• Proceedings of the Korean Society of Life Science Conference
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• 2001.06a
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• pp.67-86
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• 2001

A strain producing strongly fibrinolytic enzyme was isolated from soil and was identified to be Bacillus subtilis by biochemical and physiological characterization. The optimal culture conditions for the production of fibrinolytic enzyme was determined to be 1.0% tryptone, 1.5% soluble starch, 0.5% Peptone, 0.5% NaCl, $(NH_{4})_{3}PO_4.3H_{2}O, and MgSO_{4}.7H_{2}O.$ Initial pH and temperature were pH 8.0 and $30^{\circ}C$ , respectively, The highest enzyme production was observed at 30 hours of cultivation at $30^{\circ}C$ The fibrinolytic enzyme was purified to homogeneity by DEAE Sephadex A-50 ion exchange column chromatography, 70% ammonium sulfate precipitation, Sephadex G-200 and G-75 gel filtration column chromatography. The molecular weight of the purified enzyme was 28,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A gene encoding the fibrinolytic enzyme was cloned into a plasmid vector pBluescript, transforming E.coli XL-1 Blue. The clone was able to degrade fibrin, This indicated that the gene could encode a fibrinolytic enzyme. The nucleotide sequence of the 2.7 kb insert was determined in both direction. One open reading frame composed of 1023 nucleotides was found to be a potential protein coding region. There was the putative Shine-Dalgano sequence and TATA box upstream of the open reading frame. The homology search data in the genome database showed that both the 2.7 kb insert and 1 kb open reading frame carried no significance in the nucleotide sequence of known fibrinolytic enzyme from Bacillus serovars. The recombinant cell harboring the novel gene involved in fibrinolysis was subjected to protein purification. The molecular mass of the purified fibrinolytic enzyme was determined to be 31864 Dalton, which was highly in accordance with the molecular mass(33 kDa) of the fibrinolytic gene deduced from the insert. The fibrinolytic enzyme was Purified 50.5 folds to homogeneity in overall yield of 10.7% by DEAE Sephadex A-50 ion exchange, 85% ammonium sulfate precipitation, Sephadex G-50, Superdex 75 HR FPLC gel filtration. In conclusion, a novel fibrinolytic gene from Bacillus subtilis was identified and characterized by cloning a genomic library of Bacillus subtilis into pBleuscript. For the soybean fermented by this strain, it is found that there increased assistant protein about 20% compared to the soybean not fermented and increased about 30% according to amino acid analysis and, in particular, essential amino acid increased about 40%. When keeping this fermented soybean powder at room temperature for about 70days, it showed very high stability maintaining almost perfect activity and, therefore, it gave us great suggestion its possibility of development as a new functional food.

• Preventive Nutrition and Food Science
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• v.11 no.2
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• pp.127-132
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• 2006

Bacillus sp. strain K-l, which produces a strong fibrinolytic enzyme, was isolated from chongkukjang, a traditional Korean fermented soybean paste. The fibrinolytic enzyme was purified from chongkukjang base by using ammonium sulfate fractionation and chromatographic techniques. Purified enzyme, CK K-1 was demonstrated to be homogeneous by SDS-PAGE and isoelectric focusing electrophoresis, and has molecular mass of a 12.4 kDa and a pI of 8.0. The optimal reaction pH value and temperature were 8.0 and $40^{\circ}C$, respectively. Phenyl-methyl-sulfonyl-fluoride (PMSF; serine protease inhibitor), ethylene-diamine-tetra-acetic acid (EDTA; metallo protease inhibitor), copper ion, ferric ion and lead ion inhibited the enzyme activity. These results indicated that the fibrinolytic enzyme is a metallo-serine protease and different from nattokinase and chongkukjangkinase.

• Microbiology and Biotechnology Letters
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• v.28 no.5
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• pp.258-263
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• 2000

A fibrinolytic enzyme was purified to homogeneity from Bacillus sp. S19 using DEAE and CM column chromatograhies, and gel filtration with a recovery yield of 13%. Its molecular mass was estimated to be 42 kDa by SDS-PAGE. The pH and temperature optima were 8.0 and $40^{\circ}C$, respectively. The enzyme was stable up to $45^{\circ}C$ and over a pH range of 6-9. The N-terminal amino acid sequence of the enzyme was determined as Alsa-Gln-Asp-Ala-Thr-Val-Asn-Ile-Ser-Ala-Glu-Arg-Gln-Val-Ile. The fibrinolytic activity was increased by $Cu^{2+}$ while it was strongly inhibited by metal ions such as $Cd^{2+}$ and $Ba^{2+}$ . In addition, the enzyme was inhibited by EDTA, but not by PMSF, suggesting that it is a metallorprotease.

• Journal of Life Science
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• v.12 no.1
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• pp.43-48
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• 2002

An actinomycetes which produces fibrinolytic enzyme was isolated from soil. Characteristics of the isolated strain and the optimal conditions for the productions of fibrinolytic enzyme were summarized as follows; The fibrinolytic enzyme production strain generates gray airmycelium and had about 0.6~0.8$\times$0.4~0.8${\mu}{\textrm}{m}$ cylindrical spore, smooth surface and formed spore chain of 10~40 spores. We have identified this strain as Streptomyces sp. JK-20. This strain was able to grow up at 20~32$^{\circ}C$ and its optimum growth temperature and pH was 24$^{\circ}C$ and pH 6.0, respectively. The optimal conditions for porducing fibrinolytic enzyme; carbon source, nitrogen source, metal ions and phosphorous sources was 1% xylose, 0.5% yeast extract, 0.5% polypepton, 0.1% MgSO$_4$.7$H_2O$ and 0.1% NaH$_2$PO$_4$.2$H_2O$, respectively. This strain showed the highest productivity of fibrinolytic enzyme after the fourth day under such optimal culture conditions.

• Microbiology and Biotechnology Letters
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• v.28 no.1
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• pp.52-58
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• 2000

The effect of fivrinolytic enzyme produced from Bacillus subtilis K-54 on the thrombosis and stress in vivo was investigated. Each partially purified fibrinolytic enzyme of 4 protein casein unit was administered orally for 3 days before intravenously injection with collagen and epinephrine. In the mice group administered with the enzyme and increased life span of mice was observed in comparison with that of control. The result suggest that the enzyme may prevent the formation of thrombos in vivo. Administration of the enzyme did not influence to stress itself because 5-hydroxyindoleacetatic acid concentration of brain in the mice group with stress did not decreased after the administration of the enzyme. The value of lipid peroxide (LPO) of the liver and brain cells in the group treted with the enzyme was lower than that of control. However, protein degradation (PDP value showed no significant difference between treatment and control groups. In addition, the value of activated partial thromboplastin time (APTT), protrombin time (PT0 and antiplasmin in blood were higher in the stress group than that of the enzyme treated group.

• Journal of Life Science
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• v.8 no.6
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• pp.737-740
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• 1998

Effect of oral administration with fibrinolytic enzyme isolated from fermented anchovy(the traditional fermented food in Korea called Myulchi Jeot-gal) and its functionally active enzyme to rat, activation of plasma fibrinolysis was observed. The euglobulin fibrinolytic activities and the plasma levels of H-D-Val-Leu-Lys-pNA(S-2251) amidolysis reached a maximum at 3 hours after the administration to rat. And euglobulin Iysis time(ELT) value after oral admi-nistration showed its activity 2∼3 hours later. From the above result, it was confirmed the enzyme activity in blood by oral administration fibrinolytic enzyme through animal experiment.

• Biomedical Science Letters
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• v.8 no.4
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• pp.245-250
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• 2002

Fibrinolytic enzyme has been purified from the edible mushroom, Tricholoma sejunctum using DEAE-cellulose chromatography, Phenyl-Sepharose chromatography and Mono-S column chromatography. The apparent molecular mass of purified enzyme was estimated to be 17100 Da by SDS-polyacrylamide gel electrophoresis and 19000 Da by gel filtration, Indicating that it was a monomer. The N-terminal amino acid sequence of the enzyme was Ala-Thr-Tyr-Lys-Ile-X-Ser-Ala-Thr-His-Gln-X-X-Leu-Val. It has a pH optimum at pH 9.5, suggested that purified enzyme was a alkaline protease. The activity of purified enzyme was inhibited by EDTA and 1,10-phenanthroline, indicating that purified enzyme is a metalloprotease. The activity of purified enzyme was increased by Zn$^{2+}$ and Co$^{2+}$, however, the enzyme activity was totally inhibited by Hg$^{2+}$.

• Journal of Microbiology and Biotechnology
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• v.25 no.12
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• pp.2090-2099
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• 2015

Recently, the cardiovascular disease has been widely problematic in humans probably due to fibrin formation via the unbalanced Western style diet. Although direct (human plasmin) and indirect methods (plasminogen activators) have been available, bacterial enzyme methods have been studied because of their cheap and mass production. To detect a novel bacterial fibrinolytic enzyme, 111 bacterial strains with fibrinolytic activity were selected from kimchi. Among them, 14 strains were selected because of their stronger activity than 0.02 U of plasmin. Their 16S rRNA sequence analysis revealed that they belong to Bacillus, Leuconostoc, Propionibacterium, Weissella, Staphylococcus, and Bifidobacterium. The strain B. subtilis ZA400, with the highest fibrinolytic activity, was selected and the gene encoding fibrinolytic enzyme (bsfA) was cloned and expressed in the E. coli overexpression system. The purified enzyme was analyzed with SDS-PAGE, western blot, and MALDI-TOF analyses, showing to be 28.4 kDa. Subsequently, the BsfA was characterized to be stable under various stress conditions such as temperature (4-40oC), metal ions (Mn²⁺, Ca²⁺, K²⁺, and Mg²⁺), and inhibitors (EDTA and SDS), suggesting that BsfA could be a good candidate for development of a novel fibrinolytic enzyme for thrombosis treatment and may even be useful as a new bacterial starter for manufacturing functional fermented foods.

• Microbiology and Biotechnology Letters
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• v.31 no.3
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• pp.284-291
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• 2003

Bacillus amyloliquefaciens K-42, which produces strongly a fibrinolytic enzyme, Was isolated from Ganjang, a traditional Korean soy sauce. The fibrinolytic enzyme was purified to homogeneity by ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Sephadex A-50, gel chromatography on Sephadex G-100, and gel chromatography on Sephadex G-75 of the culture filtrate of Bacillus amyloliquefaciens K42. The purified enzyme showed the specific activity of 59.4 units per milligram, which was increased by 17.1 fold over the culture broth. And the molecular weight of purified fibrinolytic enzyme was confirmed to be about 45,000 Dalton by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme activity was relatively stable at pH 4.0-10.0 and the optimum pH was 8.0. The activity of the purified enzyme was increased by $Mg^{2+}$ , Cu$^{2+}$ but the enzyme was totally inhibited by $Ba^{2+}$ $Hg^{2+}$ In addition, the enzyme activity was potently inhibited by EDTA, EGTA and CDTA. It was concluded that the purified enzyme was a metalloprotease. And Km value was 2.03 mg/ml to fibrin.

• Journal of the Korean Society of Food Science and Nutrition
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• v.33 no.2
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• pp.439-442
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• 2004

Bacterial strains showing the firinolytic and $\beta$-glucosidase activity were screened from Doeniang. The strain of KH-15 revealed a high level of fibrinolytic and $\beta$-gluocosidase activity. The isolated bacterium was identified and desingnated as Bacillus sp. KH-15. The carbon, nitrogen and salts sgnificantly influenced te fibrinolytic enzyme and $\beta$-glucosidase production. The optimized composition of medium appeared to be 2％ glucose, 0.5% yeast extract and 0.1% calcium chloride. The optimum pH and temperature for fibrinolytic enzyme and $\beta$-glucosidase activities were pH 7∼8, 4$0^{\circ}C$ and pH 6∼8, 30∼4$0^{\circ}C$, respectively.