• 대한약침학회지
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• 제14권3호
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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.

• 대한약침학회지
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• 제14권2호
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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.

• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2003년도 춘계 학술발표대회
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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.

• 한국미생물·생명공학회지
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• 제23권1호
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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.

• 생명과학회지
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• 제16권2호
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• pp.357-359
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• 2006

대추 추출물에서 fibrin을 가수분해 하는 생리활성 물질이 존재함을 알고 이 추출물의 열과 산에 대한 안정성 및 그 특징을 조사 하였다. Fibrin plate method 를 사용하여 측정된 fibrinolytic activity 는 대추 추출물의 첨가량이 증가할수록 높았으며, 이 추출물은 $100^{\circ}C$ 에서 10분간 열처리시에 안정한것으로 나타났다. 또한 pH 2.0, 3.0, 4.0 에서 3시간 보관후 fibrinolytic activity 를 측정한 결과 control의 80% 이상의 활성을 나타내었다. 한편 투석후 (분자량 12,000) 에는 활성이 나타나지 않았으며, 이는 대추에서 추출한 fibrin 분해 생리활성 물질이 분자량이 낮은 저분자 물질임을 알 수 있었다.

• Preventive Nutrition and Food Science
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• 제7권4호
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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.

• 한국균학회지
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• 제26권4호통권87호
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• pp.583-588
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• 1998

뽕나무버섯 추출물에서 fibrinolytic enzyme을 분리 정제하였다. 이 효소의 분자량은 약 19800Da 이고 pH 7.0에서 최고의 활성을 보여주는 neutral protease로 $55^{\circ}C$에서 최대 활성을 나타냈다. EDTA에 의해 활성이 저해되는 것으로 보아 metalloprotease로 추정되며 $Hg^{+2}$의 영향을 받아 활성이 상실되었다. N-terminal amino acid 분석 결과 15번째까지의 아미노산 잔기 순서는 X-X-T-N-G-X-T-X-S-R-Q-T-T-L-V 이었으며 지금까지 알려진 것과는 다른 새로운 효소이다.

• 대한의생명과학회지
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• 제10권4호
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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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• 제25권11호
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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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• 제9권1호
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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.