Purification and characterization of a fibrinolytic subtilisin-like protease of Bacillus subtilis TP-6 from an Indonesian fermented soybean, Tempeh.
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 microM and 145 micromol 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.
Kim
Seong-Bo
SB
Department of Biotechnology, Yonsei University, 120-749, Seoul, South Korea.
Lee
Dong-Woo
DW
Cheigh
Chan-Ick
CI
Choe
Eun-Ah
EA
Lee
Sang-Jae
SJ
Hong
Young-Ho
YH
Choi
Hak-Jong
HJ
Pyun
Yu-Ryang
YR
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D005591:Chemical Fractionation; D002853:Chromatography, Liquid; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D005337:Fibrin / Q000378:metabolism*; D005341:Fibrinolysin / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D045730:Soy Foods / Q000382:microbiology*; D013379:Substrate Specificity; D020860:Subtilisin / Q000235:genetics
D004269:DNA, Bacterial; D017395:Plasminogen Activator Inhibitor 1; D005337:Fibrin; D012697:Serine Endopeptidases; D020860:Subtilisin; D005341:Fibrinolysin