• Title/Summary/Keyword: fibrinolytic enzyme activity

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Purification and Characterization of a Fibrinolytic Enzyme from Snake Venom of Macrovipera lebetina turanica

  • Kwon, Ki-Rok;Park, Do-Il;Lee, Seung-Bae;Choi, Suk-Ho
    • 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.

Purification and Characterization of Fibrinolytic Enzyme from Armillariella mellea (뽕나무버섯으로부터 Fibrinolytic enzyme의 정제 및 특성 연구)

  • Kim, Jun-Ho;Kim, Yang-Sun
    • 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.

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Optimization of the Production of Fibrinolytic Enzyme from Bacillus firmus NA-1 in Fermented Soybeans

  • Seo, Ji-Hyun;Lee, Sam-Pin
    • 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.

Purification and Characterization of Fibrinolytic Enzyme from Lepista nuda (민자주방망이버섯으로부터 혈전용해효소의 정제 및 특성 연구)

  • Kim, Jun-Ho
    • The Korean Journal of Mycology
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    • v.33 no.2
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    • pp.69-74
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    • 2005
  • Fibrinolytic enzyme has been isolated and purified from the edible mushroom, Lepista nuda. The apparent molecular mass of purified enzyme was estimated to be 34 KDa by SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of the enzyme was Tyr-Pro-Ser-Pro-Ser-His-Gln-Thr-Ala-Val-Asn-Ala-Ile-Ile-X. It has a pH optimum at $7.0.{\sim}9.5$, suggesting that the purified enzyme is an alkaline protease. It shows the maximum fibrinolytic activity at $55^{\circ}C$. The fibrinolytic activity was inhibited by phenylmethylsulfonyl fluoride, indicating that the purified enzyme is a serine protease. The activity of the purified enzyme was totally inhibited by $Hg^{2+}$.

Purification and Characterization of Fibrinolytic Enzyme Excreted by Bacillus subtilis K-54 Isolated from Chung Guk Jang. (청국장에서 분리한 Bacillus subtilis K-54가 분비하는 혈전용해효소의 정제 및 특성)

  • 유천권;서원상;이철수;강상모
    • Microbiology and Biotechnology Letters
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    • v.26 no.6
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    • pp.507-514
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    • 1998
  • The strain K-54, the best producer of fibrinolytic enzyme, was isolated from Korean traditional food Chung Guk Jang and identified as Bacillus subtilis. Fibrinolytic enzyme was purified and characterized, and its molecular weight was determined. The fibrinolytic enzyme activity was increased about 66.9 times via purification with recovery yield of 10.1%. The optimum pH and temperature of this enzyme were 11 and $65^{\circ}C$. The enzyme was stable within a pH range 8-12 and unstable at 9$0^{\circ}C$. The molecular weight was estimated to be 29,000 dalton in the form of monomer with no other subunit. The isoelectric point was calculated 8.67. N-terminal sequence was identified Ala-Gly-Ser-Val-Pro-Try-Gly-Ser. Km value of the enzyme for $\alpha$-casein was calculated to be 0.31 (3.1 mg/$m\ell$). The enzyme activity highly inhibited by PMSF at 1 nM.

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Purification and Characterization of the Fibrinolytic Enzyme Produced by Bacillus subtilis KCK-7 from Chungkookjang

  • Paik, Hyun-Dong;Lee, Si-Kyung;Heo, Seok;Kim, Soo-Young;Lee, Hyung-Hoan;Kwon, Tae-Jong
    • Journal of Microbiology and Biotechnology
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    • v.14 no.4
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    • pp.829-835
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    • 2004
  • A fibrinolytic enzyme has been found in several bacteria isolated from fermented food. This study was carried out to investigate the purification and characteristics of the fibrinolytic enzyme produced by Bacillus subtilis KCK-7 originated from Chungkookjang. The fibrinolytic enzyme was purified to homogeneity from the culture supernatant using ammonium sulfate fractionation and chromatographies on DEAE-cellulose and on Sephadex G-100. The final specific activity of the purified enzyme increased 11.0-fold, and the protein amount in the purified enzyme was about 16% of that in the culture supernatant. The molecular weight of the purified enzyme was estimated to be about 45,000 by SDS-PAGE. The optimum pH and temperature for the enzyme activity were pH 7.0 and $60^{\circ}C$, respectively. The enzyme activity was relatively stable up to $60^{\circ}C$ over the pH range of 7.0-10.0. The fibrinolytic enzyme activity increased by $Ca^{2+}$ and $Cu^{2+}$, whereas it was inhibited by $Hg^{2+}$ and $Ba^{2+}$. In addition, it was severely inhibited by PMSF and DFT. It is suggested that the purified enzyme was a serine protease for the fibrinolysis. The purified enzyme could completely hydrolyze fibrin in vitro within 8 h. Hence, it is suggested that the purified enzyme can be put into practice as an effective thrombolytic agent.

Fibrinolytic Enzyme Production by Bacillus subtilis KH-4 Isolated from Deonjang

  • Kim, J.M.;Suh, H.J.;Ahn, S.W.;Kim, M.S.;Oh, S.H.
    • 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.

Purification and Characterization of Fibrinolytic Enzyme from Tricholoma sejunctum

  • Kim, Jun-Ho
    • 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$^{2+}$.

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Isolation and Characterization of a 32-kDa Fibrinolytic Enzyme (FE-32kDa) from Gloydius blomhoffii siniticus Venom -Fibrinolytic Enzyme from Gloydius blomhoffii siniticus Venom-

  • Kim, Joung-Yoon;Lee, Seung-Bae;Kwon, Ki Rok;Choi, Suk-Ho
    • Journal of Pharmacopuncture
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    • v.17 no.1
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    • pp.44-50
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    • 2014
  • Objectives: This study was undertaken to isolate a fibrinolytic enzyme from the snake venom of Gloydius blomhoffii siniticus and to investigate its enzymatic characteristics and hemorrhagic activity 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 investigated using fibrin plate assay, protein hydrolysis analysis, and hemorrhage assay. Its amino acid composition was determined. Results: The fibrinolytic enzyme with the molecular weight of 32kDa (FE-32kDa) from Gloydius blomhoffii siniticus showed a fibrin hydrolysis zone at the concentration of 0.2 mg/mL in the fibrin plate assay. The fibrin hydrolysis activity of the enzyme was inhibited completely by ethylenediaminetetraacetic acid (EDTA), ethyleneglycoltetraacetic acid (EGTA), and 1, 10-phenanthroline, thiothreitol and cysteine, and partially by phenylmethanesulfonylfluoride (PMSF). Metal ions such as $Fe^{2+}$ and $Hg^{2+}$ inhibited the fibrin hydrolysis completely, but $Zn^{2+}$ enhanced it. FE-32kDa hydrolyzed ${\alpha}$-chain but did not hydrolyze ${\beta}$-chain and ${\gamma}$-chain of fibrinogen. High-molecular-weight polypeptides of gelatin were hydrolyzed partially into low-molecular-weight polypeptides, but the extent of hydrolysis was limited. FE-32kDa induced hemorrhage beneath back skin of mice at the dose of $2{\mu}g$. Conclusions: FE-32kDa is a ${\alpha}$-fibrin(ogen)olytic metalloprotease that requires $Zn^{2+}$ for fibrinolytic activity and causes hemorrhage, suggesting that the enzyme is not appropriate for use as a clinical pharmacopuncture.

Variation of fibrinolytic enzyme activity produced Bacillus subtilis by gene cloning (유전자 cloning에 의한 Bacillus subtilis의 fibrinolytic enzyme 활성 변화)

  • 이홍석;유천권;이철수;강상모
    • Microbiology and Biotechnology Letters
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    • v.28 no.1
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    • pp.14-20
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    • 2000
  • The transformation of Bacillus subtilis K-54 and J-10 was carried out with constructed vectors containing structure and enhancer genes of aprN and prtR, to increase their fibrinolytic enzyme activity. Bands for the aprN and prtR genes were identified from B. subtilis J-10 by PCR that was carried out with the constructed primers for the genes. In addition, the gene fragments contained promoter site based on the results of analysing their nucleotide sequence. The two gene fragments, aprN and prtR, obtained by the PCR, were, then, inserted to vector such as T-vector and E.coli/Bacillus shuttle vector. The constructed vector were designated as pAPR2 (aprN), pENC2 (prtR) and pFLA1 (aprN and prtR), respectively. The constructed vector was used for transformation of the strains of B.subtilis J-10 and B. subtilis K-54 and the fribrinolytic activity of the transformed strains was investigated. The introduction of the vector, pAPR2 and the fibrinolytic activity of the transformed strains was investigated. The introduction of the vector, pAPR2 and pFLA1, resulted in the increase of fibrinolyitic enzyme activity in B. subtilis J-10 by 27.3% and 16%, respectively. However, the introduction of pENC2 to B. subtilis J-10 did not seem to induce increase of the enzyme activity. The strain of B.subtilis K-54 transformed with pENC2 showed an increased fibrinolytic activity by 5 folds compared with that of the original strain of B. subtilis K-54.

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