• Microbiology and Biotechnology Letters
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• v.16 no.6
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• pp.522-525
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• 1988

A method to produce tissue type Plasminogen Activator (tPA) from normal human fibroblast is developed by cultivating cells in serum free media containing heparin as an inducer. Optimal dose of this inducer was 30$\mu$g/m$\ell$. The composition of serum free medium was also defined to fit to the industrial scale cultivation. 1.42 ug of tPA per 10$^5$ viable cells per ml was produced. 1.1 gram of tPA can be produced every day from this cell line under normal perfusion chemostat operations assuming that same productivity is maintained when the process is sealed up. This method could reduce pro-duction costs and simplify purification processes by using serum free medium. Tissue type PA produced from this cell line has high ability of dissolving clots, based upon fibrin lysis test showing 50mm$^2$ of clearing zones in agarose gel plate. These results were reproducible and in good agreement with results of ELISA assay. tPA from normal human cells will be safer than that from melanoma and recombinant cells in human clinical trials.

• The Korean Journal of Mycology
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• v.33 no.2
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• pp.69-74
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• 2005

Fibrinolytic enzyme has been isolated and purified from the edible mushroom, Lepista nuda. The apparent molecular mass of purified enzyme was estimated to be 34 KDa by SDS-polyacrylamide gel electrophoresis. 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. It has a pH optimum at $7.0.{\sim}9.5$, suggesting that the purified enzyme is an alkaline protease. It shows the maximum fibrinolytic activity at $55^{\circ}C$. The fibrinolytic activity was inhibited by phenylmethylsulfonyl fluoride, indicating that the purified enzyme is a serine protease. The activity of the purified enzyme was totally inhibited by $Hg^{2+}$.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.7 no.3
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• pp.476-482
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• 2006

The aim of this work was to perform the screening and identification of the bacterium, MK-15 having the activity of fibrinolytic enzyme for the commercial use. Initially, strain MK-15 was enriched and isolated from naturally fermented soybean. Morphological and various physiological characteristics of the strain MK-15 was examined. The activity of fibrinolytic enzyme derived from supernatants of test culture MK-15 was performed by fibrin plate method for solid fibrinolytic activity. As the result, the fibrinolytic activity of MK-15 grown on the soybean media was about 2.7 times greater than that of plasmin used as stardard. 16S rRNA analyses revealed that strain MK-15 was 99.9% similar to Bacillus subtilis species cluster, and the bacterium was designated as Bacillus sp. MK-15. Strain MK-15 was registered in GenBank as [DQ163021].

• KSBB Journal
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• v.9 no.1
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• pp.48-54
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• 1994

A modified amidolytic assay and a fibrin plate method were used to accurately measure the concentration of single chain urokinase type plasminogen activator (scu-PA) and two-chain urokinase type plasminogen activator (tc-PA) in the spent media. $1.65{\times}10^6$(viable cells/ml) of maximum cell density and 1670(IU/ml) of scu-PA concentration were obtained in 1% serum containing medium. The overall conversion ratio from scu-PA to tc-PA was less than 10%. In the results of batch cultivation in a spinner vessel, $4.43{\times}10^6(total cells/ml)$ of maximum cell density and 1560(IU/ml) of scu-PA concentration was observed. The maximum scu-PA concentration and specific scu-PA Productivity were obtained in 1760(IU/ml) and $3.13{\times}10^{-4}(IU/cell)$, respectively, from perfusion cultivation. The conveysion ratios from batch, fed-batch and perfusion cultivations were less than 12%, which means that about 90% of scu-PA secreted from the cells can be maintained during the cultivations.

• Korean Journal of Food Science and Technology
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• v.37 no.2
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• pp.255-260
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• 2005

To make Chungkuk-jang with enhanced fibrinolytic and immunostimulating activities, 220 strains isolated from Chungkuk-jangs were tested, and 13 Bacillus strains with excellent proteolytic and polysaccharide-producing activities were selected and tested for their fibrinolytic and immunostimulating activities using fibrin plate method and RAW 264.7 cell line, respectively. To assess macrophage activation, contents of cytokines such as tumor necrosis factor ($TNF-{\alpha}$) and $interleukin-1{\alpha}$ and nitric oxide were measured. Three strains showing highest fibrinolytic and immunostimulating activities were identified as Bacillus licheniformis (CHKJ 1249, 1326) and Bacillus subtilis (CHKJ 1339).

• 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).

• Food Science and Preservation
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• v.12 no.6
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• pp.643-649
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• 2005

This study was investigated to find out optimal medium maximizing the production of fibrinolytic enzyme by Bacillus sp. isolated from Korean fermented soybean paste, which could hydrolyze the fibrin produced through the blood coagulation mechanism in human body. Among carbon sources tested, galactose was most effective for the enzyme production, and the level of the concentration for the optimal enzyme production was $4\%$(w/v). For nitrogen sources tested, malt extract was most effective for the enzyme production, and level of the concentration for optimal enzyme production was $4\%$(w/v). For mineral sources tested, $K_2HPO_4$ was most effective for enzyme production. The enzyme was maximally produced by cultivating the organism at the liquid medium of the initial pH 6 and temperature of $40^{\circ}C$.

• BMB Reports
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• v.38 no.2
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• pp.177-181
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• 2005

Effects of common electrophoretic reagents, reducing agents ($\beta$-mercaptoethanol [BME] and DTT), denaturants (SDS and urea), and non-ionic detergent (Triton X-100), on the activity and stability of bovine plasmin (b-pln) and human plasmin (h-pln) were compared. In the presence of 0.1% SDS (w/v), all reagents completely inhibited two plns, whereas SDS (1%) and urea (1 M) denatured plns recovered their activities after removal of SDS by treatment of 2.5% Triton X-100 (v/v). However, reducing agents (0.1 M of BME and DTT) treated plns did not restore their activities. Based on a fibrin zymogram gel, five (from b-pln) and four (from h-pln) active fragments were resolved. Two plns exhibited unusual stability in concentrated SDS and Triton X-100 (final 10%) and urea (final 6 M) solutions. Two bands, heavy chain-2 (HC-2) and cleaved heavy chain-2 (CHC-2), of b-pln were completely inhibited in 0.5% SDS or 3 M urea, whereas no significant difference was found in h-pln. Interestingly, 50 kDa (cleaved heavy chain-1, CHC-1) of b-pln and two fragments, 26 kDa (light chain, LC) and 29 kDa (microplasmin, MP), of h-pln were increased by SDS in a concentration dependent manner. We also found that the inhibition of SDS against both plns was reversible.

• 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.

• Food Science and Preservation
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• v.13 no.2
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• pp.241-245
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• 2006

The biofunctional properties of traditional Doenjang supplemented with the extract of herb medicines (refer as DHM) were examined DHM divided to four groups, I, II, III and IV according to herb medicines were added. Nitrite scavenging-activities from all 4 groups of DHMs were significantly higher than that of control. Also hydrogen donating-activities from all 4 groups of DHMs were slightly higher than that of control. Antibacterial activities against pathogenic bacteria such as Streptococcus mutant and Salmonella enteritidis and fibrinolytic activity of all DHMs on fibrin plate were higher than those of control. On the other those of groups III and IV were higher in sensory score, biofunctional and antimicrobial activities than those of group I, II and control. Therefore, the usage of extract of Korean herb medicines instead of water to make Doenjang will effective in development of traditional soybean Doenjang with the biofunctional properties.