Purification and Characterization of Pullulanase from Klebsiella pnrumoniae NFB-320

  • Published : 1997.03.01


Pullulanase was produced from the Klebisella pneumonias NFB_320 with the conmposition of 0.1% pullualn 1.5% yeast extract, 0.2% $K_2$HPO$_4$ and 0.02% MgSO$_4$.7$H_2O$(pH5.5). The optimum temperature for activity of the pulluanase was 3$0^{\circ}C$ and the highest yield of the enzyme was obtained after cell growth at 3$0^{\circ}C$ for 18hr, and maintained until 24hr cultivation. The pullulanase was successively purified 52.6 folds with 7.8% yield by acetone precipitation. DEAE-cellulose column chromatography and gel fitrations. The purified enzyme hydrolyzed pullulan into maltotriose exclusively. Chemical and physical properties of purified pullulanase from Klebisella pneumonias NFB-320 were examined. The optimum pH and temperature for enzyme activity were 5.0 and 6$0^{\circ}C$, respectively. The enzyme was stable between pH4 and 7, and up 5$0^{\circ}C$. The effect of mo-dification on the rate of enzyme reaction was studies with various chemicals and metal ions. The enzyme has been found to be inactivated by I$_2$ and N-bromosussinimide(NBS), which probably indicated the involve- ment of tryptophan residues in the active center of the enzyme.