ABSTRACT
Pullulanase (EC 3.2.1.41) has been isolated and purified from white edible mushrooms by ammonium sulphate precipitation (20-70%) followed by ion exchange chromatography (DEAE-cellulose) and gel filtration (Sephadex G 75-120), with final yield (20%) and purification fold (17.8). The molecular mass of pullulanase enzyme was 112 kDa as estimated by SDS-PAGE and the pI value was 6.2. The apparent Km and Vmax values for purified pullulanse on pulluan were 0.27 mM and 0.74 μM min-1 respectively. The activity was optimum at 40○C and pH 6. Pullulanase showed moderate thermo-stability. A relative substrate specificity for hydrolysis of soluble starch, amylopectin and glycogen was 80, 60 and 30% respectively. Enzyme activity was highly activated by Fe+2, Mn+2 and Ca+2 ions, while the activity was inhibited by Hg+2 and Ag+ ions. Ethylenediaminetetraacetic acid (EDTA) and Dithiothreitol (DTT) were activated the enzyme activity. On contarary iodoacetate and sodium fluoride were inhibited the activity. HPTLC (High Performance Thin Layer Chromatography) plate showed that the purified pullulanase caused the complete hydrolysis of pullulan to maltotriose.