• Journal of Microbiology and Biotechnology
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• v.11 no.5
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• pp.845-852
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• 2001

A microorganism producing fibrinolytic enzyme was isolated from Korean traditional soybean paste and identified as Bacillus sp. KDO-13. The fibrinolytic enzyme was purified to homogeneity by ammonium sulfate fractionation, ion-exchange chromatography on DEAE-celluose, and gel chromatography on Sephadex G-100 of the culture supernatant of Bacillus sp. KDO-13. The molecular weight of the purified enzyme was estimated to be 44,000 by SDS-PAGE. The optimum pH and temperature for the enzyme activity were pH 8.0 and $50{\circ}C$, respectively. The enzyme activity was relatively stable at pH 7.0-9.0 and temperature below $50{\circ}C$. the activity of the enzyme was inhibited by $AI^{3+}$ and $Hg^{2+}$, but activated by $Co^{2+}$\;and\;Ni^{2+}. In addition, the enzyme activity was potently inhibited by EDTA and 0-phenanthroline. The purified enzyme could completely hydrolyze a fibrin substrate within 6 h in vitro, and had a low $K_m$ value for fibrin hydrolysis. It was concluded that the purified enzyme was a metalloprotease with relatively high specificity for fibrinolysis, and thus, could be applied as an effective thrombolytic agent.

• Journal of Microbiology and Biotechnology
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• v.25 no.1
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• pp.89-97
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• 2015

Bacillus subtilis HK176 with high fibrinolytic activity was isolated from cheonggukjang, a Korean fermented soyfood. A gene, aprE176, encoding the major fibrinolytic enzyme was cloned from B. subtilis HK176 and overexpressed in E. coli BL21(DE3) using plasmid pET26b(+). The specific activity of purified AprE176 was 216.8 ± 5.4 plasmin unit/mg protein and the optimum pH and temperature were pH 8.0 and 40℃, respectively. Error-prone PCR was performed for aprE176, and the PCR products were introduced into E. coli BL21(DE3) after ligation with pET26b(+). Mutants showing enhanced fibrinolytic activities were screened first using skim-milk plates and then fibrin plates. Among the mutants, M179 showed the highest activity on a fibrin plate and it had one amino acid substitution (A176T). The specific activity of M179 was 2.2-fold higher than that of the wild-type enzyme, but the catalytic efficiency (k_cat/K_m) of M179 was not different from the wild-type enzyme owing to reduced substrate affinity. Interestingly, M179 showed increased thermostability. M179 retained 36% of activity after 5 h at 45℃, whereas AprE176 retained only 11%. Molecular modeling analysis suggested that the 176^th residue of M179, threonine, was located near the cation-binding site compared with the wild type. This probably caused tight binding of M179 with Ca²⁺, whichincreased the thermostability of M179.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2003.04a
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• pp.104-104
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• 2003

In this study, seven grain seeds(sorghum maize, buckwheat, soy bean, mung bean, red bean, and barngrass) and germinated seven grain seeds were examined the fibrinolytic activity through fibrin plate assay and SDS-PAGE. The results obtained were as follows : 1. In the fibrin plate assay, the extracts of maize, barngrass, sorghum and buckwheat showed fibrinolytic activity. Especially, Maize of them showed fibrinolytic activity that was almost similar to plasmin, fibrinolytic enzyme used as a positive control. 2. In the SDS-PAGE of seven grain seeds, fibrinolytic activity was remarkably shown in mung bean and red bean. 3. In the fibrin plate assay of germinated grain seeds, buckwheat(5 mm), buckwheat(10 mm) and soy bean(10 mm) showed a level of fibrinolytic activity that was about 0.3 fold than 1.0 unit of plasmin, Also maize(10 mm) of them showed a level of fibrinolytic activity that was about 0.5 fold than 1.0 unit of plasmin. As a result, maize of grain seeds was found that it has a strong fibrinolytic activity.

• Microbiology and Biotechnology Letters
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• v.39 no.4
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• pp.330-336
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• 2011

Bacterial strains exhibiting fibrinolytic activity were screened from traditional Korean soybean sauce. The Fibrinolytic activities of the various isolated microorganism were further examined and the superior strain YJ11-21 was selected for further analyses. Gene sequence analysis of 16S rDNA of the YJ11-21 strain revealed Bacillus licheniformis. Optimal culture conditions were investigated in order to maximize the production of the fibrinolytic enzyme by YJ11-21. Amongst the carbon sources tested, glucose was the most effective for enzyme production and amongst the nitrogen sources tested, yeast extract was seen to be the most effective. A one percent addition of NaCl to the medium resulted in the highest fibrinolytic activity. Interestingly, a 10% addition of NaCl resulted in a high activity together with a high cell growth rate. Therefore, YJ11-21 is speculated of being a halotolerant. The optimum pH and temperature for enzyme production were a pH of 9.0 and $30^{\circ}C$, respectively.

• 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.

• Microbiology and Biotechnology Letters
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• v.28 no.5
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• pp.258-263
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• 2000

A fibrinolytic enzyme was purified to homogeneity from Bacillus sp. S19 using DEAE and CM column chromatograhies, and gel filtration with a recovery yield of 13%. Its molecular mass was estimated to be 42 kDa by SDS-PAGE. The pH and temperature optima were 8.0 and $40^{\circ}C$, respectively. The enzyme was stable up to $45^{\circ}C$ and over a pH range of 6-9. The N-terminal amino acid sequence of the enzyme was determined as Alsa-Gln-Asp-Ala-Thr-Val-Asn-Ile-Ser-Ala-Glu-Arg-Gln-Val-Ile. The fibrinolytic activity was increased by $Cu^{2+}$ while it was strongly inhibited by metal ions such as $Cd^{2+}$ and $Ba^{2+}$ . In addition, the enzyme was inhibited by EDTA, but not by PMSF, suggesting that it is a metallorprotease.

• Preventive Nutrition and Food Science
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• v.14 no.4
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• pp.335-342
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• 2009

Bioactive compounds were produced from carrot pomace by solid-state fermentation using Bacillus subtilis HA and Leuconostoc mesenteroides. The carrot pomace (CP) fermented by B. subtilis HA with 3% monosodium glutamate (MSG) showed higher production of various bioactive compounds, with 1.64 Pa·sn of consistency, 2.31% of mucilage content, 16.95 unit/g of fibrinolytic enzyme activity, 35.3 unit/g of proteolytic activity and 37.5 mg% of tyrosine content. The mucilage production was greatly dependent upon the concentration of MSG added. Most MSG added in CP was converted into mucilage (2.3%) including 0.83% poly-$gamma$-glutamic acid (PGA) with 1,505 kDa of molecular weight. The CP fermented secondly by Leuc. mesenteroides showed acidic pH and lower consistency. However, the fibrinolytic and proteolytic activities were increased. The secondly fermented CP showed the viable cell counts with $2.5{\time}108$ CFU/g of B. subtilis HA and $3.7{\time}109$ CFU/g of Leuc. mesenteroides, respectively. The freeze-dried fermented CP showed 2.88 Pa·sn of consistency, 24% of mucilage content and 104.9 unit/g of fibrinolytic enzyme activity, respectively. Also, the powder of fermented CP indicated viable cell counts of $8.0{\time}107$ CFU/g of B. subtilis and $4.0{\time}108$ CFU/g of Leuc. mesenteroides. Therefore, the fermented CP that was fortified with dietary fibers, fibrinolytic enzyme and probiotics could be utilized as valuable ingredients of functional foods in food or cosmetic industries.

• Microbiology and Biotechnology Letters
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• v.31 no.3
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• pp.271-276
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• 2003

Bacillus amyloliquefaciens D4, which produces a strongly fibrinolytic enzyme, was isolated from Chungkook-Jang, a traditional Korean soybean-fermented food. B. amyloliquefaciens D4 was mutated with N-methyl-N-nitro-N-nitrosoguanidine (MNNG) to yield a series of mutants with increasing levels of fibrinolytic enzyme production. After mutation, a mutant D4-7 was obtained with fibrinolytic activity about eight times stronger than the parent strain. The fibrinolytic activity of B. amyloliquefaciens D4-7, reached a maximum, when the producer was cultivated in 2% Isolated Soy Protein (ISP) broth for 48 h at $37^{\circ}C$. Compared to commercial fibrinolytic enzymes, the cell-free culture supernatant of B. amyloliquefaciens D4-7 showed stronger activity than plasmin and streptokinase. The optimum temperature and pH were $50^{\circ}C$ and 10.0 and thermostability was increased by the addition of glycerol, glucose, and NaCl.

• BMB Reports
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• v.28 no.2
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• pp.138-142
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• 1995

An anticoagulant/fibrinolytic protease was purified to homogeneity from the earthworm Lumbricus rubellus. The protein was a single chain glycoprotein of 32 kDa that exhibited strong proteolytic activity on human thrombin and fibrin clots. Proteolytic degradation of these plasma proteins by the purified enzyme occurred at a neutral pH range. Among several human plasma proteins tested as possible substrates for the protease reaction, the 32 kDa enzyme specifically hydrolyzed both thrombin and fibrin polymers without affecting other proteins, such as serum albumin, immunoglobulin, and hemoglobin. Treatment of the purified enzyme at neutral pH with either phenylmethylsulfonylfluoride or soybean trypsin inhibitor resulted in a loss of catalytic activity. The enzyme hydrolyzed the chromogenic substrate H-D-Phe-L-Pipecolyl-L-Arg-p-nitroanilide with a $K_m$ value of 1.1 ${\mu}M$ at a neutral pH. These results suggest that the anticoagulant/fibrinolytic enzyme from Lumbricus rubellus is a member of the serine protease family having a trypsin-like active site, and one of the potential clevage sites for the enzyme is the carbonyl side of arginine residues in polypeptide chains.

• Fashion & Textile Research Journal
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• v.8 no.2
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• pp.239-244
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• 2006

For studies of fibrinolytic enzyme strain K-54 was isolated from the Korean traditional food chungkook-jang. Isolated strains K-54 was identified as Bacillus subtilis. The molecular weight of fibrinolytic enzyme from B. subtilis K-54 was 27 kDa. Optimum temperature for fibrinolytic enzyme of B. subtilis K-54 was $50-70^{\circ}C$ and optimum pH for producing the enzyme of this strain was ranging from 8 to 12. Also, it was found out enzyme activity was completely inhibited by 1mM PMSF. The result indicated this enzyme was thermo-stable alkaline serine protease with strong fibrinolytic activity. The wool and silk were treated with protease of B. subtilis K-54. As a result, the property of dyeing of wool fabrics was increased. By the increasing of treatment time became smoothened. But the change of mechanical properties were not changed.