Purification of pullulanase from Hypocrea jecorina QM9414

In this study, pullulanase induction was realized for the first time at Hypocrea jecorina QM 9414 obtaining from Vienna University of Technology, In(stitute of Chemical Engineering, Division Gene Technology and Applied Biochemistry. The optimum conditions were determined for the enzyme induction. Pullulanase [EC 3.2.1.41] was purified for the first time from this microorganism. Some properties of the purified enzyme were investigated.Pullulanase was purified 11 fold from Hypocrea jecorina QM 9414 by the methods of homogenization, ammonium sulfate precipitation, DEAE cellulose and Sephadex G-100 column chromatography. Temperature of +4 °C was maintained during the purification process. The protein content was determined according to the methods of Bradford and Warburg-Christian and the enzyme activity was measured spectrophotometrically at 37 °C according to the method of Miller at 540 nm.The PAGE of the purified enzyme showed one activity band corresponding to 130,56 kDa molecular weight. SDS-PAGE showed that the enzyme was an monomer composed. The optimum temperature of enzyme was determined between at 35?C-65°C. The optimum pH of Hypocrea jecorina pullulanase was changed according to substrate. The optimum pH was found at 6.5 for amylopectin or pullulan; at 5.0 for starch. The activation energy of the reaction catalyzed by the enzyme was calculated as 0.6178 kcal.mol-1. The affinity of the enzyme against the amylopectin, starch and pullulan was investigated. Km values were for amylopectin, starch and pullulan 10.7 mg/ml, 15.5 mg/ml ve 38.4 mg/ml, respectively. Vmax values were for amylopectin, starch, and pullulan 3.3145 ?A/min, 3.3211 ?A/min ve 3.818 ?A/min, respectively.