• 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.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.9 no.3
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• pp.800-806
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• 2008

The strain HK-12 was enriched and isolated from naturally fermented soybean for the production of fibrinolytic enzyme and the proteome of this enzyme induced during the incubation period was analyzed. The activity of fibrinolytic enzyme derived from supernatants of the HK-12 culture was performed by fibrin plate method for solid fibrinolytic activity. As the result, the fibrinolytic activity of HK-12 grown on the nutrient agar media was about 2.3 times greater than that of plasmin used as standard. The purified enzyme was prepared by a series of purification process including ammonium sulfate precipitation, DEAE-cellulose, Sephadex chromatography. The molecular weight of the enzyme was determined to approximately 23kDa with SDS-PAGE. In order to examine which strain HK-12 proteins increased or decreased during the incubation period, 2-DE analysis was performed. Protein spot #1 significantly expressed on the 2-DE gel of bacteria cultivated for 36-hrs was analysed. As the result of protein sequence analysis using MALDI-TOF MS, one protein was identified as serine protein kinase (PrkA).

• 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.

• KSBB Journal
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• v.14 no.1
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• pp.103-108
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• 1999

A bacterium having strong fibrinolytic activity, S7-16 strain, was isolated from soil. The isolated bacterium was identified and named as Bacillus sp. S7-16. The optimal composition of the medium for the production of fibrinolytic enzyme by Bacillus sp. S7-16 was 0.5%(w/v) polypeptone, 0.5%(w/v) yeast extract, 0.3%(w/v) NaCl, 0.1% (w/v) $KH_2PO_4,\;0.3%(w/v)\;K_2PHO_4,\;and\;0.01%(w/v)\;MgSO_4{\cdot}7H_2O$. The optimal temperature and initial pH of the medium for the production of the enzyme were $35^{\circ}C$ and 7.0, respectively. The maximum production of the fibrinolytic enzyme was obtained after 24 hours of the incubation. Under the above conditions, the culture supernatant had strong fibrinolytic activity. Within pH4~11, the crude fibrinolytic enzyme was stable. The enzyme was stable up to $50^{\circ}C$. The optimum pH and temperature for the enzyme activity were around 7.5 and $40^{\circ}C$, respectively.

• Journal of Microbiology
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• v.44 no.6
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• pp.622-631
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• 2006

In this study we purified a fibrinolytic enzyme from Cordyceps militaris using a combination of ion-exchange chromatography on a DEAE Sephadex A-50 column, gel filtration chromatography on a Sephadex G-75 column, and FPLC on a HiLoad 16/60 Superdex 75 column. This purification protocol resulted in a 191.8-fold purification of the enzyme and a final yield of 12.9 %. The molecular mass of the purified enzyme was estimated to be 52 kDa by SDS-PAGE, fibrin-zymography, and gel filtration chromatography. The first 19 amino acid residues of the N-terminal sequence were ALTTQSNV THGLATISLRQ, which is similar to the subtilisin-like serine protease PR1J from Metarhizium anisopliae var. anisopliase. This enzyme is a neutral protease with an optimal reaction pH and temperature of 7.4 and $37^{\circ}C$, respectively. Results for the fibrinolysis pattern showed that the enzyme rapidly hydrolyzed the fibrin $\alpha$-chain followed by the $\gamma$-$\gamma$ chains. It also hydrolyzed the $\beta$-chain, but more slowly. The A$\alpha$, B$\beta$, and $\gamma$ chains of fibrinogen were also cleaved very rapidly. We found that enzyme activity was inhibited by $Cu^{2+}$ and $Co^{2+}$, but enhanced by the additions of $Ca^{2+}$ and $Mg^{2+}$ ions. Furthermore, fibrinolytic enzyme activity was potently inhibited by PMSF and APMSF. This enzyme exhibited a high specificity for the chymotrypsin substrate S-2586 indicating it's a chymotrypsin-like serine protease. The data we present suggest that the fibrinolytic enzyme derived from the edible and medicinal mushroom Cordyceps militaris has fibrin binding activity, which allows for the local activation of the fibrin degradation pathway.

• Journal of Life Science
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• v.20 no.6
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• pp.838-844
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• 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
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• v.36 no.1
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• pp.111-122
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• 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
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• v.23 no.7
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• pp.974-983
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• 2013

Bacillus amyloliquefaciens CB1 was isolated from cheonggukjang, a Korean fermented soy food. B. amyloliquefaciens CB1 secretes proteases with fibrinolytic activities. A gene homologous to aprE of Bacillus subtilis, aprECB1, was cloned from B. amyloliquefaciens CB1, and DNA sequencing showed that aprECB1 can encode a prepro-type serine protease consisting of 382 amino acids. When aprECB1 was introduced into B. subtilis WB600 using an E. coli-Bacillus shuttle vector, pHY300PLK, transformants showed fibrinolytic activity and produced a 28 kDa protein, the size expected for the mature enzyme. The 28 kDa fibrinolytic enzyme was purified from the culture supernatant of B. subtilis WB600 transformant. AprECB1 was completely inhibited by phenylmethylsulfonyl fluoride and almost completely inhibited by EDTA and EGTA, indicating that it is a serine metalloprotease. AprECB1 exhibited the highest specificity for N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, a known substrate for ${\alpha}$-chymotrypsin. $A{\alpha}$ and $B{\beta}$ chains of fibrinogen were quickly degraded by AprECB1, but the ${\gamma}$-chain was resistant.

• Food Science and Biotechnology
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• v.18 no.2
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• pp.500-505
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• 2009

The fibrinolytic enzyme, subtilisin D5, was purified from the culture supernatant of the isolated Bacillus amyloliquefaciens DJ-5. The molecular weight of subtilisin D5 was estimated to be 30 kDa. Subtilisin D5 was optimally active at pH 10.0 and $45^{\circ}C$. Subtilisin D5 had high degrading activity for the A$\alpha$-chain of human fibrinogen and hydrolyzed the $B{\beta}$-chain slowly, but did not affect the $\gamma$-chain, indicating that it is an $\alpha$-fibrinogenase. Subtilisin D5 was completely inhibited by phenylmethylsulfonyl fluoride, indicating that it belongs to the serine protease. The specific activity (F/C, fibrinolytic/caseinolytic activity) of subtilisin D5 was 2.37 and 3.52 times higher than those of subtilisin BPN' and Carlsberg, respectively. Subtilisin D5 exhibited high specificity for Meo-Suc-Arg-Pro-Tyr-pNA (S-2586), a synthetic chromogenic substrate for chymotrypsin. The first 15 amino acid residues of the N-terminal sequence of subtilisin D5 are AQSVPYGISQIKAPA; this sequence is identical to that of subtilisin NAT and subtilisin E.

• 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.