Journal
JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY
Volume 33, Issue 6, Pages 436-444Publisher
SPRINGER HEIDELBERG
DOI: 10.1007/s10295-006-0085-4
Keywords
Bacillus subtilis TP-6; fibrin clot lysis; subtilisin-like protease; Tempeh; TPase
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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 K-m and V-max for the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-pNA were 259 mu M and 145 mu mol 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 alpha- and beta-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.
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