• 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.

• International Journal of Industrial Entomology and Biomaterials
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• v.22 no.2
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• pp.83-93
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• 2011

Biochemical and enzymatic characterization for extracellular protease isolated from Cordyceps militaris cultivated on rice bran medium was investigated. C militaris produced proteolytic enzymes from 10 days after inoculation, maximum enzyme production was found at 25 days. The optimum temperature and pH of proteases production was at $25^{\circ}C$ and pH 7.0, respectively. The protease activity was observed in the four peaks (Pro-I, Pro-II, Pro-III, and Pro-IV) separated through Sephadex G-100 column chromatography. The separated protease was optimally active at $25^{\circ}C$. Optimum pH of the protease was between 7 and 8. Enzyme was also stable over at $30-80^{\circ}C$. The enzyme was highly stable in a pH range of 4-9. Protease activity was found to be slightly decreased by the addition of $Mg^{2+}$, $Mn^{2+}$, $Zn^{2+}$, $Fe^{2+}$ and $Cu^{2+}$, whereas inhibited by the addition of $Ca^{2+}$ and $Co^{2+}$ Protease activity was inhibited by protease inhibitor PMSF. On the other hand, the partially purified protease was investigated on proteolytic protease activity by zymogram gel electrophoresis using three substances (casein, gelatin and fibrin). Four active bands (F-I, FII, F-III, and F-IV) of fibrin degradation were revealed on fibrin zymogram gels. Both of F-II and FIII showed caseinolytic, fibrinolytic and gelatinolytic activities in three gels. Thermostability, pH stability, and pH-thermostability of the enzyme determined the residual fibrinolytic activity also displayed on fibrin zymogram gel. The only one enzyme (F-II) displayed over a broad range of temperature at $30-90^{\circ}C$. The FII displayed fibrinolytic activity in the pH range 3-5, but was inactivated in the range of pH 6-11. The F-I and F-III showed enzyme activity in the pH range of 6-11. In the pH-thermostability, the F-II only kept fibrinolytic activity after heating at $100^{\circ}C$ for 10, 20 and 30 min at pH 3 and pH 7, respectively. On the other hand, the F-II was retained activity until heating for 10 min under pH 11 condition. By using fibrin zymogram gel electrophoresis, extracellular fibrinolytic enzyme F-II from C. militaris showed unusual thermostable under acid and neutral conditions.

• Journal of Pharmacopuncture
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• v.16 no.2
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• pp.46-54
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• 2013

Objective: This study was undertaken to isolate a fibrin(ogen)olytic enzyme from the snake venom of Gloydius blomhoffii siniticus and to investigate the enzymatic characteristics and hemorrhagic activity of the isolated enzyme as a potential pharmacopuncture agent. Methods: The fibrinolytic enzyme was isolated by using chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fibrin plate assay. The characteristics of the enzyme were determined by using fibrin plate assay, protein hydrolysis analysis, and hemorrhage assay. Its amino acid composition was determined. Results: The fibrin(ogen)olytic enzyme with the molecular weight of 27 kDa (FE-27kDa) isolated from G. b. siniticus venom consisted of two heterogenous disulfide bond-linked polypeptides with the molecular weights of 15 kDa and 18 kDa. When more than $20{\mu}g$ of FE-27kDa was applied on the fibrin plate, fibrinolysis zone was formed as indicating its fibrinolytic activity. The fibrinolytic activity was inhibited completely by phenylmethanesulfonylfluoride (PMSF) and ethylenediaminetetraacetic acid (EDTA) and partially by thiothreitol and cysteine. Metal ions such as $Hg^{2+}$ and $Fe^{2+}$ inhibited the fibrinolytic activity completely, but $Mn^{2+}$ did not. FE-27kDa preferentially hydrolyzed ${\alpha}$-chain of fibrinogen and slowly hydrolyzed ${\beta}$-chain, but did not hydrolyze ${\gamma}$-chain. High-molecular-weight polypeptides of gelatin were hydrolyzed partially into polypeptides with molecular weights of more than 45 kDa. A dosage of more than $10{\mu}g$ of FE-27kDa per mouse was required to induce hemorrhage beneath the skin. Conclusion: FE-27kDa was a serine proteinase consisting of two heterogeneous polypeptides, hydrolyzed fibrin, fibrinogen, and gelatin, and caused hemorrhage beneath the skin of mouse. This study suggests that the potential of FE-27kDa as pharmacopuncture agent should be limited due to low fibrinolytic activity and a possible side effect of hemorrhage.

• 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 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.

• Applied Biological Chemistry
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• v.46 no.3
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• pp.176-182
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• 2003

An alkaline fibrinolytic protease-producing bacteria was isolated front Korean traditional soy sauce and identified as Bacillus subtilis K7 from the results of analyses of its morphological and physiological properties, $API^{\circledR}$, and Biolog system. The enzyme was purified by 75% ammonium sulfate fractionation, QAE-Sephadex anion and SP-Sephadex cation exchange column chromatography and Sephadex G-100 gel filtration. The specific activity of the purified enByme was 233.9 unit/mg protein and the yield of enzyme was 3.8%. The homogeneity of the purified enzyme was confirmed by polyacrylamide gel electrophoresis. Molecular mass of the enzyme was estimated about 21,500 Da by SDS-polyacrylamide get electrophoresis and gel chromatography. The optimum temperature and pH for the enzyme activity were $40^{\circ}C$ and 9.0, respectively. The enzyme was stable in a pH range of 5.0 to 12.0, and 60% of its activity was lost on heat treatment at $50^{\circ}C$ for 20 min. The activity of the purified enzyme was inhibited by the presence of $Fe^{2+},\;Ag^{2+},\;Cu6{2+}$, iodoacetate, ethylene diamine tetraacetic acid (EDTA), and trans-1,2-diaminocycloheane-N,N,N',N'-tetraacetic acid (CDTA). The results indicates that the enzyme requires a metal ion for its enzymatic activity.

• Journal of Microbiology
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• v.44 no.6
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• pp.622-631
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• 2006

In this study we purified a fibrinolytic enzyme from Cordyceps militaris using a combination of ion-exchange chromatography on a DEAE Sephadex A-50 column, gel filtration chromatography on a Sephadex G-75 column, and FPLC on a HiLoad 16/60 Superdex 75 column. This purification protocol resulted in a 191.8-fold purification of the enzyme and a final yield of 12.9 %. The molecular mass of the purified enzyme was estimated to be 52 kDa by SDS-PAGE, fibrin-zymography, and gel filtration chromatography. The first 19 amino acid residues of the N-terminal sequence were ALTTQSNV THGLATISLRQ, which is similar to the subtilisin-like serine protease PR1J from Metarhizium anisopliae var. anisopliase. This enzyme is a neutral protease with an optimal reaction pH and temperature of 7.4 and $37^{\circ}C$, respectively. Results for the fibrinolysis pattern showed that the enzyme rapidly hydrolyzed the fibrin $\alpha$-chain followed by the $\gamma$-$\gamma$ chains. It also hydrolyzed the $\beta$-chain, but more slowly. The A$\alpha$, B$\beta$, and $\gamma$ chains of fibrinogen were also cleaved very rapidly. We found that enzyme activity was inhibited by $Cu^{2+}$ and $Co^{2+}$, but enhanced by the additions of $Ca^{2+}$ and $Mg^{2+}$ ions. Furthermore, fibrinolytic enzyme activity was potently inhibited by PMSF and APMSF. This enzyme exhibited a high specificity for the chymotrypsin substrate S-2586 indicating it's a chymotrypsin-like serine protease. The data we present suggest that the fibrinolytic enzyme derived from the edible and medicinal mushroom Cordyceps militaris has fibrin binding activity, which allows for the local activation of the fibrin degradation pathway.

• Journal of Life Science
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• v.12 no.3
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• pp.357-362
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• 2002

A bacterial strain showing the fibrinolytic activity was screened from korean traditional soybean products. For the identification, the strain was investigated morphology and biochemical characteristics and it was classified to Bacillus subtilis. The strain had high fibrinolytic activity in Chungkukjang. The optimum fermentation condition of temperature and time were 37$^{\circ}C$ and 24hour. The pH in Chungkukjang was gradually alkalized during fermentation. The fibrinolytic enzyme in Chungkukjang stable at heft treatment; After heating at 6$0^{\circ}C$ and 8$0^{\circ}C$ for 30 min, the fibrinolytic activity remained 75% and 40%, respectively.

• 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.

• Journal of Microbiology and Biotechnology
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• v.25 no.9
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• pp.1449-1459
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• 2015

A novel proteolytic enzyme with fibrinolytic activity, FSP3, was purified from the recently isolated Streptomyces sp. P3, which is a novel bacterial strain isolated from soil. FSP3 was purified to electrophoretic homogeneity by ammonium sulfate precipitation, anion exchange, and gel filtration. FSP3 is considered to be a single peptide chain with a molecular mass of 44 kDa. The maximum activity of the enzyme was observed at 50℃ and pH 6.5, and the enzyme was stable between pH 6 and 8 and below 40℃. In a fibrin plate assay, FSP3 showed more potent fibrinolytic activity than urokinase, which is a clinical thrombolytic agent acting as a plasminogen activitor. The activity was strongly inhibited by the serine protease inhibitor PMSF, indicating that it is a serine protease. Additionally, metal ions showed different effects on the activity. It was significantly suppressed by Mg²⁺ and Ca²⁺ and completely inhibited by Cu²⁺, but slightly enhanced by Fe²⁺. According to LC-MS/MS results, its partial amino acid sequences are significantly dissimilar from those of previously reported fibrinolytic enzymes. The sequence of a DNA fragment encoding FSP3 contained an open reading frame of 1287 base pairs encoding 428 amino acids. FSP3 is a bifunctional enzyme in nature. It hydrolyzes the fibrin directly and activates plasminogen, which may reduce the occurrence of side effects. These results suggest that FSP3 is a novel serine protease with potential applications in thrombolytic therapy.