• Journal of the Korean Society for Precision Engineering
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• v.26 no.7
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• pp.22-30
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• 2009

• Proceedings of the Korean Society of Postharvest Science and Technology of Agricultural Products Conference
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• 2007.11a
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• pp.231-231
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• 2007

• Journal of Life Science
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• v.16 no.2 s.75
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• pp.274-281
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• 2006

A fibrinolytic enzyme gene was isolated from Bacillus subtilis BB-1 by PCR method. Primers for PCR cloning were designed according to pre-identified gene for fibrinolytic enzymes from B. subtilis. The primer sequences were 5'-CGG ATC CGT GAG AGG CAA AAA GGT G-3' and 5'-TGA ATT CTT AAT GTG CTG CTG CTT GTC C-3' as concensus sequences of the fibrinolytic genes of Bacillus species. The PCR product was 1,145 bp and the sequence homology was 99% with nattokinase gene isolated from Japanese natto. The cloned fibrinolytic gene was reconstructed in Bacillus-E. coli shuttle vector, pEB for bulk-production. The fibrinolytic enzyme was purified by FPLC from the cloned B. subtilis 168. The optimum pH and temperature of the enzyme were 7.0 and $35^{\circ}C$, respectively. The fibrinolytic enzyme did not show any activity toward to skim milk, gelatin, casein and blood agar plate. The enzyme specific polyclonal antibody was prepared in rabbit for further assays such as detection of the gene expression in plant cells. This means that the enzyme may be used for health-care such as thrombosis without any hamful effects in the blood vessel.

• Journal of Life Science
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• v.20 no.2
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• pp.183-189
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• 2010

A fibrinolytic enzyme producing Bacillus sp. J-19 was isolated from the popular Korean seasoning, pickled anchovy. The fibrinolytic enzyme was purified to homogeneity by chromatographic methods including ethanol precipitation and gel-filtration using Sephadex G-50. Compared to the crude enzyme extract, the specific activity of the enzyme increased 1021-fold with a recovery of 23%. The purified enzyme was estimated to be approximately 45 kDa by SDS-PAGE. Especially, the amidolytic activity in the presence of the synthetic substrate for serine protease (H-D-Ile-Pro-Arg-pNA, S-2288) represented approximately 17 U/mg. In addition, more than the 60% activity of the 45 kDa fibrinolytic activity was maintained in the presence of up to 30% (w/v) sodium chloride. These findings could provide a unique fibrinolytic enzyme, leading to a potential thrombolytic agent.

• 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.

• Journal of Chest Surgery
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• v.41 no.4
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• pp.484-488
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• 2008

During long-term anticoagulation treatment with using heparin in a pregnant patient with a mechanical mitral prosthesis, we observed several anticoagulation-related complications, including repeated prosthetic valve thrombosis. This was found to be caused by heparin resistance due to an anti-thrombin III deficiency. Thrombolytic therapy using urokinase or tissue plasminogen activator (tPA) was successful and safe for her as well as her baby.

• Journal of Life Science
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• v.12 no.1
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• pp.43-48
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• 2002

An actinomycetes which produces fibrinolytic enzyme was isolated from soil. Characteristics of the isolated strain and the optimal conditions for the productions of fibrinolytic enzyme were summarized as follows; The fibrinolytic enzyme production strain generates gray airmycelium and had about 0.6~0.8$\times$0.4~0.8${\mu}{\textrm}{m}$ cylindrical spore, smooth surface and formed spore chain of 10~40 spores. We have identified this strain as Streptomyces sp. JK-20. This strain was able to grow up at 20~32$^{\circ}C$ and its optimum growth temperature and pH was 24$^{\circ}C$ and pH 6.0, respectively. The optimal conditions for porducing fibrinolytic enzyme; carbon source, nitrogen source, metal ions and phosphorous sources was 1% xylose, 0.5% yeast extract, 0.5% polypepton, 0.1% MgSO$_4$.7$H_2O$ and 0.1% NaH$_2$PO$_4$.2$H_2O$, respectively. This strain showed the highest productivity of fibrinolytic enzyme after the fourth day under such optimal culture conditions.

• Journal of Life Science
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• v.11 no.6
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• pp.561-567
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• 2001

To isolate and purify fibrinolytic active substance from Sarcodon aspratus(N $H_4$)$_2$S $O_4$ precipitation, DE52 anion exchange column chromatography, Sephacryl-S 200gel filtration chromatography and Mono S cation FPLC were carried out and the characterizations of the purified enzyme were investigated. The bound active fraction on DE52 anion exchange column chromatography were eluted with 0.2 M NaCI and the fibrionlytic enzyme was purified after following Sephacryl-S200 gel fitration chromatography and Mono S cation EPLC. The specific activity of purified enzyme was 55.2 U/mg protein and increased 11.3 fold comparing crude extract and the yield was 49.5%. 12% SDS-PAGE electrophoresis and gel filtration chromatography revealed that Sarcodon aspratus fibrionloytic enzyme was highly purified and had 29.300 Da molecular weight. Enzyme activity of the purified fibrinolytic enzyme from Sarcodon aspratus was increased on higher pH and was stable until pH 10.5. On temperature dependent stability, the enzyme activity was decrease sharply but remained 25% relative activity on 8$0^{\circ}C$. This enzyme activity was inhibited by heavy metal ion, C $U^{2+}$ and $Co^{3+}$ with 68% and 38%, respectively. And also, the enzyme activity was inhibited with $Ca^{2+}$ chelator EDTA and serine protease inhibitor PMSF. These results from this study suggested that the fibrinolycit enzyme from Sarcodon aspratus is a serine protease and the enzyme activity was increased by $Ca^{2+}$ or $Mg^{2+}$ ion.n.ion.n.

• Applied Biological Chemistry
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• v.46 no.3
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• pp.176-182
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• 2003

An alkaline fibrinolytic protease-producing bacteria was isolated front Korean traditional soy sauce and identified as Bacillus subtilis K7 from the results of analyses of its morphological and physiological properties, $API^{\circledR}$, and Biolog system. The enzyme was purified by 75% ammonium sulfate fractionation, QAE-Sephadex anion and SP-Sephadex cation exchange column chromatography and Sephadex G-100 gel filtration. The specific activity of the purified enByme was 233.9 unit/mg protein and the yield of enzyme was 3.8%. The homogeneity of the purified enzyme was confirmed by polyacrylamide gel electrophoresis. Molecular mass of the enzyme was estimated about 21,500 Da by SDS-polyacrylamide get electrophoresis and gel chromatography. The optimum temperature and pH for the enzyme activity were $40^{\circ}C$ and 9.0, respectively. The enzyme was stable in a pH range of 5.0 to 12.0, and 60% of its activity was lost on heat treatment at $50^{\circ}C$ for 20 min. The activity of the purified enzyme was inhibited by the presence of $Fe^{2+},\;Ag^{2+},\;Cu6{2+}$, iodoacetate, ethylene diamine tetraacetic acid (EDTA), and trans-1,2-diaminocycloheane-N,N,N',N'-tetraacetic acid (CDTA). The results indicates that the enzyme requires a metal ion for its enzymatic activity.

• 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.