Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Identification of Three Extracellular Proteases from Bacillus subtilis KCTC 3014

  • Choi Nack-Shick (Systemic Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Department of Microbiology, Chungnam National University) ;
  • Chung Dong-Min (Systemic Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Department of Microbiology, Chungnam National University) ;
  • Ryu Chung-Hun (Systemic Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Yoon Kab-Seog (Systemic Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Maeng Pil-Jae (Department of Microbiology, Chungnam National University) ;
  • Kim Seung-Ho (Systemic Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology)
  • Published : 2006.03.01
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Abstract

Three extracellular proteases (Vpr, peptidase T, and subtilisin) were identified from the culture supernatant of Bacillus subtilis KCTC 3014. All the proteins were partially purified as a mature form by using a DEAE-cellulose ion-exchange column chromatography. Their activities were determined by using zymography and densitometry. The relative molecular masses of Vpr and peptidase T (PepT) were determined to be 68 and 48 kDa by SDS-PAGE and zymography, respectively. However, subtilisin formed a 'binding mode' at the top of the separating gel. After denaturation by boiling at $100^{\circ}C$ for 5 min, its molecular mass was determined to be 29 kDa, whereas its activity was lost. The optimal pH of Vpr, PepT, and subtilisin were 9.0, 6.0-7.0, and 7.0-8.0, respectively. The optimal temperature of Vpr, PepT, and subtilisin was 40, 50, and $40^{\circ}C$, respectively. Inhibitor test revealed that Vpr and subtilisin were serine proteases and that PepT was a metalloprotease. Interestingly, we found that Vpr showed no enzyme activity on a 2DE zymogram gel. Three genes, vpr, pepT, and apr (encoding subtilisin protein), were cloned and their nucleotide and deduced amino acid sequences were determined.

Keywords

References

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