Methods: The enzymes were purified from a bacterial cell extract by HPLC (DEAE-5PW, G4000SW/XL and G2000SW/XL columns), and by affinity chromatography arginine-Sepharose 4B. Enzymatic activity in samples was determined by monitoring hydrolysis of chromogenic synthetic peptides according to the method of Erlanger et al. The amino acid sequence of the purified enzymes was performed on the Sequencer (Model 473A, Applied Biosystems, Foster-City, CA).
Results: Molecular weights of purified enzymes, as revealed by SDS-PAGE analysis were approximately 108, 92, 74, 64 and 57 kDa. The enzymes cleaved synthetic peptides containing arginine or lysine, but not native proteins such as casein and gelatin. They were found to be localized in cytoplasm. The optimal activity of these enzymes was obtained at 35-38oC and pH 8.2-8.6. Enzymes were inhibited by serine protease inhibitors such as antipain, leupeptin, elastatiol and TLCK.
Conclusion: Results of this study indicated that the trypsin-like enzymes produced by T. forsythensis had different properties as compared with those produced by other putative periodontopathogenic bacteria.