• Journal of Pharmacopuncture
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• v.14 no.3
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• pp.71-79
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• 2011

Objectives : This study was undertaken to identify fibrinolytic proteases from Gloydius blomhoffii siniticus venom and to characterize a major fibrinolytic protease purified from the venom. Methods : The venom was subjected to chromatography using columns of Q-Sepharose and Sephadex G-75. The molecular weights of fibrinolytic proteases showing fibrinolytic zone in fibrin plate assay were determined in SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) The effects of inhibitors and metal ions on fibrinolytic protease and the proteolysis patterns of fibrinogen, gelatin, and bovine serum albumin were investigated. Results : 1) The fibrinolytic fractions of the three peaks isolated from Gloydius blomhoffii siniticus venom contained two polypeptides of 46 and 59 kDa and three polypeptides of 32, 18, and 15 kDa and a major polypeptide of 54 kDa, respectively. 2) The fibrinolytic activity of the purified protease of 54 kDA was inhibited by metal chelators, such as EDTA, EGTA, and 1,10-phenanthroline, and disulfhydryl-reducing compounds, such as dithiothreitol and cysteine. 3) Calcium chloride promoted the fibrinolytic activity of the protease, but mercuric chloride and cobalt(II) chloride inhibited it. 4) The fibrinolytic protease cleaved preferentially A${\alpha}$-chain and slowly B${\beta}$-chain of fibrinogen. It also hydrolyzed gelatin but not bovine serum albumin. Conclusions : The Gloydius blomhoffii siniticus venom contained more than three fibrinolytic proteases. The major fibrinolytic protease was a metalloprotease which hydrolyzed both fibrinogen and gelatin, but not bovine serum albumin.

• Journal of Pharmacopuncture
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• v.14 no.2
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• pp.5-14
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• 2011

Objectives: Fibrinolytic enzyme preparations were isolated from the snake venom of Macrovipera lebetica turanica in this study. Methods: The purity of the preparations was determined using SDS-PAGE and the enzymic characteristics of the purified fibrinolytic enzyme were determined. Results: 1. All of the two preparations with fibrinolytic activity obtained from the snake venom of M. l. turanicat contained the major polypeptide with the molecular weight of 27,500. One of the preparation showed purified fibrinolytic enzyme. 2. The purified fibrinolytic enzyme hydrolyzed ${\alpha}$-chain of fibrinogen faster than ${\beta}$-chain but not ${\gamma}$-chain. 3. The fibrinolytic activity was inhibited completely by EDTA, EGTA, 1,10-phenanthroline, and dithiothreitol. 4. The fibrinolytic activity was inhibited completely by calcium chloride, iron(III) chloride, mercuric chloride, and cobalt (II) chloride. 5. The fibrinolysis zone formed after addition of zinc sulfate was smaller but clearer than the control. Conclusions: These results suggested that the fibrinolytic enzyme purifed from the snake venom of M. l turanica was a metalloprotease containing dithiol group.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2003.04a
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• pp.104-104
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• 2003

In this study, seven grain seeds(sorghum maize, buckwheat, soy bean, mung bean, red bean, and barngrass) and germinated seven grain seeds were examined the fibrinolytic activity through fibrin plate assay and SDS-PAGE. The results obtained were as follows : 1. In the fibrin plate assay, the extracts of maize, barngrass, sorghum and buckwheat showed fibrinolytic activity. Especially, Maize of them showed fibrinolytic activity that was almost similar to plasmin, fibrinolytic enzyme used as a positive control. 2. In the SDS-PAGE of seven grain seeds, fibrinolytic activity was remarkably shown in mung bean and red bean. 3. In the fibrin plate assay of germinated grain seeds, buckwheat(5 mm), buckwheat(10 mm) and soy bean(10 mm) showed a level of fibrinolytic activity that was about 0.3 fold than 1.0 unit of plasmin, Also maize(10 mm) of them showed a level of fibrinolytic activity that was about 0.5 fold than 1.0 unit of plasmin. As a result, maize of grain seeds was found that it has a strong fibrinolytic activity.

• Microbiology and Biotechnology Letters
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• v.23 no.1
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• pp.1-5
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• 1995

Bacterial strains showing the fibrinolytic activity were screened from Chungkook-jang and Natto (Japanese traditional soy food). The strains isolated from Natto revealed a high level of fibrinolytic activity, wherease nearly half of the isolates from Chungkook-jang did not show the relevant activity. However, one strain isolated from Chungkook-jang showed the highest fibrinolytic activity (1.84 plasmin unit), and subsequently identified as Bacillus species. The fibrinolytic strain was designated as Bacillus sp. CK 11-4.

• Journal of Life Science
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• v.16 no.2 s.75
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• pp.357-359
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• 2006

Crude extract prepared from jujube (Zizyphus mauritiana) possseses fibrinolytic activity hydrolyzing fibrin. A clear transparent region is observed where fibrin is hydrolyzed, and its diameter increased as the added jujube extract increased. The fibrinolytic activity of jujube extract seems to be heat and acid stable since its activity was retained after heat treatment at $100^{\circ}C$ for 10 min or an acid treatment by incubating at pH 2.0, 3.0, and 4.0 for 3 hours. But the jujube extract dialyzed with a cellulose membrane with molecular weight cutoff of 12,000 and 2,000 lost its activity, which suggests the fibrinolytic activity might be compounds of low molecular weight.

• Preventive Nutrition and Food Science
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• v.7 no.4
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• pp.417-420
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• 2002

A strong fibrin-specific fibrinolytic enzyme was produced from Bacillus subtilis KH-4 isolated from Deonjang, a Korean fermented soybean paste similar to Japanese miso. The addition of glucose as a carbon source resulted in the highest levels of caseinolytic and fibrinolytic activities. Likewise, the addition of yeast extract as the nitrogen source resulted in the highest caseinolytic and fibrinolytic activities (3473.2 unit and 47.4 munit, respectively), It was observed that out of all metal ion sources only calcium (chloride) enhanced caseinolytic and fibrinolytic activities, with increases of 4949.3 unit and 58.2 unit/mg, respectively. The optimal temperature for the production of the enzyme was found to be 4$0^{\circ}C$ in the optimal medium (glucose 20 g, yeast extract 5 g, CaCl$_2$l g, and NaCl 2 g). The maximum fibrinolytic activity was observed at the late stationary phase. B. subtilis KH-4 produced a fibrinolytic enzyme at 4$0^{\circ}C$, after 30 h growth, which increased up to 54 h and then remained constant. These results suggest that Deonjang has potential as a source of physiologically active anti-thromotic enzymes.

• The Korean Journal of Mycology
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• v.26 no.4 s.87
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• pp.583-588
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• 1998

A fibrinolytic enzyme has been isolated from the edible honey mushroom, Armillariella mellea and purified. The apparent molecular mass of purified enzyme was estimated to be 19800Da by SDS polyacryl amide gel electrophoresis and 19900Da by gel filtration, indicating that it was a monomer. The enzyme was optimal at pH 7, suggesting that the purified enzyme was a neutral proteinase. It shows the maximum fibrinolytic activity at $55^{\circ}C$, is completely inactivated above $65^{\circ}C$, and still indicates 40% of activity at $37^{\circ}C$. The fibrinolytic activity has been decreased by the addition of EDTA. Fifteen amino acid sequence was determined by protein sequencing techniques.

• Biomedical Science Letters
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• v.10 no.4
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• pp.415-420
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• 2004

Fibrinolytic substance was purified from the Wooltalikong (Phaseolus ssp.), using DEAE-cellulose chromatography, Sephadex G-150 gel-filtration, and FPLC gel-filtration. The substance has a molecular weight of 5262.70 Da as measured by MALD-TOF mass spectrometry. It has a pH optimum at pH 6.0. The fibrinolytic activity of purified substance was inhibited by EDTA and 1,10-phenanthroline and slightly decreased by PMSF and pepstatin A. It shows the maximum fibrinolytic activity at 40℃ and the substance was stable up to 50℃. The activity of the substance was increased by Zn/sup 2+/ and was totally inhibited by Hg/sup 2+/. This study revealed that Wooltalikong could be a good source of fibrinolytic products due to its small molecular size and heat-resistant ability.

• Journal of Microbiology and Biotechnology
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• v.25 no.11
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• pp.1863-1870
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• 2015

Fibrinolytic enzyme genes (aprE2, aprE176, and aprE179) were introduced into the Bacillus subtilis 168 chromosome without any antibiotic resistance gene. An integration vector, pDG1662, was used to deliver the genes into the amyE site of B. subtilis 168. Integrants, SJ3-5nc, SJ176nc, and SJ179nc, were obtained after two successive homologous recombinations. The integration of each fibrinolytic gene into the middle of the amyE site was confirmed by phenotypes (Amy-, Spec^S) and colony PCR results for these strains. The fibrinolytic activities of the integrants were higher than that of B. subtilis 168 by at least 3.2-fold when grown in LB broth. Cheonggukjang was prepared by inoculating each of B. subtilis 168, SJ3-5nc, SJ176nc, and SJ179nc, and the fibrinolytic activity of cheonggukjang was 4.6 ± 0.7, 10.8 ± 0.9, 7.0 ± 0.6, and 8.0 ± 0.2 (U/g of cheonggukjang), respectively at 72 h. These results showed that construction of B. subtilis strains with enhanced fibrinolytic activities is possible by integration of a strong fibrinolytic gene via a marker-free manner.

• Preventive Nutrition and Food Science
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• v.9 no.1
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• pp.14-20
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• 2004

Bacillus strains capable of producing fibrinolytic enzyme were isolated from traditional fermented Korean soybean paste and Japanese fermented soybean (Natto). Among the 16 strains, a selected Bacillus sp. was identified as bacillus firmus, with 80.7％ homology, by API kit analysis. Seed starter or B. firmus NA-1 was prepared with 5％ soymilk prepared from micronized soybean powder. To produce fibrinolytic enzyme by B. firmus NA-1 the liquid culture was performed with NB broth (pH 7.0) fortified with 1％ galactose, 0.1% tryptone, and 0.5% $K_2$HPO$_4$, by shaking with 180 rpm at 37$^{\circ}C$. Fibrinolytic enzyme activity reached the highest value at 7.8 unit/mL (plasmin unit) after fermentation for 72 hr. The crude fibrinolytic enzyme showed higher relative activity in the range of pH 7.0∼9.0. The activity of crude fibrinolytic enzyme was well maintained even after concentration by the vacuum evaporation at 5$0^{\circ}C$ for 1 hr.