Purification and biochemical characterization of a nattokinase by conversion of shrimp shell with Bacillus subtilis TKU007.
BSN1, a nattokinase, was purified from the culture supernatant of Bacillus subtilis TKU007 with shrimp shell wastes as the sole carbon/nitrogen source. The BSN1 was purified to homogeneity by three-step procedure with a 515-fold increase in specific activity and 12% recovery. The molecular masses of BSN1 determined by SDS-PAGE and gel filtrations were approximately 30 kDa and 28 kDa, respectively. The results of peptide mass mapping showed that four tryptic peptides of BSN1 were identical to the nattokinase from B. subtilis (GenBank accession number gi14422313) with 37% sequence coverage. The N-terminal amino acid sequence of the first 12 amino acids of BSN1 was AQSVPYGISQIK. The optimum pH, optimum temperature, pH stability, and thermal stability of BSN1 were 8, 40 °C, pH 4-11, and less than 50°C, respectively. BSN1 was inhibited completely by PMSF, indicating that the BSN1 was a serine protease. Using this method, B. subtilis TKU007 produces a nattokinase/fibrinolytic enzyme and this enzyme may be considered as a new source for thrombolytic agents.
Wang
San-Lang
SL
Life Sciences Development Center, Tamkang University, Taipei 25137, Taiwan. sabulo@mail.tku.edu.tw
Wu
Ying-Ying
YY
Liang
Tzu-Wen
TW
D000595:Amino Acid Sequence; D000818:Animals; D001412:Bacillus subtilis / Q000201:enzymology*; D001483:Base Sequence; D033364:Decapoda / Q000737:chemistry*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013379:Substrate Specificity; D013381:Subtilisins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D013696:Temperature
D013381:Subtilisins; C053771:nattokinase