Purification, identification and functional characterization of glucansucrase from Leuconostoc dextranicum NRRL B-1146

In the present study the polyethylene glycols of different molecular weights were screened to purify exo-cellular glucansucrase from Leuconostoc dextranicum NRRL B-1146. The glucan produced by this enzyme is unique in that it contains α-(1–6) and α-(1–4) linkages which have clinical applications. The cell free extract was subjected to fractionation by PEG-200, 400, 4000 and 6000. The 30% (w/v) PEG-400 gave glucansucrase with maximum specific activity of 4.5 U/mg with 7.5 fold purification in a single step. Further purification of PEG-400 purified glucansucrase by gel-filtration gave glucansucrase with specific activity of 9 U/mg with 15 fold purification. The purified glucansucrase confirmed the presence of glucan, after in-situ activity detection by Periodic Acid Schiff staining when run under non-denaturing on SDS-PAGE gels. The activity bands corresponded to the native and active form of the purified glucansucrase of approximately, 205 kDa molecular size, that appeared on the denaturing gels stained with Coomassie Brilliant Blue. The purified glucansucrase gave a K_m value of 18.7 mM and a V _m of 9.4 U/mg. The addition of 0.8 mM EDTA led to 50% loss of enzyme activity. Urea displayed deactivating effect on glucansucrase at all concentrations. Tween 80 provided stability to the enzyme against activity losses.