• Journal of Life Science
• /
• v.19 no.10
• /
• pp.1478-1483
• /
• 2009

A solid-state culture based on grain materials was attempted to produce a fibrinolytic enzyme for blood circulation improvement using Bacillus subtilis BK-17. The spore, rather than vegetative cell inoculation, of B. subtilis BK-17 on the solid-state culture was effective in the production of a fibrinolytic enzyme. Maximum spore production was obtained with a SFM medium (0.8% nutrient broth, 0.05% yeast extract, $10^{-1}$ M $MgCl_2$, $10^{-3}$ M $FeCl_3$, $10^{-4}$ $MnCl_2$, $10^{-5}$ M dipicolic acid, pH 6.5). Optimal pH and temperature were pH 6 and $30^{\circ}C$, respectively. The spore production reached a maximum at 60 hours of incubation. Bacillus subtilis BK-17 on the mung bean solid-state culture produced greater fibrinolytic activity, and less activity was seen in other grains such as kidney bean, soybean and corn. Protein and lipid contents of fermented soybeans were about 10 - 30% more than those of unfermented soybeans. Amino acid content was also 5 - 20% more than that of unfermented soybeans. These results indicated that fermented solid-state culture medium, fermented soybean in this case, can be utilized as a food supplement.

• Microbiology and Biotechnology Letters
• /
• v.41 no.1
• /
• pp.33-43
• /
• 2013

A bacterium producing a fibrinolytic enzyme was isolated from Cheonggukjang. The bacterium was identified as a strain of Bacillus amyloliquefaciens by 16S rDNA analysis and designated as B. amyloliquefaciens HC188. The optimum culture medium appeared to be one containing 0.5% (w/v) maltose and 0.5% (w/v) soytone. Bacterial growth in the optimal medium at $37^{\circ}C$ reached the stationary phase after 27 h of incubation and the fibrinolytic enzyme showed optimum activity at 24 h. The enzyme was purified by 20-80% ammonium sulfate precipitation, CM Sepharose fast flow ion exchange chromatography, and Sephacryl S-200HR column chromatography. Its specific activity was 38359.3 units/mg protein and the yield was 5.5% of the total activity of the crude extracts. The molecular weight was 24.7 kDa and the amino acids of the N-terminal sequence were AQSVPYGVSQIKAPA. The fibrinolytic enzyme activity had an optimum temperature of $40^{\circ}C$ and an optimum pH of 8.0, and the enzyme was stable in the ranges $20-40^{\circ}C$ and pH 6.0-8.0. Enzyme activity was increased by $Ca^{2+}$ and $Co^{2+}$ but inhibited by $Cu^{2+}$, EDTA, and PMSF. It is suggested that the purified enzyme is a metallo-serine protease.

• Journal of Life Science
• /
• v.15 no.3 s.70
• /
• pp.434-438
• /
• 2005

In the previous study, we isolated a myulchikinase (MK), which has fibrinolytic activity and cytotoxicity to the tumor cell line, from myl- chi-jeot-gal. In this study, the effect of NaCl concentration, metallic ions, pH, temperature, and plasminogen on the activity of MK was analysed. The MK activity was maintained at least $80\%$ activity up to $30\%$ NaCl, which indicates that the enzyme may be halotolerant. The optimal pH and temperature were 8 and $40^{\circ}C$, respectively. The fibrinolytic activity of MK was completely inhibited with 0.5 mM $Hg^{2+}$ and inhibited to $50^{\circ}C$ with 1 mM $Cu^{2+}\;and\;Zn^{2+}$. The MK showed strong activity in plasminogen- rich fibrin plate but not in plasminogen-free fibrin plate. The result indicates that the MK may be a plasminogen activator type fibrinolytic enzyme.

• Journal of Life Science
• /
• v.20 no.6
• /
• pp.838-844
• /
• 2010

A fibrinolytic enzyme of Streptomyces corcohrussi from soil sediment was purified by chromatography using DEAE-Sephadex A-50 and Sephadex G-50. The analysis of SDS-polyacrylamide gel suggested that the purified enzyme is a homogeneous protein and the molecular mass is approximately 34 kDa. The purified enzyme showed activity of 0.8 U/ml in a plasminogen-rich fibrin plate, while its activity in a plasminogen-free fibrin plate was only 0.36 U/ml. These results suggested that the purified enzyme acts as a plasminogen activator. The fibrinolytic activity of the enzyme under the supplementation of protease inhibitors, $\varepsilon$-ACA, t-AMCHA and mercuric chloride in the enzyme reaction was less than 24%, indicating that it could be modulated by the plasmin and/or fibrinogen inhibitors involved in the fibrinogen-to-fibrin converting process. As time passed, $Zn^{2+}$, a heavy metal ion, inhibited the activity to 34.1%. The optimum temperature of the purified enzyme was approximately $50^{\circ}C$ and over 92% of the enzyme activity was maintained between pH 5.0 and 8.0. Therefore, our results provide a potential fibrinolytic enzyme as a noble thrombolytic agent from S. corcohrussi.

• Korean Journal of Agricultural Science
• /
• v.36 no.1
• /
• pp.111-122
• /
• 2009

When the cattail pollen was identified by using fibrinolytic agents, we found that the fibrinolytic activity was controlled by an enzyme. Therefore, for determining the fibrinolytic activity of cattail pollen, the fibrinolytic enzyme in cattail pollen was purified by gel filtration using DEAE-cellulose, Sephadex G-150 and HPLC. Also, its purity was certified by polyacrylamide gel electrophoresis, and its physico-chemical properties, such as pH and temperature stabilities and effects of metal, inhibitors and substrates, were examined. The specific activity, purification fold, and molecular weight of the enzyme were 38U/mg, 86.4,and 75kDa, respectively. The optimum pH for the purified enzyme was at 4.0 and it was stable at pH 4.0-6.0. The optimum temperature was $55^{\circ}C$ and it was stable at $30-60^{\circ}C$. But the enzyme began to be inactivated at $70^{\circ}C$ and its activity was totally lost at temperatures above $80^{\circ}C$. As for substrate specificity, the enzyme was most effective in dissolving fibrin, followed by whole casein, ${\kappa}$-casein, ${\alpha}$-casein, ${\beta}$-casein, and BSA. With casein as the substrate, Km value was found to be 0.44mM and the enzyme showed a high affinity for casein. As for the metal ions affecting enzyme activity, $K^+$, $Na^+$, and $Mg^{2+}$ had no effect on enzyme reaction while $Zn^{2+}$ and $Fe^{2+}$ showed potent inhibitory activity. Judging from the fact that the purified enzyme was also strongly inhibited by PMSF, iodoacetic acid, and SDA, it assumed to be a serine protease.

• Journal of Microbiology and Biotechnology
• /
• v.30 no.11
• /
• pp.1720-1728
• /
• 2020

We have previously characterized AprESJ4, the major fibrinolytic enzyme from Bacillus subtilis SJ4 (Yao et al., 2019). During that study, we observed a 68 kDa protein with fibrinolytic activity. In this study, we cloned the gene (vprSJ4) encoding the 68 kDa protein, a mature Vpr and minor protease secreted by Bacillus species. vprSJ4 encodes a preproenzyme consisting of 810 amino acids (aa) including signal sequence (28 aa) and prosequence (132 aa). The mature enzyme (650 aa) has a predicted molecular weight of 68,467.35. Unlike Vprs from other B. subtilis strains, VprSJ4 has 4 additional amino acids (DEFA) at the C-terminus. vprSJ4 was overexpressed in Escherichia coli. PreproVprSJ4 was localized in inclusion bodies, and subjected to in vitro renaturation and purification by an affinity column. SDS-PAGE and western blot showed that autoprocessing of preproVprSJ4 occurred and 68 kDa and smaller proteins were produced. The optimum pH and temperature of the recombinant VprSJ4 were pH 7.0 and 40℃, respectively. Kinetic parameters of recombinant VprSJ4 were measured by using an artificial substrate, N-succinyl-ala-ala-pro-phe-p-nitroanilide. Coexpression of vprSJ4 and aprESJ4 using pHY300PLK increased the fibrinolytic activity a further 117% when compared with aprESJ4 single expression using the same vector in B. subtilis WB600.

• Korean Journal of Microbiology
• /
• v.45 no.4
• /
• pp.377-384
• /
• 2009

Among 27 Bacillus sp. isolated from Meju, a traditional Korean soybean fermented food, a strain MJ-226 was selected due to its strong fibrinolytic activity, and it was identified to be Bacillus subtilis MJ-226 according to morphological and biochemical characterization and sugar utilization. The fibrinolytic enzyme of B. subtilis MJ-226 was maximally produced by cultivating in the Tryptic Soy Broth (TSB) for 24~26 h at $37^{\circ}C$, and the enzymes activity was promoted with adding glucose, fructose, peptone or yeast extract to TSB. The fibrinolytic enzyme was stable at the range of pH from 6.0 to 8.0, and between 35 and $40^{\circ}C$. Also, when the crude enzyme was exposed to various metal ions and chemical inhibitors for 12 h, the enzyme stability was maintained by $MnSO_4$, $CaCl_2$, KCl, and NaCl. However, the stability was destroyed by treatment with $CuSO_4$, $MgSO_4$, $ZnSO_4$, $FeSO_4$, and $BaCl_2$, and the enzyme was unstable in the presence of chemical inhibitors such as iodoacetic acid, leupeptin, phenylmethanesulphonyl fluoride (PMSF), sodium dodecyl sulfate (SDS), thiourea, trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) and ethylenediaminetetraacetic acid (EDTA).

• Journal of Life Science
• /
• v.17 no.5 s.85
• /
• pp.606-612
• /
• 2007

Fibrin clots remained in blood vessels can be one of the serious factor caused cardiovascular disease, such as ischemia, infarction and necrosis The development of an antithrombotic and thrombolysis solvent is necessary to prevent and treat these diseases. In this study, the fibirinolytic protease was prepared from the maggots of Protaetia brevitarsis using ammonium sulfate fractionation and desalting column. The optimum pH and temperature for the enzyme activity were pH 9.0 and $50^{\circ}C$, respectively. The enzyme activity was relatively stable at pH 7.0-9.0 and temperature below $60^{\circ}C$. The activity of the enzyme was strongly inhibited by phenylmethanesulfonyl fluoride. And the activity of the enzyme was inhibited by $Ca^{2+}\;and\;Zn^{2+}$, but it was not by $Mg^{2+}\;and\;Fe^{2+}$ ions. In these experimental results, we have speculated that the enzyme derived from maggots of Protaetia hrevitarsis is a serine protease with a strong fibrinolytic activity.

• Korean journal of food and cookery science
• /
• v.19 no.3
• /
• pp.263-270
• /
• 2003

This study was conducted to investigate the isoflavone (daidzein and genistein) contents, and the antioxidative and fibrinolytic activities of red and mung beans. Daidzein was not found in either the red or mung beans. The genistein contents of the red and mung beans were 40.7 and 27.8 mg/kg, respectively. Both samples had very high electron donating abilities and SOD-like activities. Fibrinolytic activities were not detected in the crude mung bean extract, but fibrinolytic substances were purified or activated under various pH conditions and with heat treatments. With heat treatment at 100 $^{\circ}C$ for 10 min, the specific activity was increased 4.61 fold. This study revealed that, although red and mung beans were poor in isoflavone, they could be good sources for functional products due to their strong antioxidative activities and heat- and acid-resistant proteolytic abilities.

• Korean Journal of Food Science and Technology
• /
• v.34 no.3
• /
• pp.498-504
• /
• 2002

Isoflavone (daidzein and genistein) contents, and antioxidative and fibrinolytic activities of seven commercial Korean cooking-with-rice soybeans, including Seonbikong, Chungtae, Gangnamkong, Whangtae, Geodoo, Seolitae, and Wooltalikong were investigated. Daidzein and genistein were not found in Selitae, nor was didzein in Gangnamkong and Geodoo. Total daidzein and genistein levels in Chungtae and Whangtae were 500 and 1550 mg per kg, respectively. Wooltalikong, Whangtae, and Gangnamkong had very high electron donation abilities, over 90%, but Seonbikong and Chungtae showed significantly lower activities. SOD-like activities were also the highest in Gangnamkong and Wooltalikong. Fibrinolytic activities in Seonbikong, Whangtae, and Gangnamkong were similarly strong. Fibrinolytic substances purified from protease inhibitors or activated under various pH or heat treatment conditions, were different among the soybean varieties. This study revealed that, although several cooking-with-rice soybeans were poor in isoflavones, Wooltalikong and Gangnamkong could be good sources for functional products due to their strong antioxidative activities, and heat- and acid-resistant proteolytic abilities.