• Microbiology and Biotechnology Letters
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• v.26 no.3
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• pp.226-231
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• 1998

The medium optimization was investigated to maximize the production of fibrinolytic enzyme by Bacillus subtilis KCK-7 isolated from Chungkookjang, which could hydrolyze the fibrin produced through the blood coagulation mechanism in human body. The simultaneous addition of 5% soluble starch and 0.5% cellobiose to the medium as carbon sources resulted in the highest production of the fibrinolytic enzyme. Likewise, the optimized composition of medium appeared to be 0.5% peptone, 0.3% beef extract, 0.5% cellobiose, 5% soluble starch, 2% raw soybean meal and 0.02% Na$_2$HPO$_4$. In addition, the fibrinolytic enzyme production by Bacillus subtilis KCK-7 reached to the maximum level after the cultivation for 48 hr, using the optimized medium.

• Journal of Microbiology and Biotechnology
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• v.12 no.4
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• pp.649-656
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• 2002

Fibrinolytic microorganisms were screened from 42 samples of Korean fermented food (7 kinds of Chungook-jang, 14 kinds of commercial Doen-Jang, 5 kinds of home-made Doen-jang, and 16 kinds of Jeot-gal), 15 samples of Japanese fermented food (5 kinds of home-made soybean paste, and 10 kinds of Natto), and 19 samples of Indonesian fermented food (Tempe) as well as starters of Meju (500 microflora from Korea, and 22 from China). Initially, 11 isolates with strong fibrinolytic activity were selected for further characterization. The fibrinolytic activity of the 11 isolates ranged from 89 to 199% of standard plasmin. Four strains, M5l from Korean fermented food (Meju), I 1-1, I 1-4, and I 5-1 from Indonesian fermented food (Tempe), were chosen based on the degree of activity and reproducibility, and identified as Staphylococcus sciuri, Citrobacter or Enterobacter, Enterococcus faecalis, and Bacillus subtilis, respectively. The first two isolates are pathogenic stains while the latter two are considered as GRAS (Generally Recognized As Safe). Fibrinolytic activity of E. faecalis, characterized and designated as BRCA-5, reached a maximum, when the producer was cultivated in Ml7 broth supplemented with 1.0% glucose for 5 h at 37$^{\circ}C$ with shaking at 180 rpm. Compared to commercial fibrinolytic enzymes, the cell-free culture supernatant of 5. faecaiis BRCA-5 showed stronger activity than plasmin and streptokinase, but similar degree of specific activity as nattokinase and urokinase, aud it also demonstrated anticoagulant and antiplatelet activity ex vivo. These features of E. faecalis make it an attractive agent as a biomaterial for health-promoting foods.

• Journal of Chest Surgery
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• v.22 no.4
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• pp.525-547
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• 1989

Hemorrhagic tendency observed in open heart surgery patients has been attributed, among other causes, to increased fibrinolytic activity during extracorporeal circulation. But the exact mechanism of enhanced fibrinolytic activity which occurs during extracorporeal circulation is still unknown. So, we studied and compared the changes of parameters of fibrinolytic and protein C system according to time obtained from the plasma of 31 adult open heart surgery patients[EGG group] and 10 adult general thoracic surgery patients[control group], in order to confirm the hypothesis that the activated protein C system might affect the fibrinolytic system during extracorporeal circulation. In ECC group, the nature of the enhanced fibrinolytic activity that evolved during extracorporeal circulation was characterized by significant increase in fibrin degradation products[P < 0.01] and significant decrease in plasminogen and alpha2-antiplasmin[P < 0.05, P < 0.01] in spite of adequate amount of heparin administration. These changes were most pronounced in the early phase of extracorporeal circulation and normalized after termination of extracorporeal circulation. The results of these observations were the same after volume correction with the value of hematocrit. The change of volume corrected protein C ratio during extracorporeal circulation revealed similar pattern to those of plasminogen and alpha2-antiplasmin [P < 0.01], but volume corrected ratio of free protein S showed significant increase after the commencement of extracorporeal circulation then decreased after extracorporeal circulation. Although the above mentioned changes occur similarly in both bubble type oxygenator-used and membrane oxygenator-used patients groups, but the degree of decrease was more severe in membrane oxygenator-used patients group [P < 0.01] and showed much slower recovery to reach to the preextracorporeal circulation level. These results confirm the hypothesis that the enhanced fibrinolysis during extracorporeal circulation might be caused by the activation of protein C system and the activation is possibly linked to the appearance of thrombin from contact activation of blood after wide exposure to the synthetic surfaces of extracorporeal circuit. Key words: Extracorporeal circulation, Enhanced fibrinolysis, Protein C system.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.7 no.3
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• pp.476-482
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• 2006

The aim of this work was to perform the screening and identification of the bacterium, MK-15 having the activity of fibrinolytic enzyme for the commercial use. Initially, strain MK-15 was enriched and isolated from naturally fermented soybean. Morphological and various physiological characteristics of the strain MK-15 was examined. The activity of fibrinolytic enzyme derived from supernatants of test culture MK-15 was performed by fibrin plate method for solid fibrinolytic activity. As the result, the fibrinolytic activity of MK-15 grown on the soybean media was about 2.7 times greater than that of plasmin used as stardard. 16S rRNA analyses revealed that strain MK-15 was 99.9% similar to Bacillus subtilis species cluster, and the bacterium was designated as Bacillus sp. MK-15. Strain MK-15 was registered in GenBank as [DQ163021].

• Korean Journal of Food Science and Technology
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• v.31 no.1
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• pp.219-223
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• 1999

Various bacterial strains that secret extracellular fibrinolytic enzyme were screened from kimchi, a traditional vegetable fermented food in Korea. Three microbes of them were identified to be Bacillus amyloliquefaciens, Bacillus brevis and Micrococcus luteus strains according to Bergey's Manual of Systematic Bacteriology. It was found that B. amyloliquefaciens, B. brevis and M. luteus produced 2.58, 1.48 and 2.03 plasmin unit/mL of fibrinolytic enzyme, respectively. All extracellular proteases showing the fibrinolytic activity were confirmed by SDS-PAGE and fibrin zymography assay and we propose that some of the fibrinolytic enzymes from this work are novel enzymes.

• 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.

• Journal of Microbiology and Biotechnology
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• v.27 no.1
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• pp.9-18
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• 2017

Nine bacilli with fibrinolytic activities were isolated from doenjang, a traditional Korean fermented soy food. Among them, RSB34 showed the strongest activity and was identified as Bacillus amyloliquefaciens by 16S rRNA and recA gene sequencing. During growth on LB up to 96 h, RSB34 showed the highest fibrinolytic activity ($83.23mU/{\mu}l$) at 48 h. Three bands of 23, 27, and 42 kDa in size were observed when the culture supernatant was analyzed by SDS-PAGE and 27 and 42 kDa bands by fibrin zymography. The gene encoding the 27 kDa fibrinolytic enzyme AprE34 was cloned by PCR. BLAST analyses confirmed that the gene was a homolog to genes encoding AprE-type proteases. aprE34 was overexpressed in Escherichia coli BL21(DE3) using pET26b(+). Recombinant AprE34 was purified and examined for its properties. The $K_m$ and $V_{max}$ values of recombinant AprE34 were $0.131{\pm}0.026mM$ and $16.551{\pm}0.316{\mu}M/l/min$, respectively, when measured using an artificial substrate, N-succinyl-ala-ala-pro-phe-p-nitroanilide. aprE34 was overexpressed in B. subtilis WB600 using pHY300PLK. B. subtilis transformants harboring pHYRSB34 (pHY300PLK with aprE34) showed higher fibrinolytic activity than B. amyloliquefaciens RSB34.

• 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.

• Korean Journal of Food Science and Technology
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• v.31 no.2
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• pp.553-557
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• 1999

Five fungi (mushrooms), Daedaleopsis styracina, Trichaptum abietium, Coriolus versicolor, Pisolithus tinctorius and Tricholomopsis decora, were screened and examined the fibrinolytic activity and specificity. The extracts of mushrooms showed a level of fibrinolytic activity that was about 3-4 times higher than that of plasmin 1.0 unit. In particular, Pisolithus tinctorius of them showed the greatest enzyme activity (4.71 plasmin unit/mL) by fibrin plate assay, and the highest specificity (1.32 plasmin unit/mL) using chromogenic substrate (N-p-Tosyl-Gly-Pro-Lys p-nitroanilide) by Tricholomopsis decora. And the same molecular mass 54 and 61kDa showing the fibrinolytic activity obtained from all fruiting bodies were confirmed, and it was found that Trichaptum abietium and Tricholomopsis decora have a strong fibrinolytic enzyme with an apparent size of 100 kDa and 84 kDa, respectively on SDS-fibrin zymography activity assay.

• The Korean Journal of Food And Nutrition
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• v.18 no.1
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• pp.39-47
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• 2005

Using the bacteria isolated from Chungkookjang, Bacillus sublilis MG410 which is excellent in fibrinolytic enzyme activity was isolated. In increase the high production of fibrinolytic enzyme from Bacillus sublilis MG410, the effect of various carbon sources, nitrogen sources, inorganic sources, the initial pH of medium were investigated. The most effective carbon and nitrogen sources were founded cellobiose 0.5%(w/v) and soybean meal 2%(w/v) respectively. None of inorganic sources examined had any detectable stimulating effect on fibrinolytic enzyme production except Na₂HPO₄·12H₂O. The initial optimum pH for fibrinolytic enzyme production ranged from 5∼6 and agitation speed was effect at 150rpm. In jar fermentor experiments under optimal culture conditions, the activity of fibrinolytic enzyme reached about 5.050 unit after 48hours.