• The Korean Journal of Mycology
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• v.35 no.2
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• pp.128-132
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• 2007

The aim of this study was to investigate the physiological functionalities of 60 wild mushroom methanol extracts. We determined their fibrinolytic and ${\alpha}-glucosidase$ inhibitory activities. Among them, 11 mushrooms showed fibrinolytic activity. In particular, Armillaria sp. showed the greatest enzyme activity (4.2 plasmin units/ml) in a fibrin plate assay. The fibrinolytic activities of Amanita virosa. Gymnophilus sp. and Lepista nuda were 2.3, and the activities of Amanita citrina and Cortinarius alboviolaceus were 2.1 and 2.0 plasmin units, respectively. In a ${\alpha}-glucosidase$ inhibitory activity test, Naematoloma fasciculare showed the greatest inhibitory activity at 98.5%. The ${\alpha}-glucosidase$ inhibitory activities of Suillus luteus and Gomphidius maculatus were 66.6% and 61.5%, respectively. This study suggests that both Armillaria sp. and N. fasciculare can be used as candidates for the development of new medicine effective to cardiovascular diseases and biofunctional food.

• Applied Biological Chemistry
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• v.42 no.1
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• pp.6-11
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• 1999

The bacteria which could hydrolyze the fibrin produced through the blood coagulation mechanism in the human body, were isolated from soybean paste. The KDO-13 strain was selected among the isolated bacteria as the best strain for fibrinolytic activity. It was spore forming and Gram positive. $C_{15:0}$ anteiso fatty acid, $C_{15:0}$ iso fatty acid and $C_{15:0}$ anteiso fatty acid were 47.7, 13.5 and 13.6%, respectively as major component among its cellular fatty acid composition. It showed the similarity of 57.7%, compared with standard strain. It was thus identified to be Bacillus atrophaeus according to Bergey's manual of systematic bacteriology and its fatty acid profiles of gas chromatography. The optimum culture temperature and pH were $37^{\circ}$ and 6 for the production of fibrinolytic enzyme by Bacillus atrophaeus KDO-13.

• 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 Preservation
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• v.13 no.1
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• pp.77-82
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• 2006

This study was carried out to investigate the Cheongbukjang fermentation characteristics and to select the strain showing a fibrinolytic activity. Fibrinolytic activity of 5 strains isolated from the commercial Chungkukjang was tested N2 strain showed the highest activity $(41.7\%)$ while N3 and N5 had similar activity $(27.8\%)$ compared to plasmin 1 unit/mL The selected N2 strain was determined as B. subtilis with $90.1\%$ homology by API kit analysis. Quality characteristics of Cheongbukjang fermented by 6 kinds of strains were tested Among 3 strains cultured, B. subtilis (KCTC 3014) showed the highest viscous substance, fibrinolytic activity and amino type nitrogen content. After isolated, B. subtilis N2 showed the highest viscous substance, fibrinolytic activity and amino type nitrogen content. Optimum steam-time for Cheongbukjang fermented by B. subtilis (KCTC 3014) and B. subtilis N2 was 45 min while optimum fermentation-time was 20 hr.

• 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).

• Microbiology and Biotechnology Letters
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• v.42 no.2
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• pp.184-189
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• 2014

Yeast strains demonstrating fibrinolytic activity were isolated from traditional fermented soybean in Gangwon province, Korea. The AFY-1 strain isolated from fermented soybean paste showed the highest fibrinolytic activity (3.5 U/mg protein) corresponding to a 1.75 fold higher fibrinolytic activity compared with the plasmin (2.0 U/mg protein). The optimum temperature for the growth of AFY-1 strain was $32^{\circ}C$. Analysis of 18S rRNA gene sequence and carbon source utilization pattern indicated that the AFY-1 strain shares the highest homology (99%) with Saccharomycetales sp.

• 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).

• Microbiology and Biotechnology Letters
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• v.49 no.3
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• pp.289-297
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• 2021

Bacillus velezensis CJ1, showing significant fibrinolytic activity, was isolated from Myeolchi Jeotgal, a popular Korean fermented seafood. When B. velezensis CJ1 was grown on four different culture media, the culture on the Luria-Bertani (LB) broth showed the highest fibrinolytic activity (102.94 mU/μl) at 48 h. LB was also the best medium for growth. SDS-PAGE of culture supernatant showed four major bands, 38, 35, 27, and 22 kDa in size. Fibrin zymography showed four active bands, 50, 47, 40, and 30 kDa in size. A gene homologous to aprE of the Bacillus species was cloned by PCR. DNA sequencing showed that aprECJ1 can encode a protease consisting of 382 amino acids. The translated amino acid sequence of AprECJ1 showed high identity values with those of B. velezensis strains and other Bacillus species. The aprECJ1 gene was introduced into B. subtilis WB600 using an E. coli-Bacillus shuttle vector, pHY300PLK, and overexpressed. A 27 kDa band corresponding to the mature form of AprECJ1 was produced and confirmed by SDS-PAGE and fibrin zymography. B. subtilis WB600 [pHYaprECJ1] showed 1.8-fold higher fibrinolytic activity than B. velezensis CJ1 at 48 h.

• Journal of Microbiology and Biotechnology
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• v.31 no.2
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• pp.327-337
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• 2021

Fibrinolytic enzymes with a direct mechanism of action and safer properties are currently requested for thrombolytic therapy. This paper reports on a new enzyme capable of degrading blood clots directly without impairing blood coagulation. This enzyme is also non-cytotoxic and constitutes an alternative to other thrombolytic enzymes known to cause undesired side effects. Twenty-four Bacillus isolates were screened for production of fibrinolytic enzymes using a fibrin agar plate. Based on produced activity, isolate S127e was selected and identified as B. subtilis using the 16S rDNA gene sequence. This strain is of biotechnological interest for producing high fibrinolytic yield and consequently has potential in the industrial field. The purified fibrinolytic enzyme has a molecular mass of 27.3 kDa, a predicted pI of 6.6, and a maximal affinity for Ala-Ala-Pro-Phe. This enzyme was almost completely inhibited by chymostatin with optimal activity at 48℃ and pH 7. Specific subtilisin features were found in the gene sequence, indicating that this enzyme belongs to the BPN group of the S8 subtilisin family and was assigned as AprE127. This subtilisin increased thromboplastin time by 3.7% (37.6 to 39 s) and prothrombin time by 3.2% (12.6 to 13 s), both within normal ranges. In a whole blood euglobulin assay, this enzyme did not impair coagulation but reduced lysis time significantly. Moreover, in an in vitro assay, AprE127 completely dissolved a thrombus of about 1 cc within 50 min and, in vivo, reduced a thrombus prompted in a rat tail by 11.4% in 24 h compared to non-treated animals.

• KSBB Journal
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• v.13 no.5
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• pp.491-496
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• 1998

The production of fibrinolytic enzyme from Bacillus subtilis BK-17 was studied in the shake flask cultures. The important medium components studied include nitrogen source, carbon source and inorganic salts. The environmental conditions include initial pH, temperature, shaking speed and working volume. Among various N-sources, C-sources and inorganic salts tested, soybean flour, D-glucose and Na2HPO4 gave the best results, and their optimal concentrations were 1.5%, 0.5% and 0.05%, respectively. The optimal pH and temperature were 9.0 and 37$^{\circ}C$. With decreasing working volume in the range of 25∼100ml in the 250ml flask or increasing shaking speed in the range of 100∼300rpm, the enzyme production was greatly enhanced. The enzyme activity under the optimal conditions was about 1400I.U./ml with urokinase as a standard.