the degradation ring was 2 cm or more were selected secondarily. Among the second screened strains, the best strain was named WRD-2. 1 is a photograph of a degradation ring showing the activity of WRD-2 derived thrombolytic enzymes. In order to maintain extracellular protease activity, it was used while subcultured in skim milk medium.

The isolated strain WRD-2 was positive in Gram staining, spores were present in spore staining and had a circular colony.

For identification of WRD-2, it was identified as API 50CHB kit by the Biotechnology Research Center Gene Center and identified as Bacillus. Bacillus sp. It was named WRD-2 and deposited with the institution under accession number KCTC 0869BP on 2 October 2000. The physiological characteristics of the microbial strains of the present invention are shown in Table 1.

Physiological Characteristics of WRD-2 of the Genus Bacillus 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 + Glycogen + D-Mannose + Xylitol - L-Sorbose - β-Gentibiose + Rhamnose - D-Turanose - Dulcitol - D-Lyxose - Inositol + D-Tagatose - Manitol + D-Fucose - Sorbitol + L-Fucose - α-Methyl-D-mannoside - D-Arabitol - α-Methyl-D-glucoside + L-Arabitol - N-Acetyl glucosamine - Gluconate - Amygdaline + 2-Keto gluconate - Arbutine + 5-Keto gluconate - Esculine +

Example 2: Cell growth and enzyme activity according to incubation time

In the following Examples, LB (Luria-Bertani) medium (Bactotryptone 10g / L, yeast extract 5g / L, NaCl 5g / L) was used as a medium to examine the culture conditions of the strain WRD-2 of the present invention. Cell growth was measured at 660 nm using a spectrophotometer (HP 8452A) at 3 hours intervals using LB medium. On the other hand, quantitative analysis of protein was performed by Lowry, OH et al., 1951, Protein Measurement with the folinphenol reagent.J. Biol. Che m. 193: 265-275 using Bovine serum albumin as a standard protein. Was measured according to

In order to measure cell growth and protease activity according to the incubation time, 3% of WRD-2 preculture was inoculated in 100 ml of LB medium and incubated at 30 ° C at 180 rpm for 2 hours. Cell growth and enzyme activity were measured. The enzyme activity was measured by the method of Hagihara et al. (Hagihara, B., H. et al., 1958, Crystalline bacterial proteinase. 1. Preparation of crystalline proteinase of Bacillus subtilis . J. Biochem . 45 : 185 -194). That is, 1 mM CaCl ₂ and 0.6% skim milk casein were mixed in 50 mM sodium borate-NaOH buffer (pH 10.4) and used as a substrate solution.The reaction was performed after mixing 0.5 ml of substrate solution and 0.1 ml of enzyme solution. It carried out for 10 minutes at ° C. Thereafter, the reaction of the reaction solution was stopped using an ice bath, and 0.5 ml of 5% trichloroacetic acid was added to the reaction solution, which was allowed to stand at room temperature for 20 minutes, followed by centrifugation at 10,000 × g for 10 minutes. Absorbance was measured at 280 nm. One unit (U) of enzyme activity was determined by the amount of enzyme required to increase the absorbance 0.002 per minute.

The results of investigating cell growth and protease activity of each strain of WRD-2 according to the present invention are shown in FIG. 2. Cell growth began to increase rapidly from 6 hours of cultivation, the highest at 15 hours of cultivation, and then decreased again. The results were similar to the cell growth and protease activity according to the incubation time of the WRD-1 genus previously isolated from Bacillus (OK M. et al., Characterization of Extracellular protease of Bacillus sp.WRD-1 Isolated from Soil.Kor . J. Appl Microbiol.Biotechnol.vol 28. No 6. 329-333 (2000)). In view of the above results, the results after 15 hours of incubation were adopted in the experiment.

Example 3: Cell Growth and Protease Activity According to Initial pH Change

After adjusting the initial pH of the medium to 4-10, 3% each of the WRD-2 preculture was inoculated and incubated at 30 ° C. for 15 hours, and then cell growth and protease activity were measured. The results are shown in FIG.

When the initial pH was adjusted to 5-9, cell growth was generally good, but the enzyme activity started to increase from pH 4 to maximum at pH 6, and when the initial pH was adjusted to 6-8, cell growth and protease activity were observed. This was up.

Example 4: Cell Growth and Protease Activity According to Carbon Sources

Glucose, maltose, sucrose, lactose and galactose were added to the LB medium at 3% concentrations, and cultured for 15 hours with shaking at 180 rpm at 30 ° C., and cell growth and protease activity were measured. The results are shown in Table 2 below.                     

Activity of Cell Growth and Protease Activity According to Carbon Source of WRD-2 of the Genus Bacillus Carbon source Cell growth (absorbance at 660 nm) Protease Activity (U / mL) Glucose 1.5048 37.92 Sucrose 1.4377 33.55 Maltose 1.9754 64.02 Galactose 2.4788 45.32 Lactose 2.2741 28.41

Cell growth according to the carbon source used was the best when using galactose, the enzyme activity was the highest with 64.02U / ㎖ using maltose. Considering that the activity of protease was the highest when maltose was used as a carbon source, the concentration of maltose was 0%, 1%, 2%, 3%, and 4%, followed by proteolysis after 15 hours incubation at 30 ° C The activity of the enzyme was measured. As a result, when the concentration of maltose was 3%, the activity of protease was the highest compared to other concentrations. The results are shown in FIG.

Example 5: Cell Growth and Protease Activity According to Nitrogen Sources

Instead of 1% tryptone as a nitrogen source in LB medium, the same concentration of peptone, yeast extract, malt extract, NaNO ₃ , (NH ₄ ) ₂ SO ₄ , NH ₄ Cl, casein, and soybean was replaced at 180 ° C at 30 ° C. After incubation for 15 hours with shaking, cell growth and enzyme activity were measured. The results are shown in Table 3.

Cell Growth and Protease Activity According to Nitrogen Sources of WRD-2 of the Genus Bacillus Nitrogen source Cell growth (absorbance at 660 nm) Protease Activity (U / mL) Big head 2.5293 82.16 Malt Extract 2.0473 45.43 peptone 1.9754 45.25 Yeast extract 2.6183 91.88 casein 2.3713 80.20 (NH ₄ ) ₂ SO ₄ 0.7317 20.88 NH ₄ Cl 1.0840 11.02

Cell growth and protease activity were generally good when using complex nitrogen sources. In particular, cell growth was highest in the medium containing yeast extract, and the enzyme activity was the highest as 91.88 U / mL in the medium containing yeast extract. The enzyme activity was relatively low when the nitrogen source in the ammonia form was used, which is the same as reported by Himelbloom et al. (Himelbloom, BH and HM Hassen. 1986. Effect of cysteine on growth, protease production, and catalase activity of Pseudomonase fluorescens) Appl.Environ.Microbiol . 51: 418-421).

Considering that yeast extract was the highest when protease activity was used as a nitrogen source, the concentration of yeast extract was 0%, 1%, 2%, 3%, 4%, 5%, and cultured for 15 hours at 30 ° C. The activity of the protease was then measured. As a result, when the concentration of yeast extract was 4%, the activity of protease was the highest compared to other concentrations. The results are shown in FIG.

Example 6 Preparation of Coenzyme Solution and Enzyme Activity of Coenzyme Solution According to pH and Temperature

In order to measure the activity of WRD-2-derived protease according to pH and temperature, 3% of the preculture was inoculated into 200 ml of LB liquid medium and incubated at 180 ° C. for 15 hours at 30 ° C. To prepare a crude enzyme solution from this culture solution, the culture solution was centrifuged at 10,000 x g for 20 minutes, and then the supernatant was taken as a crude enzyme solution. In order to determine the enzyme activity of the crude enzyme solution according to pH, 0.02M citric acid-sodium phosphate dibasic buffer to adjust the pH to 4.0 to 7.0, to adjust the pH to 7.0 to 8.0 0.02M sodium phosphate monobasic sodium phosphate dibasic buffer 0.02M Tris-HCl buffer to adjust pH 8.0 to 9.0, 0.02M to adjust pH 9.0 to 11.0 Glycine-NaOH buffer was used. 0.5 ml of the casein (w / v) substrate solution for each pH concentration was mixed and reacted at 37 ° C. for 1 hour, and then enzyme activity was measured. In order to measure the enzyme activity of the crude enzyme solution according to the temperature change, the pH was adjusted to pH 9.0 having the optimum enzyme activity of the WRD-2 crude enzyme solution, and then mixed with the crude enzyme solution and 0.6% casein (w / v) substrate solution. Enzyme activity was measured by reaction at 20 ° C, 30 ° C, 40 ° C, 50 ° C, 60 ° C and 70 ° C. The results are shown in FIGS. 6 and 7.

In the relatively wide pH range of pH 6-9, the activity of the protease was 70% or more, and the reaction optimum pH was 6. Therefore, it was found that the protease of the crude enzyme solution was neutral. On the other hand, the crude enzyme solution showed a high enzyme activity of 80% or more at 20 ~ 60 ℃, the optimum reaction temperature was 40 ℃.

Example 7: Confirmation of thrombolytic ability

In order to confirm the thrombolytic ability of the strain of the present invention, WRD-2, the culture solution was centrifuged and diluted in a ratio of 7: 3 with Tris buffer (pH 7.2, 20 mM). 50 µl and 100 µl of this enzyme dilution were dropped onto a fibrin plate prepared in advance, and reacted for 4 hours at 37 ° C. in an incubator, and the degradation area was measured from the diameter of the degradation ring. When the enzyme solution was dropped on the fibrin plate, the enzyme solution was dropped on a 8 mm diameter paper disk.

Fibrin plates were prepared by administering thrombin to fibrinogen. That is, 0.06 g of fibrinogen was added to 10 ml of 0.1 M borate buffer (pH7.5), and the mixture was left to stand at 37 ° C. for 2 hours to completely dissolve. Dissolved fibrinogen was dispensed in a petri dish by 10ml, and 40ml of thrombin (5,000units, manufactured by SIGMA, USA) per 10ml of fibrinogen was gently shaken up, down, left and right so as to spread evenly over the petri dish (Astrup, T., Mullertz, S., The fibrin plate method for estimating fibrinolytic activity.Arch Biochem. Biophys., 40 , 346 (1952)). The results are shown in FIG.

The thrombolytic ability of WRD-2 using fibrin plates tended to increase proportionally to the area of the dissociated rings formed at two-hour intervals up to eight hours, such as Lee Si Kyung et al., 1998, Medium Optimization for Fibrinolytic. .... enzyme Production by Bacillus subtilis KCK-7 Isolated from Korean Traditional Chungkookjang Kor J. Appl Microbiol Biotechnol 26:. 226 - 231) and roads, etc. (Kil Ji Oeun, etc., 1998, Production and Characterization of Fibrinoloytic enzyme: Optimal Codition for Production of the Enzyme from Bacillus sp.KP -6408 Isolated from Chungkook-jang.J . Korean Soc.Food Sci.Nutr . 27 : 51-56 ).

As described above, the Bacillus genus WRD-2 of the present invention isolated from the soil as described through the above example has very high thrombolytic ability by producing protease having a high enzyme activity of 90% or more at 20-40 ° C. at a relatively low temperature. Therefore, the thrombolytic composition comprising the WRD-2 and the extracellular protease produced by the strain as an active ingredient is a very useful invention in the pharmaceutical industry.

Claims (2)

Novel Bacillus sp. WRD-2 ( Bacillus sp. WRD-2, KCTC 0869BP) isolated from soil producing extracellular protease. A thrombolytic composition comprising a supernatant obtained by centrifuging Bacillus sp. WRD-2 (KCTC 0869BP) strain culture medium isolated from soil as an active ingredient.

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