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

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Journal of Industrial Microbiology and Biotechnology

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 K m and V max 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.

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Acknowledgements

This work [R11–1994-03405001-0] was supported by the Korea Science and Engineering Foundation (KOSEF). We gratefully acknowledge Prof. Jerry Eichler for editing the manuscript.

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Author notes

  1. Dong-Woo Lee

    Present address: Department of Biology, University of Pennsylvania, Philadelphia, PA, 19104, USA

  2. Eun-Ah Choe

    Present address: Department of Human Genetics, Emory University, Atlanta, GA, 30322, USA

Authors and Affiliations

  1. Department of Biotechnology, Yonsei University, 120-749, Seoul, South Korea

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

  2. Department of Pathology, University of Chicago, Chicago, IL, 60637, USA

    Hak-Jong Choi

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  1. Seong-Bo Kim
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Correspondence to Yu-Ryang Pyun.

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Seong-Bo Kim and Dong-Woo Lee contributed equally to the work.

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Kim, SB., Lee, DW., Cheigh, CI. et al. Purification and characterization of a fibrinolytic subtilisin-like protease of Bacillus subtilis TP-6 from an Indonesian fermented soybean, Tempeh. J IND MICROBIOL BIOTECHNOL 33, 436–444 (2006). https://doi.org/10.1007/s10295-006-0085-4

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  • DOI: https://doi.org/10.1007/s10295-006-0085-4

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