• Microbiology and Biotechnology Letters
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• v.49 no.3
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• pp.289-297
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• 2021

Bacillus velezensis CJ1, showing significant fibrinolytic activity, was isolated from Myeolchi Jeotgal, a popular Korean fermented seafood. When B. velezensis CJ1 was grown on four different culture media, the culture on the Luria-Bertani (LB) broth showed the highest fibrinolytic activity (102.94 mU/μl) at 48 h. LB was also the best medium for growth. SDS-PAGE of culture supernatant showed four major bands, 38, 35, 27, and 22 kDa in size. Fibrin zymography showed four active bands, 50, 47, 40, and 30 kDa in size. A gene homologous to aprE of the Bacillus species was cloned by PCR. DNA sequencing showed that aprECJ1 can encode a protease consisting of 382 amino acids. The translated amino acid sequence of AprECJ1 showed high identity values with those of B. velezensis strains and other Bacillus species. The aprECJ1 gene was introduced into B. subtilis WB600 using an E. coli-Bacillus shuttle vector, pHY300PLK, and overexpressed. A 27 kDa band corresponding to the mature form of AprECJ1 was produced and confirmed by SDS-PAGE and fibrin zymography. B. subtilis WB600 [pHYaprECJ1] showed 1.8-fold higher fibrinolytic activity than B. velezensis CJ1 at 48 h.

• Journal of Life Science
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• v.15 no.4 s.71
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• pp.513-521
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• 2005

A bacterium producing five fibrinolytic isozymes was isolated from black bean chung kuk. The bacterium was identified as Bacillus subtilis BB-1 by 16s rDNA sequence homology search. A gene out of five fibrinolytic genes in the Bacillus subtilis BB-1 was cloned by shot-gun method. A Cla I DNA fragment of B. subtilis BB-1 chromosome was cloned in to pBluescript II SK(-) and showed the fibrinolytic activity to bacterial cells. The Cla I DNA fragment was sequenced and the sequences did not show homology with gene for protease or fibrinolytic enzyme genes in other organisms. The Cla I DNA fragment was reduced to 2,142 bp by activity-guided PCR cloning method. The optimum pH and temperature of the enzyme were 5.0 and $35^{\circ}C$, respectively. Substrate specificity of the fibrinolytic enzyme was detected in skim milk, casein, gelatin and blood agar plates. The activity of the enzyme was not detected with these substrates. Taken together, this enzyme is a new fibrinolytic enzyme and may be used to prevent thrombosis and arteriosclerosis.

• Journal of Life Science
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• v.11 no.4
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• pp.304-310
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• 2001

In order to produce a high quality and functional black bean Chungkugjang, a bacterium which has potent enzyme activities(protease:124.8 U/$m\ell$, $\alpha$-amylase: 78.2U/$m\ell$, glucoamylase; 13.9U/$m\ell$, ), was isolated by using the halo zone method and identified by morphological, cultural and biochemical properties, and then finally confirmed by GP-microplate identification system as Bacillus megatrium SMY-212. The isolated SMY-212 system was also high in the formation of mucoid material and fibrinolytic activity.

• Preventive Nutrition and Food Science
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• v.17 no.1
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• pp.64-70
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• 2012

Bacilli with fibrinolytic activities were isolated from traditionally-prepared Meju and some of these strains showed strong antifungal activities. One isolate, MJ1-4, showed the strongest antifungal activity. MJ1-4 and other isolates were identified as B. amyloliquefaciens strains by recA gene sequencing and RAPD-PCR results. B. amyloliqufaciens MJ1-4 efficiently inhibited an Aspergillus spp.-producing aflatoxin B1 ($AFB_1$) and a Penicillium spp.-producing ochratoxin (OTA) in addition to other fungi. Antifungal activity of B. amyloliquefaciens MJ1-4 culture reached its maximum (40 AU/mg protein) in LB or TSB medium around 48 hr at $37^{\circ}C$. Antifungal activity of the concentrated culture supernatant was not decreased significantly by protease treatments, implying that the antifungal substance might not be a simple peptide or protein. Considering its antifungal and fibrinolytic activities together, B. amyloliquefaciens MJ1-4 can serve as a starter for fermented soyfoods such as Cheonggukjang and Doenjang.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1994.04a
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• pp.279-279
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• 1994

Fibrinolytic proteases, piscivorase I (PI) and piscivorase II (PII), were isolated from Agkistrodon piscivorus piscivorus (eastern cotonmouth moccasin) venom using gel filtration on Bio-Gel P100 and ion-exchange chromatography on CM-Sepharose. The molecular welghts of two proteases were approximately 23400 and 29000. Their isoelectric points 6.6 and 8.5, respectively. The partial amino acid sequences of PI were characterized by tryptic digestion. PI readily cleaves the A${\alpha}$-and B${\beta}$-chaln of fibronogen, but PII rapidly cleaves A${\alpha}$-chain and more slowly the B${\beta}$-chain, They were activated by Ca$\^$2+/, Mg$\^$2+/ and Ba$\^$2+/, but inhibited by Zn$\^$2+/, Cu$\^$2+/ and Mn$\^$2+/. Two enzymes were also inhibited by cysten, ${\beta}$-mercapto -ethanol, and by metal chelators such as EDTA and EGTA, but not by benzamidine, PMSF, soybean trypsin inhibitor and aprotinin. They did not act like thrombin, plasmin and kallikrein, using specific chromogenllc substrates. Two protease did not induce platelet aggregation. PI showed low hemorrhagic activity at dosage of 50 $\mu\textrm{g}$.

• Korean Journal of Food Science and Technology
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• v.43 no.6
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• pp.735-741
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• 2011

Bealmijang is a short-term fermented regional product that is prepared with soybean and extra ingredients. In this study, starter strain candidates were screened from Bealmijang for fermented soybean paste products. Twenty one bacterial strains producing extracellular enzymes (amylase, cellulase, protease, xylanase and lipase) were isolated from Bealmijang, buckwheat sokseongjang. The isolates were assessed for fibrinolytic and antibacterial activities, and salt tolerance. Strain HJ18-4, identified as Bacillus subtilis (AB601598) by biochemical properties (89.6%) and 16S rDNA sequencing (100%), showed the highest enzymatic, fibrinolytic, and antibacterial activities among the isolates. Although the growth of HJ18-4 was inhibited by the increase of NaCl concentration, the growth still exceeded that of B. subtilis KACC 10114 at 5% and 10% NaCl. These results suggest that B. subtilis HJ18-4 is suitable as a starter for soybean paste manufacture.

• Journal of Microbiology and Biotechnology
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• v.24 no.2
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• pp.245-253
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• 2014

A fibrinolytic enzyme was produced by an edible mushroom of Pleurotus ostreatus using submerged culture fermentation. The enzyme was purified from the culture supernatant by applying a combination of freeze-thaw treatment, ammonium sulfate precipitation, hydrophobic interaction, and gel filtration chromatographies. The enzyme was purified by a 147-fold, with a yield of 7.54%. The molecular masses of the enzyme an determined by gel filtration and SDS-PAGE were 13.6 and 18.2 kDa, respectively. The isoelectric point of the enzyme was 8.52. It hydrolyzed fibrinogen by cleaving the ${\alpha}$ and ${\beta}$ chains of fibrinogen followed by the ${\gamma}$ chains, and also activated plasminogen into plasmin. The enzyme was optimally active at $45^{\circ}C$ and pH 7.4. The enzyme activity was completely inhibited by EDTA, whereas protease inhibitors of TPCK, SBTI, PMSF, aprotinin and pepstatin showed no inhibition on its activity. The partial amino acid sequences of the enzyme as determined by Q-TOF2 were ATFVGCSATR, GGTLIHESSHFTR, and YTTWFGTFVTSR. These sequences showed a high degree of homology with those of metallo-endopeptidases from P. ostreatus and Armillaria mellea. The purified enzyme can also be applied as a natural agent for oral fibrinolytic therapy or prevention of thrombosis.

• The Korean Journal of Mycology
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• v.28 no.1
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• pp.60-65
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• 2000

Two fibrinolytic enzymes were purified from the fruiting bodies of Tricholoma saponaceum. The enzymes have a molecular weight of 18(FE-1) and 18.2(FE-2) kDa, respectively, and include $Zn^{2+}$ ion as determined by ICP/MS. The N-terminal amino acid sequence of the two enzymes were exactly the same: A-L-Y-V-G-X-S-P-X-Q-Q-S-L-L-V. The activity of FE-1 was highly inhibited by EDTA and 1,10-phenanthroline, indicating that the enzyme is a metalloprotease. The activity of FE-1 was slightly increased by $Mg^{2+},\;Zn^{2+},\;Fe^{2+}\;and\; Co^{2+}$, however, the enzyme activity was totally inhibited by $Hg^{2+}$. Addition of $Zn^{2+}\;and\;Co^{2+}$ reversed the inhibition caused by 1,10-phenanthroline. It has a pH optimum at pH 7.5, suggested that FE-1 was a neutral protease. It shows the maximum fibrinolytic activity at $55^{\circ}C$, is completely inactivated above at $65^{\circ}C$.

• Food Science and Biotechnology
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• v.18 no.2
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• pp.500-505
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• 2009

The fibrinolytic enzyme, subtilisin D5, was purified from the culture supernatant of the isolated Bacillus amyloliquefaciens DJ-5. The molecular weight of subtilisin D5 was estimated to be 30 kDa. Subtilisin D5 was optimally active at pH 10.0 and $45^{\circ}C$. Subtilisin D5 had high degrading activity for the A$\alpha$-chain of human fibrinogen and hydrolyzed the $B{\beta}$-chain slowly, but did not affect the $\gamma$-chain, indicating that it is an $\alpha$-fibrinogenase. Subtilisin D5 was completely inhibited by phenylmethylsulfonyl fluoride, indicating that it belongs to the serine protease. The specific activity (F/C, fibrinolytic/caseinolytic activity) of subtilisin D5 was 2.37 and 3.52 times higher than those of subtilisin BPN' and Carlsberg, respectively. Subtilisin D5 exhibited high specificity for Meo-Suc-Arg-Pro-Tyr-pNA (S-2586), a synthetic chromogenic substrate for chymotrypsin. The first 15 amino acid residues of the N-terminal sequence of subtilisin D5 are AQSVPYGISQIKAPA; this sequence is identical to that of subtilisin NAT and subtilisin E.

• Biomolecules & Therapeutics
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• v.3 no.2
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• pp.165-170
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• 1995

The biochemical properties of the fibrinolytic protease of 50,800 Da isolated from the venom of Kgdistrodon blomhoffi brevicaudus were characterized. The enzyme hydrolyzed the carboxyl side of arginine in the synthetic chromogenic peptides, N-Benzoyl-Phe-Val-Arg-pNA and N-p-Tosyl-Gly-Pro-Arg-pNA, and the enzyme activity was inhibited by phenylmethylsulfonylfluoride indicating that the enzyme belongs to the serine protease family. The pretense showed maximum activity at pH 7.5 and inhibited by ZnCl$_2$, CuSO$_4$, but not by soybean trypsin inhibitor, pepstatin A, 2-mercaptoethanol and EDTA. The fm value determined with N-p-Tosyl-Gly-Pro-Arg-pNA was 0.2 mM. The thrombolytic activity of the purified enzyme was evaluated by platelet aggregation test in rabbits. While the platelet count ratio in blood of the rabbits injected with thrombin alone declined from 1.0 to 0.6 within 7 min and maintained around 0.6 for 24 hours thereafter, the ratio rapidly recovered from around 0.6 to 0.8 in 1 hr, to 1.0 in 24 hrs when the rabbits were sequentially treated with thrombin and the purified enzyme. The result showed that the serine protease from A. blomhoffi brevicoudus of 50,800 Da had a thrombolytic activity in vivo and the enzyme might be developed as a therapuetic agent for the treatment of thrombic disease.