• Archives of Pharmacal Research
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• v.28 no.7
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• pp.816-822
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• 2005

Catharsius protease-2 (CPM-2) was isolated from the body of dung beetles, Catharsius molossus, using a three step purification process (ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, and affinity chromatography on DEAE Affi-Gel blue). The purified CPM-2, having a molecular weight of 24 kDa, was assessed homogeneously by SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of CPM-2 was composed of X Val Gin Asp Phe Val Glu Glu lie Leu. CPM-2 was inactivated by $Cu^{2+}\;and\;Zn^{2+}$ and strongly inhibited by typical serine proteinase inhibitors such as TLCK, soybean trypsin inhibitor, aprotinin, benzamidine, and ${\alpha}_1$-antitrypsin. However, EDTA, EGTA, cysteine, $\beta$-mercaptoethanol, E64, and elastatinal had little effect on enzyme activity. In addition, antiplasmin and antithrombin III were not sensitive to CPM-2. Based on the results of a fibrinolytic activity test, CPM-2 readily cleaved $A{\alpha}-$ and $B{\beta}$-chains of fibrinogen and fibrin, and y-chain of fibrinogen more slowly. The nonspecific action of the enzyme resulted in extensive hydrolysis, releasing a variety of fibrinopeptides of fibrinogen and fibrin. Polyclonal antibodies of CPM-2 were reactive to the native form of antigen. The ELISA was applied to detect quantities, in nanograms, of the antigen in CPM-2 protein.

• Korean Journal of Food Science and Technology
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• v.44 no.5
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• pp.600-606
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• 2012

A Bacillus sp. that produces fibrinolytic enzyme was isolated from Cheonggukjang, a traditional Korean soybean-fermented food. According to 16S rRNA gene base sequencing, the bacillus was identified as a variety of Bacillus subtilis, and named Bacillus subtilis IDCC 9204. Fibrinolytic enzyme NK-IL9204 was stable up to $60^{\circ}C$ and within pH range of 5-10. Purified NK-IL9204 was detected through fibrin zymography. The molecular weight and isoelectric point of the enzyme were estimated to be 27.7 kDa and 6.7 by SDS-PAGE and 2D electrophoresis, respectively. Its amino acid sequence was similar to that of nattokinase (identities 99.5%) and different from that of nattokinase BPN (identities 86.4%). The plasma fibrinolytic activity of NK-IL9204 was measured by euglobulin clot lysis times (ECLT). The NK-IL9204 was orally administered to SD rats for 3 weeks (1,000 FU/rat/day). The ECLT was significantly shortened by supplementation of NK-IL9204.

• Biomolecules & Therapeutics
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• v.3 no.2
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• pp.159-164
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• 1995

Fibrinolytic and fibrinogenolytic activities of the venoms from the Korean snakes, Agkistrodon caliginosus, nosus, Agkistrodon saxatilis and Agkistrodon blomhoffi brevicaudus were compared by fibrin-plate method and polyacrylamide gel electrophoresis, respectively. The venom from A. blomhoffi brevicaudus showed the highest degree of fibrin(ogen)olytic activity, and a protease with the fibrin(open)olytic activity was purified by p-amino-benzamidine affinity chromatography and DEAE ion-exchange chromatography. The purified enzyme had a molecular weight of 50,800 and a capability to degrade the B$\beta$-chain of fibrinogen preferentially to the $A\alpha$-chain, but not the ${\gamma}$-chain. Fibrinolytic activity of the purified enzyme was approximately 3.8 plasmin unit/mg protein.

• Journal of Microbiology and Biotechnology
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• v.24 no.7
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• pp.969-978
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• 2014

The aprE2 gene with its prosequence from Bacillus subtilis CH3-5 was overexpressed in Escherichia coli BL21(DE3) by using plasmid pET26b(+). After IPTG induction, active and mature AprE2 was produced when cells were grown at $20^{\circ}C$, whereas inactive and insoluble enzyme was produced in a large amount when cells were grown at $37^{\circ}C$. The insoluble fraction was resuspended with 6 M guanidine-HCl and dialyzed against 2 M Tris-HCl (pH 7.0) or 0.5 M sodium acetate (pH 7.0) buffer. Then active AprE2 was regenerated and purified by a Ni-NTA column. Purified AprE2 from the soluble fraction had a specific activity of $1,069.4{\pm}42.4U/mg$ protein, higher than that from the renatured insoluble fraction. However, more active AprE2 was obtained by renaturation of the insoluble fraction. AprE2 was most stable at pH 7 and $40^{\circ}C$, respectively. The fibrinolytic activity of AprE2 was inhibited by PMSF, but not by EDTA and metal ions. AprE2 degraded $A{\alpha}$ and $B{\beta}$ chains of fibrinogen quickly, but not the ${\gamma}$-chain. AprE2 exhibited the highest specificity for N-succinyl-Ala-Ala-Pro-Phe-pNA. The $K_m$ and $k_{cat}/K_m$ of AprE2 was 0.56 mM and $3.10{\times}10^4S^{-1}M^{-1}$, respectively.

• Journal of Sasang Constitutional Medicine
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• v.19 no.1
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• pp.160-170
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• 2007

1. Objectives This study was performed to find the activities and characteristics of purified thrombolytic enzymes from Holotrichia extracts. 2. Methods In the first time, a coarse enzyme fluid was made by using the freedried Holotrichia extracts. After manufacturing total soluble proteins and purifing enzymes, it was evauluated the activities and characteristics of this enzyme's dissolving capability to fibrin and thrombus. This study was taken using azocasein assay, fibrin-plate method, native-PAGE and fibrin zymography. 3. Results A soluble proteins were efficiently extracted form freezedried Holotrichia extracts. And, this purified enzyme had a ten times fibrinolytic capability compare with ustulation Holotrichia sample. In native PAGE and fibrin zymography, Holotrichia extracts showed the respectable fibrinolytic activity. Also, It had higher thrombolytic activities compared with general thrombolytic enzyme 'plasmin'. In experiment of various protease inhibitors of the purified enzyme from Holotrichia extracts on the azocaseinolytic activity, the enzyme was strongly inhibited by EDTA ${\cdot}$ EGTA, and weakly by APMSF ${\cdot}$ PMSF ${\cdot}$ TPCK. 4. Conclusion Holotrichia extracts has the thrombolytic activities, and it will operate directly th fibrin-clot and thrombus.

• Proceedings of the KOSOMBE Conference
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• v.1998 no.11
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• pp.227-228
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• 1998

Lumbrokinase, potent fibrinolytic enzyme purified from earthworm, was immobilized onto polyurethane valves using photoreaction, photoreactive polyallyl-amino as a photoreactive linker. For evaluation of blood compatibility, lumbrokinase immobilized polymer valves were assembled into the total artificial heart (TAH). This TAH was implanted to 60kg healthy lamb for 1-3 days with the cardiac output 5 L/min. In the control lamb, the valves were untreated, in ore other, only valves on the right were treated, and in the remaining animal, only those on the left. To facilitate the thrombus formation, low doses of heparin were administered. For evaluation of the immobilized lumbrokinase, thrombus formation, proteolytic and fibrinolytic activity was measured. This data shows that lumbrokinase-treated polyurethane valves lead to decreased thrombus formation in vivo, and that their biocompatibility is therefore higher than that of untreated valves.

• Journal of Microbiology and Biotechnology
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• v.16 no.2
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• pp.264-271
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• 2006

Two fibrinolytic enzymes were purified from the culture supernatant of Fomitella fraxinea mycelia by ion-exchange and gel filtration chromatographies, and were designated as F. fraxenia proteases 1 and 2 (FFP1 and FFP2). The apparent molecular masses of the enzymes were estimated to be 32 kDa and 42 kDa, respectively, by SDS-PAGE and gel filtration chromatography. Both enzymes had the same optimal temperature ($40^{\circ}C$), but different pH optima (10.0 and 5.0 for FFP1 and FFP2, respectively). FFP1 was relatively stable at pH 7.0-9.0 and temperature below $30^{\circ}C$, whereas FFP2 was very stable in the pH range of 4-11 and temperature below $40^{\circ}C$. FFPI activity was completely inhibited by phenylmethylsulfonyl fluoride (PMSF) and aprotinin, indicating that this enzyme is a serine protease. The activity of FFP2 was enhanced by the addition of $CO^{2+}$ and $Zn^{2+}$ and inhibited by $Cu^{2+},\;Ni^{2+}$, and $Hg^{2+}$. Furthermore, FFP2 activity was strongly inhibited by EDTA and 1,10-phenanthroline, implying that the enzyme is a metalloprotease. Both enzymes readily hydrolyzed fibrinogen, preferentially digesting the $A{\alpha}$- and $B{\beta}$-chains of fibrinogen over ${\gamma}$-chain. FFP1 showed broad substrate specificity for synthetic substrates, but FFP2 did not. $K_{m}$ and $V_{max}$ values of FFP1 for a synthetic substrate, N-succinyl-Ala-Ala-Pro-Phe-pNA, were 0.213 mM and 39.68 units/ml, respectively. The first 15 amino acids of the N-terminal sequences of both enzymes were APXXPXGPWGPQRIS and ARPP(G)VDGQ(R,I)SK(L)ETLPE, respectively.

• Journal of Microbiology and Biotechnology
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• v.9 no.5
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• pp.675-678
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• 1999

Fibrinolytic protease activity was detected in the fruit body of Pleurotus sajor-caju using a fibrin plate method. Two fibrinolytic activities (FPI and Ⅱ) were found at the regions of 14.5 and 86.0 kDa by using gel-filtration column chromatography. FPⅡ was identified as an alkaline protease, whereas FPⅠ was a neutral protease. Both were inhibited by phenanthrolin and EDTA, suggesting that they are metalloprotease. Inactivated enzyme activities were restored by adding Co/sup 2+/ or Zn/sup 2+/. Iodoacetate inhibited FPⅠ, but not FPⅡ. Both enzymes cleaved B/sub β/ and γ chains of the human fibrinogen. FPⅡ showed a preference to hydrophobic and bulky residues of nitroanilidine compounds as substrates, whereas FPⅠ preferred positively charged residues.

• Journal of the Korean Society of Food Science and Nutrition
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• v.39 no.5
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• pp.655-661
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• 2010

The nutraceutical role of fermented omija (Schizandra chinensis) beverage (FOB) was determined through the analysis of fibrinolytic and alcohol metabolizing activities, nitrite scavenging activity, and angiotensin converting enzyme and $\alpha$-glucosidase inhibitory effects. Firstly, FOB increased fibrinolytic activity in a dose-dependent manner and indicated angiotensin converting enzyme inhibitory activity of 94.8% at 20% FOB (0.6 mg/mL). In addition, the inhibitory activities of FOB on $\alpha$-amylase and $\alpha$-glucosidase were determined to be 100% at 100% FOB (3 mg/mL) and 49% at 60% FOB (1.8 mg/mL), respectively. Nitrite scavenging activity of FOB was about 96.1%, 72.3%, and 68.3% on pH 1.2, 3.0, and 6.0 at 100% FOB, respectively. To determine influence of FOB on alcohol metabolism, the generating activities of reduced-nicotinamide adenine dinucleotide (NADH) by alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) were measured. Facilitating rate of ADH activity was 70.3% at 50% FOB, but ALDH activity was not affected. These results revealed that FOB has a strong alcohol metabolizing activity, and fibrinolytic and nitrite scavenging activities and exhibits the angiotensin converting enzyme, $\alpha$-amylase, and $\alpha$-glucosidase inhibitory activities.

• Korean Journal of Food Science and Technology
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• v.34 no.2
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• pp.283-289
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• 2002

This study was performed to search for potential microorganism that has rapid fermenting and physiological function from anchovy sauce. We isolated three bacterial strains, JM-1, JM-2, and JM-3 with proteolytic and fibrinolytic activity from anchovy sauce. Among the 3 bacterial strains, JM-3 showed the strongest proteolytic and fibrinolytic activity. Bacterial strain JM-3 was gram-positive rod, motile and formed endospore. The 16S rRNA of bacterial strain JM-3 was amplified by PCR and then its sequence was determined by ABI 310 genetic analyzer. The 16S rRNA sequence of bacterial strain JM-3 was compared to BLAST DNA database and identified to Bacillus subtilis with 99% of homology. The optimum temperature, pH and NaCl concentration for growth of B. subtilis JM-3 were $40^{\circ}C$, 5.0 and 0%, respectively. The optimum temperature, pH and NaCl concentration for proteolytic and fibrinolytic enzyme production of B. subtilis JM-3 were same as optimum conditions for growth. At 20% of NaCl concentration which is common NaCl concentration of fish sauce, B. subtilis JM-3 showed about 60% of proteolytic and fibrinolytic activity of 0% NaCl concentration. From above results, we found that B. subtilis JM-3 will be able to used for starter of functional fish sauce.