Purification and characterization of a fibrinolytic subtilisin-like protease of Bacillus subtilis TP-6 from an Indonesian fermented soybean, Tempeh

Seong Bo Kim, Dong Woo Lee, Chan Ick Cheigh, Eun Ah Choe, Sang Jae Lee, Young Ho Hong, Hak Jong Choi, Yu Ryang Pyun

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64 Citations (Scopus)

Abstract

We have isolated a bacterium (TP-6) from the Indonesian fermented soybean, Tempeh, which produces a strong fibrinolytic protease and was identified as Bacillus subtilis. The protease (TPase) was purified to homogeneity by ammonium sulfate fractionation and octyl sepharose and SP sepharose chromatography. The N-terminal amino acid sequence of the 27.5 kDa enzyme was determined, and the encoding gene was cloned and sequenced. The result demonstrates that TPase is a serine protease of the subtilisin family consisting of 275 amino acid residues in its mature form. Its apparent Km and Vmax for the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-pNA were 259 μM and 145 μmol mg-1 min-1, respectively. The fibrinogen degradation pattern generated by TPase as a function of time was similar to that obtained with plasmin. In addition, N-terminal amino acid sequence analysis of the fibrinogen degradation products demonstrated that TPase cleaves Glu (or Asp) near hydrophobic acids as a P1 site in the α- and β-chains of fibrinogen to generate fragments D′, E′, and D′ similar to those generated by plasmin. On plasminogen-rich fibrin plates, TPase did not seem to activate fibrin clot lysis. Moreover, the enzyme converted the active plasminogen activator inhibitor-1 to the latent form.

Original languageEnglish
Pages (from-to)436-444
Number of pages9
JournalJournal of Industrial Microbiology and Biotechnology
Volume33
Issue number6
DOIs
Publication statusPublished - 2006 Jun

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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