• International Journal of Industrial Entomology and Biomaterials
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• v.22 no.2
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• pp.83-93
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• 2011

Biochemical and enzymatic characterization for extracellular protease isolated from Cordyceps militaris cultivated on rice bran medium was investigated. C militaris produced proteolytic enzymes from 10 days after inoculation, maximum enzyme production was found at 25 days. The optimum temperature and pH of proteases production was at $25^{\circ}C$ and pH 7.0, respectively. The protease activity was observed in the four peaks (Pro-I, Pro-II, Pro-III, and Pro-IV) separated through Sephadex G-100 column chromatography. The separated protease was optimally active at $25^{\circ}C$. Optimum pH of the protease was between 7 and 8. Enzyme was also stable over at $30-80^{\circ}C$. The enzyme was highly stable in a pH range of 4-9. Protease activity was found to be slightly decreased by the addition of $Mg^{2+}$, $Mn^{2+}$, $Zn^{2+}$, $Fe^{2+}$ and $Cu^{2+}$, whereas inhibited by the addition of $Ca^{2+}$ and $Co^{2+}$ Protease activity was inhibited by protease inhibitor PMSF. On the other hand, the partially purified protease was investigated on proteolytic protease activity by zymogram gel electrophoresis using three substances (casein, gelatin and fibrin). Four active bands (F-I, FII, F-III, and F-IV) of fibrin degradation were revealed on fibrin zymogram gels. Both of F-II and FIII showed caseinolytic, fibrinolytic and gelatinolytic activities in three gels. Thermostability, pH stability, and pH-thermostability of the enzyme determined the residual fibrinolytic activity also displayed on fibrin zymogram gel. The only one enzyme (F-II) displayed over a broad range of temperature at $30-90^{\circ}C$. The FII displayed fibrinolytic activity in the pH range 3-5, but was inactivated in the range of pH 6-11. The F-I and F-III showed enzyme activity in the pH range of 6-11. In the pH-thermostability, the F-II only kept fibrinolytic activity after heating at $100^{\circ}C$ for 10, 20 and 30 min at pH 3 and pH 7, respectively. On the other hand, the F-II was retained activity until heating for 10 min under pH 11 condition. By using fibrin zymogram gel electrophoresis, extracellular fibrinolytic enzyme F-II from C. militaris showed unusual thermostable under acid and neutral conditions.

• Microbiology and Biotechnology Letters
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• v.48 no.4
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• pp.439-446
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• 2020

Two Bacillus strains, K3 and K208, both demonstrating strong fibrinolytic activities were isolated from Kimchi, a traditional Korean preparation of fermented vegetables. Isolates were subjected to various molecular biology based identification methods including RAPD-PCR and identified as B. subtilis and B. velezensis, respectively. Tryptic soy broth (TSB) was found to best maintain both the growth and the fibrinolytic activity of these strains. Culture supernatants were analyzed by SDS-PAGE and fibrin zymography, and the results indicate that a 40 and 27 kDa band seem to be responsible for the fibrinolytic activities of these two isolates and the 27 kDa band was subsequently identified as the mature form of AprE, the major fibrinolytic enzyme. Thus the aprE genes were cloned and the translated amino acid sequences demonstrated 99.3% identity with each other, and 86.5% identity with BsfA, a fibrinolytic enzyme from B. subtilis ZA400 also isolated from Kimchi, and AprE2, a fibrinolytic enzyme from B. subtilis CH3-5 isolated from Cheonggukjang, a traditional Korean fermented soy. Given this B. subtilis K3 and B. velezensis K208 may be promising starter cultures in the production of fermented foods.

• 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.

• Applied Biological Chemistry
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• v.46 no.3
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• pp.176-182
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• 2003

An alkaline fibrinolytic protease-producing bacteria was isolated front Korean traditional soy sauce and identified as Bacillus subtilis K7 from the results of analyses of its morphological and physiological properties, $API^{\circledR}$, and Biolog system. The enzyme was purified by 75% ammonium sulfate fractionation, QAE-Sephadex anion and SP-Sephadex cation exchange column chromatography and Sephadex G-100 gel filtration. The specific activity of the purified enByme was 233.9 unit/mg protein and the yield of enzyme was 3.8%. The homogeneity of the purified enzyme was confirmed by polyacrylamide gel electrophoresis. Molecular mass of the enzyme was estimated about 21,500 Da by SDS-polyacrylamide get electrophoresis and gel chromatography. The optimum temperature and pH for the enzyme activity were $40^{\circ}C$ and 9.0, respectively. The enzyme was stable in a pH range of 5.0 to 12.0, and 60% of its activity was lost on heat treatment at $50^{\circ}C$ for 20 min. The activity of the purified enzyme was inhibited by the presence of $Fe^{2+},\;Ag^{2+},\;Cu6{2+}$, iodoacetate, ethylene diamine tetraacetic acid (EDTA), and trans-1,2-diaminocycloheane-N,N,N',N'-tetraacetic acid (CDTA). The results indicates that the enzyme requires a metal ion for its enzymatic activity.

• Journal of Life Science
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• v.16 no.2 s.75
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• pp.274-281
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• 2006

A fibrinolytic enzyme gene was isolated from Bacillus subtilis BB-1 by PCR method. Primers for PCR cloning were designed according to pre-identified gene for fibrinolytic enzymes from B. subtilis. The primer sequences were 5'-CGG ATC CGT GAG AGG CAA AAA GGT G-3' and 5'-TGA ATT CTT AAT GTG CTG CTG CTT GTC C-3' as concensus sequences of the fibrinolytic genes of Bacillus species. The PCR product was 1,145 bp and the sequence homology was 99% with nattokinase gene isolated from Japanese natto. The cloned fibrinolytic gene was reconstructed in Bacillus-E. coli shuttle vector, pEB for bulk-production. The fibrinolytic enzyme was purified by FPLC from the cloned B. subtilis 168. The optimum pH and temperature of the enzyme were 7.0 and $35^{\circ}C$, respectively. The fibrinolytic enzyme did not show any activity toward to skim milk, gelatin, casein and blood agar plate. The enzyme specific polyclonal antibody was prepared in rabbit for further assays such as detection of the gene expression in plant cells. This means that the enzyme may be used for health-care such as thrombosis without any hamful effects in the blood vessel.

• Korean Journal of Food Science and Technology
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• v.31 no.1
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• pp.219-223
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• 1999

Various bacterial strains that secret extracellular fibrinolytic enzyme were screened from kimchi, a traditional vegetable fermented food in Korea. Three microbes of them were identified to be Bacillus amyloliquefaciens, Bacillus brevis and Micrococcus luteus strains according to Bergey's Manual of Systematic Bacteriology. It was found that B. amyloliquefaciens, B. brevis and M. luteus produced 2.58, 1.48 and 2.03 plasmin unit/mL of fibrinolytic enzyme, respectively. All extracellular proteases showing the fibrinolytic activity were confirmed by SDS-PAGE and fibrin zymography assay and we propose that some of the fibrinolytic enzymes from this work are novel enzymes.

• Journal of Microbiology and Biotechnology
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• v.17 no.6
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• pp.1018-1023
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• 2007

Bacillus subtilis CH3-5 was isolated from cheonggukjang prepared according to traditional methods. CH3-5 secreted at least four different fibrinolytic proteases (63, 47, 29, and 20 kDa) into the culture medium. A fibrinolytic enzyme gene, aprE2, encoding a 29kDa enzyme was cloned from the genomic DNA of CH3-5, and the DNA sequence determined. aprE2 was overexpressed in heterologous B. subtilis strains deficient in extracellular proteases using a E. coli-Bacillus shuttle vector. A 29 kDa AprE2 band was observed and AprE2 seemed to exhibit higher activities towards fibrin rather than casein.

• 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.

• Korean Journal of Food Science and Technology
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• v.31 no.6
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• pp.1648-1653
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• 1999

A strain of potential producer of fibrinolytic enzyme was isolated from shrimp Jeot-Gal, a tiny salted shrimps, and identified as Bacillus sp.. The preliminary experiment showed an enzyme yield of 18 U/mL in medium for screening. The carbon, nitrogen and salts significantly influenced the fibrinolytic enzyme production. An optimized medium containing 2% skim milk, 2% soluble starch and 3% NaCl (pH 7.5) after 72 hrs fermentation time at $37^{\circ}C$ yielded 3-fold increase in enzyme production, 62 U/mL.

• Journal of Microbiology and Biotechnology
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• v.25 no.9
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• pp.1449-1459
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• 2015

A novel proteolytic enzyme with fibrinolytic activity, FSP3, was purified from the recently isolated Streptomyces sp. P3, which is a novel bacterial strain isolated from soil. FSP3 was purified to electrophoretic homogeneity by ammonium sulfate precipitation, anion exchange, and gel filtration. FSP3 is considered to be a single peptide chain with a molecular mass of 44 kDa. The maximum activity of the enzyme was observed at 50℃ and pH 6.5, and the enzyme was stable between pH 6 and 8 and below 40℃. In a fibrin plate assay, FSP3 showed more potent fibrinolytic activity than urokinase, which is a clinical thrombolytic agent acting as a plasminogen activitor. The activity was strongly inhibited by the serine protease inhibitor PMSF, indicating that it is a serine protease. Additionally, metal ions showed different effects on the activity. It was significantly suppressed by Mg²⁺ and Ca²⁺ and completely inhibited by Cu²⁺, but slightly enhanced by Fe²⁺. According to LC-MS/MS results, its partial amino acid sequences are significantly dissimilar from those of previously reported fibrinolytic enzymes. The sequence of a DNA fragment encoding FSP3 contained an open reading frame of 1287 base pairs encoding 428 amino acids. FSP3 is a bifunctional enzyme in nature. It hydrolyzes the fibrin directly and activates plasminogen, which may reduce the occurrence of side effects. These results suggest that FSP3 is a novel serine protease with potential applications in thrombolytic therapy.