• Journal of Microbiology and Biotechnology
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• v.17 no.8
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• pp.1271-1283
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• 2007

A fibrinolytic protease (PoFE) was purified from the cultured mycelia of the edible oyster mushroom Pleurotus ostreatus, using a combination of various chromatographies. The purification protocol resulted in an 876-fold purification of the enzyme, with a final yield of 6.5%. The apparent molecular mass of the purified enzyme was estimated to be 32 kDa by SDS-PAGE, fibrin-zymography, and size exclusion using FPLC. The optimal reaction pH value and temperature were pH 6.5 and $35^{\circ}C$, respectively. PoFE effectively hydrolyzed fibrinogen, preferentially digesting the $A{\alpha}$-chain and the $B{\beta}$-chain over the ${\gamma}$-chain. Enzyme activity was enhanced by the addition of $Ca^{2+},\;Zn^{2+},\;and\;Mg^{2+}$ ions. Furthermore, PoFE activity was potently inhibited by EDTA, and it was found to exhibit a higher specificity for the chromogenic substrate S-2586 for chymotrypsin, indicating that the enzyme is a chymotrypsin-like metalloprotease. The first 19 amino acid residues of the N-terminal sequence were ALRKGGAAALNIYSVGFTS, which is extremely similar to the metalloprotease purified from the fruiting body of P. ostreatus. In addition, we cloned the PoFE protein, encoding gene, and its nucleotide sequence was determined. The cDNA of cloned PoFE is 867 nucleotides long and consists of an open reading frame encoding 288 amino acid residues. Its cDNA showed a high degree of homology with PoMEP from P. ostreatus fruiting body. The mycelia of P. ostreatus may thus represent a potential source of new therapeutic agents to treat thrombosis.

• Toxicological Research
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• v.17
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• pp.97-104
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• 2001

Three types of proteases (MEF-1, MEF-2 and MEF-3) were purified from the egg cases of Ten-odera sinensis using ammonium sulfate fractionation, gel filtration on Bio-Gel P-60 and affinity chromatography on DEAE Affi-Gel blue gel. The proteases were assessed homogeneous by SDS-polyacrylamide gel electrophoresis and have molecular weight of 31,500, 32,900 and 35,600 Da, respectively. The N-terminal regions of the primary structure were compared and they were found to be different each other. MEFs readily digested the $A\alpha$ - and B$\beta$-chains of fibrinogen and more slowly the ${\gamma}$-chain. The action of the enzymes resulted in extensive hydrolysis of fibrinogen and fibrin, releasing a variety of fibrinopeptides. MEF-1 was inactivated by Cu$^{2+}$ and Zn$^{2+}$ and inhibited by PMSF and chymostatin. MEF-2 was inhibited by PMSF, TLCK. soybean trypsin inhibitor. MEF-3 was only inhibited by PMSF and chymostatin. Antiplasmin was not sensitive to MEF-1 but antithrombin III inhibited the enzymatic activity qf MEF-1. MEF-2 specifically bound to anti plasmin Among the chromogenic protease substrates, the most sensitive one to the hydrolysis of MEFs was benzoyl-Phe-Val-Arg-p-nitroanilide with maximal activity at pH 7.0 and 3$0^{\circ}C$. MEF-1 preferentially cleaved the oxidized B-chain of insulin between Leu15 and Tyr16. In contrast, MEF-2 specifically cleaved the peptide bond between Arg23 and Gly24. D-dimer concentrations increased on incubation of cross-linked fibrin with MEF-1, indicating the enzyme has a strong fibrinolytic activity.ity.

• Journal of Life Science
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• v.20 no.1
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• pp.82-89
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• 2010

This study was investigated to analyze the contents of flavonoid compounds and the effects of fermentation on the physiological activities of medical plants, also known as SanYa (SY). Antioxidative activity of the fermented SanYa (FSY) was measured by using DPPH radical scavenging and SOD-like activity. DPPH radical scavenging and SOD-like activity of FSY were 94.3% and 45.0%, respectively. Nitric oxide (NO) synthesis was increased 11 times through the addition of FSY. However, NO production of the macrophages RAW264.7 cells stimulated with lipopolysaccharide (LPS) was reduced to 56% through the addition of FSY. FSY showed fibrinolytic activity and indicated about 69.8% and 73.7% of xanthine oxidase and angiotensin converting enzyme inhibitory activities, respectively. These results suggested that FSY plays a significant role in fibrinolytic activity and have strong xanthine oxidase and angiotensin converting enzyme inhibitory activities.

• Biomolecules & Therapeutics
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• v.3 no.2
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• pp.165-170
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• 1995

The biochemical properties of the fibrinolytic protease of 50,800 Da isolated from the venom of Kgdistrodon blomhoffi brevicaudus were characterized. The enzyme hydrolyzed the carboxyl side of arginine in the synthetic chromogenic peptides, N-Benzoyl-Phe-Val-Arg-pNA and N-p-Tosyl-Gly-Pro-Arg-pNA, and the enzyme activity was inhibited by phenylmethylsulfonylfluoride indicating that the enzyme belongs to the serine protease family. The pretense showed maximum activity at pH 7.5 and inhibited by ZnCl$_2$, CuSO$_4$, but not by soybean trypsin inhibitor, pepstatin A, 2-mercaptoethanol and EDTA. The fm value determined with N-p-Tosyl-Gly-Pro-Arg-pNA was 0.2 mM. The thrombolytic activity of the purified enzyme was evaluated by platelet aggregation test in rabbits. While the platelet count ratio in blood of the rabbits injected with thrombin alone declined from 1.0 to 0.6 within 7 min and maintained around 0.6 for 24 hours thereafter, the ratio rapidly recovered from around 0.6 to 0.8 in 1 hr, to 1.0 in 24 hrs when the rabbits were sequentially treated with thrombin and the purified enzyme. The result showed that the serine protease from A. blomhoffi brevicoudus of 50,800 Da had a thrombolytic activity in vivo and the enzyme might be developed as a therapuetic agent for the treatment of thrombic disease.

• The Korean Journal of Mycology
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• v.33 no.1
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• pp.11-17
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• 2005

To increase the fibrinolytic activity and production of mycelium, extracts of 7 plant species were supplemented to the growth media of Armillaria mellea, and mycelial growth and enzymatic activity in the mycelium extracts of A. mellea were estimated. The mycelial production of A. mellea was slightly increased by adding ASH-R, UDVN or RGR extract, whereas KG extract significantly affected the growth. Supplement of ASH-S, UDVN and RGR extracts increased proteolytic activity from 36.8 to 46.1% Fibrinolytic activity was increased to $50{\sim}65%$ by supplement with RVS, ASH-S and RGR extracts, respectively. Enzyme extracts of the fungus grown with RGR extract supplement degraded all chains of fibrinogen within 2 hours, whereas control was required 3 hours. Degradation of fibrin fragments by the enzyme extracts was also observed through microscopy.

• Journal of Microbiology and Biotechnology
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• v.15 no.1
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• pp.72-79
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• 2005

A new Bacillus peptidase, bacillopeptidase DJ-2 (bpDJ-2), with molecular mass of 42 kDa and isoelectric point (pI) of 3.5- 3.7, was purified to homogeneity from Bacillus sp. DJ-2 isolated from Doen-Jang, a traditional Korean soybean fermented food. The enzyme was identified as an extracellular serine fibrinolytic protease. The optimal conditions for the reaction were pH 9.0 and $60^{\circ}C$. The first 18 amino acid residues of the N-terminal amino acid sequence of bpDJ-2 were TDGVEWNVDQIDAPKAW, which is identical to that of bacillopeptidase F (bpf). However, based on their Nterminal amino acid sequence, molecular size, and pI, it is different from that of bpf and extracellular 90 kDa. The whole (2,541 bp, full-bpDJ-2) and mature (1,956 bp, mature-bpDJ-2) genes were cloned, and its nucleotide sequence and deduced amino acid sequence were determined. The expressed proteins, full-bpDJ-2 and mature-bpDJ-2, were detected on SDSPAGE at expected sizes of 92 and 68 kDa, respectively.

• Proceedings of the Korean Society of Life Science Conference
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• 2007.10a
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• pp.87.1-87.1
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• 2007

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• Korean Journal of Food Science and Technology
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• v.43 no.6
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• pp.735-741
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• 2011

Bealmijang is a short-term fermented regional product that is prepared with soybean and extra ingredients. In this study, starter strain candidates were screened from Bealmijang for fermented soybean paste products. Twenty one bacterial strains producing extracellular enzymes (amylase, cellulase, protease, xylanase and lipase) were isolated from Bealmijang, buckwheat sokseongjang. The isolates were assessed for fibrinolytic and antibacterial activities, and salt tolerance. Strain HJ18-4, identified as Bacillus subtilis (AB601598) by biochemical properties (89.6%) and 16S rDNA sequencing (100%), showed the highest enzymatic, fibrinolytic, and antibacterial activities among the isolates. Although the growth of HJ18-4 was inhibited by the increase of NaCl concentration, the growth still exceeded that of B. subtilis KACC 10114 at 5% and 10% NaCl. These results suggest that B. subtilis HJ18-4 is suitable as a starter for soybean paste manufacture.

• KSBB Journal
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• v.16 no.2
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• pp.153-157
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• 2001

Alcohol fermentation conditions for the production of plum wine were investigated and further, sensory evaluation and physiological functionalities of the plum wines were also determined and compared with those of plum liquors made by soaking plums in a mixture of commercial soju and 10% sugar for 15, 30, 60 and 120 days. Ethanol was produced maximally when 5% Saccharomyces cerevisiae was added to red plum juices and fermented at 25$^{\circ}C$ for 5 days. Angiotensin-converting enzyme inhibitory activity and fibrinolytic activity of the red plum wine were better than those of the plum liquors. However, the antioxidant activity, the SOD-like activity and the tyrosinase inhibitory activity of the plum liquors were better than those of the red plum wine. On comparing the red plum wine and the various kinds of plum liquors, the red plum wine was shown to be more acceptable by sensory evaluation.

• Food Science and Biotechnology
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• v.14 no.6
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• pp.772-777
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• 2005

The goal of this study was to screen and characterize the physiological functions of Korean traditional wines (TW) and liquors (TL). Forty-two TW and TL were collected and evaluated for quality and cardiovascular activities. Ethanol content ranged from $9.0%{\sim}41%$, and pH ranged from $3.0{\sim}7.8$, and they also contained 0.01% to 0.67% of total acid. Samples contained a maximum of 2.0% of crude protein and $0.1%{\sim}14.0%$ of reducing sugar. Commercial CM-wine showed the highest antihypertensive angiotensin I-converting enzyme (ACE) inhibitory activity, 85.9%. The greatest fibrinolytic activity and platelet aggregation inhibitory activity were also found in commercial CM-wine (31.8U) and commercial SS2-wine (38.6 %), respectively. Commercial SHBI-liquor showed the highest HMG-CoA reductase inhibitory activity, 78%. The ACE inhibitor from commercial CM-wine was a peptide compound and also showed an antihypertensive effect in spontaneous hypertensive rats at a dosage of 1.5 mg/kg.