• Journal of Microbiology and Biotechnology
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• 제11권6호
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• pp.1111-1114
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• 2001

An extracellular fibrinolytic-enzyme-producing bacterium was isolated from Doen-Jang, a Korean traditional fermented flood, and identified as Bacillus sp. DJ based on its morphology and cellular fatty acid composition. The total extracellular fibrinase (EF) from Bacillus sp. DJ was analyzed using three fibrin zymographic techniques, SDS-fibrin zymography (SDS-FZ), isoelectrofocucing-fibrin zymographs(IEF-FZ), and a two-dimensional SDS-fibrin zymographic analysis (2D SDS-FZ). As a result, the EP map of Bacillus sp. DJ was established. The results suggest that the 2D SDS-FZ method will be a useful tool for the proteomic approach for many other bacterial pretenses.

• Journal of Microbiology and Biotechnology
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• 제15권1호
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• pp.35-39
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• 2005

To identify the activity of recombinant subtilisins (subtilisin BPN' and subtilisin Carlsberg), three different zymography methods, SDS-fibrin zymography (SDS-FZ), reverse fibrin zymography (RFZ), and isoelectric focusingfibrin zymography (IEF-FZ), were used. The recombinant subtilisins BPN' and Carlsberg did not migrate into the electrophoretic field based on a Laemmli buffer system, instead forming a "binding mode" at the top part of the separating gels with the SDS-FZ and RFZ techniques. Yet, this problem was resolved when using IEF-FZ with a pH range from 3 to 10. In addition, all these methods enabled the activity of a recombinant pro-subtilisin DJ-4 to be detected without a refolding pathway.

• BMB Reports
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• 제35권2호
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• pp.236-238
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• 2002

Based on the zymography analysis, Bacillus sp. DJ-4 (screened from Doen-Jang, a Korean traditional fermented food) secretes seven extracellular fibrinolytic enzymes (EFEs; 68, 64, 55, 45, 33, 27, and 13 kDa) in culture broth. These seven EFEs were analyzed by newly applied SDS-fibrin zymography combined with gradient polyacrylamide (SDS-FZGP). This improved gel system was used with a 5-20% acrylamide gradient in a fibrin zymogram gel for the separation of proteins with molecular masses from below 10kDa to over 100kDa on one gel plate. Using this system, high molecular weight bands (HMWBs) were clearly and sharply resolved. We also examined the relationship between an acrylamide concentration and the enzymatic activity of EFE using densitometric analysis.

• BMB Reports
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• 제34권2호
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• pp.134-138
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• 2001

By adding sodium chloride (2.5%) into a Bacillus amyloliquefaciens DJ-4 culture broth, two serine-type fibrinolytic proteases with a molecular weight of 29 (subtilisin DJ-4) and 38-kDa were stimulated on the SDS-fibrin zymogram or inhibitor gels. B. amyloliquefaciens DJ-4 showed the highest proteolytic activity (5.52 plasmin NIH unit/ml) on the fibrin plate based on the molar ratio when cells were subjected to the 2.5% NaCl. Using a fibrin plate, the secreted protease from this strain in the presence of 5% NaCl showed that about 49% of the enzyme's activity remained after incubation at $60^{\circ}C$ for 30 min, but as the salt concentration was increased (10% NaCl) the activity nearly disappeared (0.14 plasmin NIH unit/ml). However, through a fibrin zymography assay, three fibrinolytic enzymes (38, 53 and 80-kDa) from the cells in the presence of 10% NaCl were detected. Also, two salt-activated serine-type fibrinolytic professes (29 and 38kDa) showed thermostability from 65 to $70^{\circ}C$ for 30 min. Furthermore, these professes also showed stability, pH 6-11. In particular, 29-kDa (subtilisin DJ-4) was very stable in the pH range of 4-11 at $4^{\circ}C$ for 48 h.

• BMB Reports
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• 제37권3호
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• pp.298-303
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• 2004

In general, a SYPRO Ruby dye is well known as a sensitive fluorescence-based method for detecting proteins by one-or two-dimensional SDS-PAGE (1-DE or 2-DE). Based on the SYPRO Ruby dye system, the combined two-dimensional fibrin zymography (2-D FZ) with SYPRO Ruby staining was newly developed to identify the Bacillus sp. proteases. Namely, complex protein mixtures from Bacillus sp. DJ-4, which were screened from Doen-Jang (Korean traditional fermented food), showed activity on the zymogram gel. The gel spots on the SYPRO Ruby gel, which corresponded to the active spots showing on the 2-D FZ gel, were analyzed by a matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometric analysis. Five intracellular fibrinolytic enzymes of Bacillus sp. DJ-4 were detected through 2-D FZ. The gel spots on the SYPRO Ruby dye stained 2-D gel corresponding to 2-D FZ were then analyzed by MALID TOF MS. Three of the five gel spots proved to be quite similar to the ATP-dependent protease, extracellular neutral metalloprotease, and protease of Bacillus subtilis. Also, the extracellular proteases of Bacillus sp. DJ-4 employing this combined system were identified on three gels (e.g., casein, fibrin, and gelatin) and the proteolytic maps were established. This combined system of 2-D zymography and SYPRO Ruby dye should be useful for searching the specific protease from complex protein mixtures of many other sources (e.g., yeast and cancer cell lines).

• 한국식품과학회지
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• 제31권2호
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• pp.553-557
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• 1999

혈전용해능을 가진 균류 (버섯류) 5종 Daedaleopsis styracina, Trichaptum abietium, Coriolus versicolor, Pisolithus tinctorius 그리고 Tricholomopsis decora 등을 선발하여 혈전용해효소 활성을 측정하였고, 기질 특이성을 조사하였다. 버섯 추출액을 조제하여 혈전 용해도를 측정한 결과 plasmin 1.0 unit 보다 $3{\sim}4$배의 높은 활성을 보였으며, 그 중 Pisolithus tinctorius이 가장 높은 활성(4.71 plasmin unit)을 나타내었고, Tricholomopsis decora가 Plasmin에 대한 기질 N-p-Tosyl-Gly-Pro-Lys p-nitroanilide에 대해 가장 높은 특이성(1.32 plasmin unit)을 보였다. 버섯의 균사체로부터 추출한 효소를 SDS-fibrin zymography 활성확인법에 의해 분석한 결과 분자량이 54와 61 kDa의 공통된 활성을 가지는 단백질을 확인하였으며, Trichaptum abietium는 100 kDa의 그리고 Tricholomopsis decora는 84 kDa의 강한 활성을 가지는 혈전용해효소를 확인하였다.

• 사상체질의학회지
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• 제19권1호
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• pp.160-170
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• 2007

1. Objectives This study was performed to find the activities and characteristics of purified thrombolytic enzymes from Holotrichia extracts. 2. Methods In the first time, a coarse enzyme fluid was made by using the freedried Holotrichia extracts. After manufacturing total soluble proteins and purifing enzymes, it was evauluated the activities and characteristics of this enzyme's dissolving capability to fibrin and thrombus. This study was taken using azocasein assay, fibrin-plate method, native-PAGE and fibrin zymography. 3. Results A soluble proteins were efficiently extracted form freezedried Holotrichia extracts. And, this purified enzyme had a ten times fibrinolytic capability compare with ustulation Holotrichia sample. In native PAGE and fibrin zymography, Holotrichia extracts showed the respectable fibrinolytic activity. Also, It had higher thrombolytic activities compared with general thrombolytic enzyme 'plasmin'. In experiment of various protease inhibitors of the purified enzyme from Holotrichia extracts on the azocaseinolytic activity, the enzyme was strongly inhibited by EDTA ${\cdot}$ EGTA, and weakly by APMSF ${\cdot}$ PMSF ${\cdot}$ TPCK. 4. Conclusion Holotrichia extracts has the thrombolytic activities, and it will operate directly th fibrin-clot and thrombus.

• 한국약용작물학회지
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• 제14권4호
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• pp.195-201
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• 2006

미생물 및 동물에 비해 식물에서는 혈전용해효소에 대한 연구가 매우 부족한 실정이며, 기존의 혈전용해효소가 가지는 혈전에 대한 비특이적, 부작용, 고가 등의 단점을 해결할 수 있는 새로운 혈전용해효소의 개발을 위하여 동백의 종자, 종피, 유엽 그리고 성엽으로부터 추출된 수용성 단백질의 혈전용해활성을 조사하였다. 각각의 동백부위로부터 추출된 조효소 용액은 기존 혈전 용해효소인 plasmin과 양성 대조군으로 하여 비교하여 fibrin agarose plate로 확인한 결과 fibrin clot을 효과적으로 분해하였다. 그 중 단백질 분해효소의 활성이 다른 부위보다 20-33배로 높았던 동백종자와 종피의 수용성 추출물의 혈전용해활성은 양성 대조군인 plasmin과 비교하여 1.6-2.0배의 높은 활성을 나타내었다. 전체 수용성 단백질은 30-80%황산암모늄을 이용하여 농축하였으며 혈전용해효소는 fibrin zymography를 수행하여 확인하였다. SDS-PAGE에 의하여 동백유엽의 혈전용해효소 분자량을 측정한 결과 45 kDa으로 단일 polypeptide임을 확인하였으며, 각종 pretense 저해제에 의한 영향을 조사한 결과 PMSF,그리고 TLCK에 강력하게 저해되는 것으로 보아 동백유엽의 혈전용해효소는 trypsin과 유사한 serine protease의 하나로 생각되었다. 그러나 EDTA와 DTT처리에 의해서는 효소활성의 저해가 두드러지게 나타나지 않고 오히려 증진된 양상을 확인할 수 있었다. 또한, 효소 활성에 미치는 pH 및 온도의 효과는 약간의 산성쪽에 가까운 pH 5.5와 $30^{\circ}C$에서는 최적의 활성을 나타내었으며, $45^{\circ}C$ 이상의 온도에서는 효소활성이 급격히 감소하였다. 이상의 모든 결과로 볼 때 동백유엽의 혈전용해효소는 trypsin과 유사한 serine protease에 속하는 혈전용해효소임을 확인할 수 있었다.

• BMB Reports
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• 제34권6호
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• pp.531-536
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• 2001

Three zymographic techniques using casein, fibrin, and gelatin as substrates in SDS-PAGE were compared based on three aspects: (1) The proteolytic pattern of extracellular enzymes from the three bacterial strains, Bacillus sp. DJ-1, DJ-2, and DJ-3. (2) The enzymatic sensitivity of their activity on zymogram gels. (3) The stability of stained zymogram gels with Coomassie brilliant blue in the destaining solution. There was no significant difference on the pattern of extracellular enzymes from the three strains. The bands in the fibrin gel were clearer and more distinct from the extensive destaining process. It was also shown that the gelatin gel revealed the highest enzymatic sensitivity among the three gels, based on the densitometric analysis. In the casein gel, a trace that could be mistaken as a proteolytic band appeared around 40-50 kDa.

• 한국식품과학회지
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• 제31권1호
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• pp.219-223
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• 1999

우리나라의 전통 발효식품인 김치에서 혈전용해 효소를 생산하는 미생물을 분리하고 그 중 Bacillus amyloliquefaciens, Bacillus brevis, Micrococcus luteus의 세 종을 Bergey's manual of systematic bacteriology등에 의하여 동정하였다. 분리된 미생물을 효소 유도 배지에서 배양한 결과 B. amyloliquefaciens는 2.58 plasmin unit/mL, B. brevis는 1.48 plasmin unit/mL, 그리고 M. luteus는 2.03 plasmin unit/mL의 혈전용해효소 생산능을 보여 주였다. 각 균주에서 생산된 세포의 단백질을 SDS-PAGE와 fibrin zymography assay에 의해 분석한 결과 B. brevis와 M. luteus에서는 서로 다른 분자량을 가진 $3{\sim}4$개의 혈전용해 효소가 존재하였으며 B. amyloliquefaciens에서는 분자량이 약 29 kDa인 단일 band의 혈전용해 효소가 생산되었음을 확인하였다.