• Microbiology and Biotechnology Letters
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• v.47 no.4
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• pp.522-529
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• 2019

A Bacillus strain, SJ4, exhibiting strong fibrinolytic activity was isolated from saeu (shrimp, Acetes chinensis) jeotgal, a Korean traditional fermented food and was identified as B. subtilis. The B. subtilis SJ4 strain can grow at a NaCl concentration of up to 15% (w/v). The fibrinolytic activity of B. subtilis SJ4 (152.0 U/ml) cultured in Luria-Bertani (LB) broth for 48 h at 37℃ with aeration was higher than that of B. subtilis SJ4 cultured in TSB (124.5 U/ml) under same culture conditions. The major proteins in the LB culture supernatant of B. subtilis SJ4 were analyzed by SDS-PAGE, which revealed three major bands (23, 25, and 28 kDa). The band (23 kDa) with strong fibrinolytic activity, analyzed on fibrin zymogram, was observed at 60-96 h of cultivation. The aprESJ4 gene encoding the major fibrinolytic enzyme, AprESJ4, was cloned by PCR. The aprESJ4 gene sequence exhibited high similarities with the fibrinolytic gene sequences of other Bacillus species. The amino acid sequence of AprESJ4 exhibited 98.9 and 98.4% similarity with subtilisin NAT and AprE2 of B. subtilis, respectively. Hence, B. subtilis SJ4 can be a potential starter culture for jeotgal products.

• Food Science and Biotechnology
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• v.15 no.5
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• pp.704-709
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• 2006

The mucilage production and tyrosine content in soy milk cake (SMC) fermented by Bacillus firmus NA-1, Bacillus subtilis GT-D, and B. subtilis KU-A was improved by fortification with 10% defatted soybean flour. The fibrinolytic activity and consistency of the SMC were drastically increased by solid-state fermentation for 1 day. However, the consistency of the fermented SMC gradually decreased during fermentation for 3 days. Furthermore, the tyrosine content of the freeze-dried powder of SMC fermented by three Bacillus sp. was 9 times higher than that of unfermented SMC. The soybean proteins, including the 7S and 11S subunits, were partially digested during alkaline fermentation, producing lower molecular-weight peptides. The fibrinolytic enzyme produced in SMC fermented by B. firmus NA-l and B. subtilis KU-A exhibited higher thermal stability than that of B. subtilis GT-D fermentation. The powder obtained from B. subtilis GT-D fermentation had an ${\alpha}$-amylase activity and lower consistency compared to those of B. firmus NA-1 and B. subtilis KU-A. In addition, this powder contained 6.3% moisture content, 27% crude protein content and 9 units of fibrinolytic activity and proteolytic activity.

• Journal of Microbiology and Biotechnology
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• v.21 no.11
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• pp.1166-1173
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• 2011

Meju is a traditional Korean fermented soy product used as a key element for soy sauce and doenjang. Bacilli with antimicrobial activity were isolated from meju prepared by traditional methods at Sunchang county, Jeollabukdo, Korea. Six isolates were identified as Bacillus amyloliquefaciens by recA gene sequencing and RAPD-PCR. One isolate, B. amyloliquefaciens MJ5-41, showed the strongest fibrinolytic activity. A 27 kDa active fibrinolytic enzyme, AprE5-41, was purified from the culture supernatant of MJ5-41 grown on LB by chromatographic methods. The optimum pH and temperature for purified AprE5-41 were 7.0 and $45^{\circ}C$, respectively. AprE5-41 quickly degraded $A{\alpha}$ and $B{\beta}$ chains but not the ${\gamma}$-chain of fibrinogen. AprE5-41 exhibited the highest specificity for N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide, a known substrate for ${\alpha}$-chymotrypsin, cathepsin G, and subtilisin BPN'. The structural gene, aprE5-41, was cloned by PCR and successfully expressed in B. subtilis.

• KSBB Journal
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• v.23 no.3
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• pp.251-256
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• 2008

The aim of this work was to investigate the fibrinolytic activity and characterization of Bacillus licheniformis HK-12, which produces the fibrinolytic enzyme excreted from naturally fermented Chungkook-Jang. Initially, the physiological and biochemical characteristics of strain HK-12 was examined. Both physiological analysis using BIOLOG system and phylogenetic analysis using 16S rRNA sequencing were performed to identify the strain, and the strain could be assigned to Bacillus licheniformis, designated as B. lichenformis HK-12, and registered in GenBank as [EU288193]. Phylogenetic analysis of B. licheniformis HK-12 was plotted based on 16S rRNA sequence comparisons. During the incubation period of B. licheniformis HK-12, the changes of bacterial growth, fibrinolytic activity, and pH were monitored. As the results, after 36 hours of incubation, the maximum fibinolytic activity was about 2.25 times than that of plasmin used as standard. Optimal conditions on the growth of B. licheniformis HK-12 associated with the fibrinolytic activity was initial pH 7.0 and 40$^{\circ}C$, respectively.

• The Korean Journal of Mycology
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• v.36 no.2
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• pp.183-188
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• 2008

A fibrinolytic serine protease was purified from the fruiting bodies of an edible mushroom, Albatrellus confluens. The enzyme had a molecular mass of 30086.41 Da, as measured by MALDI-TOF mass spectrometry. The N-terminal amino acid sequence of the enzyme was Glu-Thr-Val-Thr-Glu-Thr-Thr-Ala -Pro-Trp-Gly-Leu-Ser-Arg-Ile. It displayed optimal activity at $50^{\circ}C$ and within a pH range of $8.0{\sim}10.0$, suggesting that the enzyme is an alkaline protease. The enzyme was stable up to $30^{\circ}C$. The enzyme displayed a strong substrate specificity for the synthetic peptide, N-Suc-Ala-Ala-Pro-Phe pNA. The enzyme activity was completely inhibited by addition of PMSF, indicating that the enzyme is a serine protease. No inhibition was observed following addition of E-64, pepstatin, or EDTA. The activity of the purified enzyme was decreased in the presence $Fe^{2+}$ or $Co^{2+}$, and the enzyme was completely inhibited by addition of $Hg^{2+}$. From these results, we propose that Albatrellus confluens could be used for biofunctional foods development and has potential therapeutic value for the treatment of vascular diseases.

• Proceedings of the KOSOMBE Conference
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• v.1997 no.05
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• pp.51-54
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• 1997

Lumbrokinase, potent fibrinolytic enzyme purified from earthworm, was immobilized onto the total artificial heart valves using photoreaction. This valve were implanted into the lamb for three days. After experiments, thrombus was observed in the untreated valves whereas no thrombus was observed in the lumbrokinase immobilized valves. The fibrinolytic activity and proteolytic activity of the implanted valve was examined. The fibrinolytic activity of the valve was remained after the implantation. The lumbrokinase could be a suitable fibrinolytic agents in the vascular contacting devices to reduce the thrombus.

• Microbiology and Biotechnology Letters
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• v.30 no.4
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• pp.325-331
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• 2002

investigated to maximize the production of fibrinolytic enzyme from Fomitella fraxinea mycelia. Among the tested media, Coriolus versicolor medium (CVM) showed the highest production for the enzyme. 2% galactose, 0.6% yeast extract and 0.1% $NaNO_3$, 0.1% $K_2HPO_4$, and 0.05% $MgSO_4$.$7H_2O$ as carbon, nitrogen, phosphorus, and inorganic salt sources resulted in the maximum level of the enzyme activity, respectively. The enzyme production from F. fraxinea was reached to highest level after the cultivation for 10 days at $25^{\circ}C$ and pH 9. The enzyme activity of culture supernatant was most active at $40^{\circ}C$ and pH 10. The activity of the enzyme was inhibited by phenylmethylsulfonylfluoride and aprotinin, suggesting that it is a serine protease.

• Biomedical Science Letters
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• v.6 no.4
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• pp.261-268
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• 2000

Fibrinolytic enzyme (FE-2) was purified from the fruiting bodies of Tricholoma saponaceum using DEAE-Cellulose chromatography and Mono-S column chromatography, The enzyme has a molecular weight of 18.23 kDa and include Zn$^{2+}$ ion as found by ICP/MS. The N-terminal amino acid sequence of the enzyme was A-L-Y-V-G-X-S-P-X-Q-Q-S-L-L-V It has a pH optimum at pH 7.5, suggested that FE-2 was a neutral pretense. The activity of FE-2 was highly inhibited by EDTA and 1,10-phenanthroline, indicating that the enzyme is a metalloprotease. The activity of FE-2 was increased by $Mg^{2+}$, Zn$^{2+}$, Fe$^{2+}$, and Co$^{2+}$, but the enzyme activity was totally inhibited by Hg$^{2+}$. No inhibition was found with PMSF, E-64, pepstatin and 2-mercaptoethanol. The enzyme hydrolyzed both $A\alpha$ and B$\beta$ chains of human fibrinogen. The $\gamma$ chain was resistant to hydrolysis by FE-2.

• KSBB Journal
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• v.21 no.6 s.101
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• pp.433-438
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• 2006

A fibrinolytic enzyme gene (BCF-1) was subcloned to the pEB vector which is high expression vector in the Bacillus host. The enzyme was purified by using FPLC after ammonium sulfate precipitation. The enzyme was oral-administrated to the rat and checked the bleeding time, blood clotting time and fibrinolytic effect of the serum. In the bleeding time retardation test, it was longer about 1.7 fold in the feeding rat than without feeding. The serum of rat feeded with the enzyme had the fibrinolytic activity from 1 hour to 3 hours after oral-administration. After 3 hours from feeding, the fibrinolytic activity was decreased gradually. Also blood clotting time after bleeding was longer than that of control rat. The enzyme could be detected at band of 30,000 Da in the blood by western blotting. The enzyme was not harmful to the all internal organs of the rats. Taken together, the enzyme originated from B. subtilis BB-1 can be a candidate to develop the drug for thrombosis, arteriosclerosis and myocardial infarction.

• Biomedical Science Letters
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• v.10 no.4
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• pp.347-351
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• 2004

Maggot fibrolase (MsMg-1) was purified from the maggots of Mimela splendems using ammonium sulfate fractionation, DEAE Affi-gel affinity chromatography. This protease had a molecular weight of 85 kDa as determined by SDS-polyacrylarnide gel electrophoresis under reducing conditions. It showed strong proteolytic and fibrinolytic activities. The purified enzyme was strongly inhibited by phenylmethanesulfonyl fluoride, Mn/sup 2+/, and Zn/sup 2+/ but it was not by EDTA, EGT, Mg/sup 2+/, Ca/sup 2+/, and Li/sup 2+/ ions. In these experimental results, we have speculated that MsMg-1 is a serine protease with a strong fibrinolytic activity.