Fructosyltransferases (FTF) are extra cellular enzymes of mutans streptococci and play important role in bacteria pathogenesis and physiology. They catalyse the formation of extracellular polysaccharide (fructans) using sucrose as substrate. FTF are associated with dental biofilm formation by mediating adhesion of specific bacteria.
Non-dialyzable material (NDM) derived from cranberry juice, inhibits enzymatic activity of FTF and affects Streptococcus mutans biofilm formation suggesting FTF-NDM interaction.
The aim of our study was to monitor the real time binding of NDM to immobilized fructosyltransferase using surface plasmon resonance (SRP) technique and concomitantly estimate its immediate effect on FTF enzymatic activity.
Methods:
Purified recombinant FTF was immobilized on sensor chip using the BIAcore 3000 system. NDM at various concentrations was added to the immobilized enzyme alone, or together, or prior to adding sucrose in order to monitor its binding to and its effect on FTF enzymatic activity in real time.
Results:
Our results showed that NDM firmly attaches to immobilized enzyme in dose dependent manner. Pronounced inhibitory effect of NDM (200 µg/ml) on FTF activity was detected when NDM was either allowed to first bind to immobilized enzyme prior to exposure of substrate (90% of inhibition compare to control) or when added together with the substrate (80% of inhibition compare to control). The inhibition was immediate and irreversible.
Conclusion:
The real time assay shows that one of the modes of NDM antibiofilm action is immediate and irreversible binding of the cranberry material to the immobilized enzyme associated with significant reduction of its fructosyltransferase enzymatic activity.