• Journal of Life Science
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• v.16 no.2 s.75
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• pp.360-364
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• 2006

Fibrin clots of blood vessels are one of the serious factor caused cardiovascular disease. The development of a antithrombotic and thrombolysis solvent is necessary to prevent and treat these diseases. It has been reported that a strong fibrin-specific fibrinolytic enzyme was produced from a Korean fermented soybean paste similar to Japanese miso. We have been screened the known or novel fibrinolytic enzymes by activity-based and sequence-based screening from soil DNA metagenome library containing all kinds of environmental genomic DNA. The activity-based screening was determined the protease activity on 0.5% skim milk. For sequence-based screening, we designed a set of primer expanding gene sequence of fibrinolytic enzyme, performed PCR and selected clones showing the expected size of amplicons from metagenome library. Transformation of the gene encoding fibrinolytic enzyme was carried out with commercial vectors and their transformants were selected. Finally, we found 15 positive clones from metagenome library. Then each of sequences were analyzed and identified as similar or known the clones of nattokinase. We are going to perform full sequence of each clones, ligate with expression vector, transform into competent cells and then determine activity of expressed enzymes.

• Biomedical Science Letters
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• v.12 no.3
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• pp.225-231
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• 2006

Fibrinolytic enzyme was purified from the fruiting bodies of Lepista nuda, using DEAE-Cellulose chromatography, Phenyl Sepharose chromatography, and Mono-S column chromatography. The substance has a molecular weight of 30006.62 Da as measured by MALD-TOF mass spectrometry. The N-terminal amino acid sequence of the enzyme was Tyr-Pro-Ser-Pro-Ser-His-Gln-Thr-Ala-Val-Asn-Ala-Ile-Ile-X. The activity of the enzyme was inhibited by PMSF, indicating that the enzyme is a serine protease. No inhibition was found with E-64, pepstatin, and EDTA. It has broad substrate specificity for synthetic peptides. The enzyme was stable up to $30^{\circ}C$. The enzyme hydrolyzes both Aa and y chains of human fibrinogen but did not show any reactivity for $B{\beta}$ chain of human fibrinogen.

• Journal of Life Science
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• v.23 no.2
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• pp.259-266
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• 2013

To isolate the fibrinolytic enzyme, 268 strains from 21 samples were morphologically isolated from Cheonggukjang collected from Korea and Japan. Among the 268 strains, protease-producing bacteria were isolated in nutrient agar medium including 1% skimmed milk. As a result of this, 22 strains were isolated. Apiweb site was used to identify these strains based on their biochemical properties. In addition, 16S rRNA sequencing was performed to identify the strain. Most of the identified strains were Bacillus subtilis and B. amyloliquefaciens. Fibrinolytic enzyme activity was measured with the fibrin plate method. Five strains were finally selected: A2-14, A2-20, C1-05, C1-09, and F2-01. Of those five strains, the A2-20 strain, which is close to B. amyloliquefaciens, showed the strongest fibrinolytic activity. The fibrinolytic enzyme produced by the A2-20 strain was partially purified from culture supernatant by gel filtration and ion exchange chromatography. The optimal pH and temperature values of the partially purified enzyme were 7.0 and $35^{\circ}C$, respectively. Purified protein analysis was carried out with SDS-PAGE and zymography. A genetic analysis was also conducted by PCR based on the consensus sequence of fibrinolytic enzyme. Corresponding genes with a partial sequence of the A2-20 strain were identified.

• Journal of Microbiology and Biotechnology
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• v.17 no.9
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• pp.1469-1476
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• 2007

Jeot-gal is a traditional Korean fermented seafood and has long been used for seasoning. We isolated 188 strains from shrimp, anchovy, and yellow corvina Jeot-gal, and screened sixteen strains that showed strong fibrinolytic activities on a fibrin plate. Among those strains, the strain that had the largest halo zone was chosen and identified as Bacillus licheniformis by using 16S rDNA sequencing and an API CHB kit. The fibrinolytic activity of Bacillus licheniformis was characterized and designated as bpKJ-31. The active component of bpKJ-31 was identified as a 37 kDa protein, designated bacillopeptidase F, by internal peptide mapping and N-terminal sequencing. The optimum activity of bpKJ-31 was shown at pH 9 and $40^{\circ}C$, with a chromogenic substrate for plasmin. It had high degrading activity for the $B{\beta}$-chain and $A{\alpha}$-chain of fibrin(ogen), and also acted on thrombin, but not skim milk and casein. The amidolytic activity of bpKJ-31 was inhibited by 1 mM phenylmethanesulfonyl fluoride, but 1 mM EDTA did not affect the enzyme activity, indicating that bpKJ-31 is an alkaline serine protease, like a plasmin. The bpKJ-31 showed approximately 14.3% higher fibrinolytic activity than the plasmin. These features of bpKJ-31 make it attractive as a health-promoting biomaterial.

• Journal of Life Science
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• v.11 no.6
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• pp.561-567
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• 2001

To isolate and purify fibrinolytic active substance from Sarcodon aspratus(N $H_4$)$_2$S $O_4$ precipitation, DE52 anion exchange column chromatography, Sephacryl-S 200gel filtration chromatography and Mono S cation FPLC were carried out and the characterizations of the purified enzyme were investigated. The bound active fraction on DE52 anion exchange column chromatography were eluted with 0.2 M NaCI and the fibrionlytic enzyme was purified after following Sephacryl-S200 gel fitration chromatography and Mono S cation EPLC. The specific activity of purified enzyme was 55.2 U/mg protein and increased 11.3 fold comparing crude extract and the yield was 49.5%. 12% SDS-PAGE electrophoresis and gel filtration chromatography revealed that Sarcodon aspratus fibrionloytic enzyme was highly purified and had 29.300 Da molecular weight. Enzyme activity of the purified fibrinolytic enzyme from Sarcodon aspratus was increased on higher pH and was stable until pH 10.5. On temperature dependent stability, the enzyme activity was decrease sharply but remained 25% relative activity on 8$0^{\circ}C$. This enzyme activity was inhibited by heavy metal ion, C $U^{2+}$ and $Co^{3+}$ with 68% and 38%, respectively. And also, the enzyme activity was inhibited with $Ca^{2+}$ chelator EDTA and serine protease inhibitor PMSF. These results from this study suggested that the fibrinolycit enzyme from Sarcodon aspratus is a serine protease and the enzyme activity was increased by $Ca^{2+}$ or $Mg^{2+}$ ion.n.ion.n.

• Preventive Nutrition and Food Science
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• v.19 no.3
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• pp.213-219
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• 2014

Bacillus pumilus 2.g isolated from gembus, an Indonesian fermented soybean cake, secretes several proteases that have strong fibrinolytic activities. A fibrinolytic enzyme with an apparent molecular weight of 20 kDa was purified from the culture supernatant of B. pumilus 2.g by sequential application of ammonium sulfate precipitation, ion-exchange chromatography, and hydrophobic chromatography. The partially purified enzyme was stable between pH 5 and pH 9 and temperature of less than $60^{\circ}C$. Fibrinolytic activity was increased by 5 mM $MgCl_2$ and 5 mM $CaCl_2$ but inhibited by 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM sodium dodecyl sulfate (SDS), and 1 mM ethylenediaminetetraacetic acid (EDTA). The partially purified enzyme quickly degraded the ${\alpha}$ and ${\beta}$ chains of fibrinogen but was unable to degrade the ${\gamma}$ chain.

• Journal of Microbiology and Biotechnology
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• v.24 no.2
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• pp.245-253
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• 2014

A fibrinolytic enzyme was produced by an edible mushroom of Pleurotus ostreatus using submerged culture fermentation. The enzyme was purified from the culture supernatant by applying a combination of freeze-thaw treatment, ammonium sulfate precipitation, hydrophobic interaction, and gel filtration chromatographies. The enzyme was purified by a 147-fold, with a yield of 7.54%. The molecular masses of the enzyme an determined by gel filtration and SDS-PAGE were 13.6 and 18.2 kDa, respectively. The isoelectric point of the enzyme was 8.52. It hydrolyzed fibrinogen by cleaving the ${\alpha}$ and ${\beta}$ chains of fibrinogen followed by the ${\gamma}$ chains, and also activated plasminogen into plasmin. The enzyme was optimally active at $45^{\circ}C$ and pH 7.4. The enzyme activity was completely inhibited by EDTA, whereas protease inhibitors of TPCK, SBTI, PMSF, aprotinin and pepstatin showed no inhibition on its activity. The partial amino acid sequences of the enzyme as determined by Q-TOF2 were ATFVGCSATR, GGTLIHESSHFTR, and YTTWFGTFVTSR. These sequences showed a high degree of homology with those of metallo-endopeptidases from P. ostreatus and Armillaria mellea. The purified enzyme can also be applied as a natural agent for oral fibrinolytic therapy or prevention of thrombosis.

• Biomedical Science Letters
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• v.13 no.3
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• pp.245-249
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• 2007

We investigated the fibrinolytic and ${\alpha}-glucosidase$ inhibitory activity of 55 wild mushroom methanol extracts. Among them, 14 mushrooms showed fibrinolytic activity. In particular, Amanita virgineoides showed the greatest enzyme activity (3.9 plasmin units/ml) by a fibrin plate assay. The fibrinolytic activities of Suillus pictus and Polypolellus varius were 3.8 plasmin units, and the activity of Gomphus fujisanensis was 2.8 plasmin units. Leccinum extremiorientale and Xerocomus nigromaculatus had the same activity with 2.3 plasmin units. In a ${\alpha}-glucosidase$ inhibitory activity test, Lactarius sp. showed the greatest inhibitory activity at 97.3%. The ${\alpha}-glucosidase$ inhibitory activities of Clitocybe odora, Xerocomus nigromaculatus, Melanoleuca melaleuca, Suillus pictus, and Gyroporus castaneus were 84.3%, 77.9%, 74.6%, 68.7%, and 65.4%, respectively. According to the results, because Suillus pictus and Xerocomus nigromaculatus have strong fibrinolytic and ${\alpha}-glucosidase$ inhibitory activities, the two mushrooms will be used as materials for the development of new biofunctional food.

• The Korean Journal of Soil Zoology
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• v.4 no.2
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• pp.101-106
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• 1999

Fibrinolytic enzyme is thought to be involved in extracellular matrix remodeling during regeneration. We investigated the expression and characterization fibrinolytic enzyme activity during earthworm tail regeneration. Electrophoretic analysis of fibrinolytic enzymes induced during regeneration revealed that at least seven types of fibrinolytic enzymes were expressed, which had molecular weight of 12, 19, 23, 27, 32, 45 and 58 kDa, respectively. These fibrinolytic enzyme activities were dramatically increased within 1 day after amputation. These activities were maintained by 7 days postamputation, followed by decrease to control level from 14 days after amputation. Alltypes of fibrinolytic enzyme activities were inhibited by treatment of PMSF and aprotinin, and were insensitive to EDTA and exogenous Ca$^{2+}$. These results indicate that the fibrinolytic enzymes are serineproteinase. Other characteristics including specificities for extracellular matrix proteins are under investigation. Based on these results, we are trying to find out the relationship among expression of proteinases, extracellular matrix remodeling, and dedifferentiation, which are believed to be essential processes during regeneration.

• Journal of Microbiology and Biotechnology
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• v.31 no.6
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• pp.833-839
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• 2021

Bacillus subtilis CH3-5 isolated from cheonggukjang secretes a 28 kDa protease with a strong fibrinolytic activity. Its gene, aprE3-5, was cloned and expressed in a heterologous host (Jeong et al., 2007). In this study, the promoter of aprE3-5 was replaced with other stronger promoters (P_cry3A, P₁₀, P_SG1, P_srfA) of Bacillus spp. using PCR. The constructed chimeric genes were cloned into pHY300PLK vector, and then introduced into B. subtilis WB600. The P10 promoter conferred the highest fibrinolytic activity, i.e., 1.7-fold higher than that conferred by the original promoter. Overproduction of the 28 kDa protease was confirmed using SDS-PAGE and fibrin zymography. RT-qPCR analysis showed that aprE3-5 expression was 2.0-fold higher with the P10 promoter than with the original promoter. Change of the initiation codon from GTG to ATG further increased the fibrinolytic activity. The highest aprE3-5 expression was observed when two copies of the P₁₀ promoter were placed in tandem upstream of the ATG initiation codon. The construct with P10 promoter and ATG and the construct with two copies of P10 promoter in tandem and ATG exhibited 117% and 148% higher fibrinolytic activity, respectively, than that exhibited by the construct containing P10 promoter and GTG. These results confirmed that significant overproduction of a fibrinolytic enzyme can be achieved by suitable promoter modification, and this approach may have applications in the industrial production of AprE3-5 and related fibrinolytic enzymes.