Determination of pullulanase activity in different plant samples, purification and immobilization of pullulanase

In this study, firstly, the pullulanase activity of the extensively consumed vegetables and fruits in our country was determined. According to the enzyme tests on several plants it was decided to use globe artichoke as pullulanase source. Pullulanase was firstly purified from the leaves of globe artichoke and the properties of the enzyme were investigated. Purified pullulanase was immobilized on various supports by adsorption method. Characterization of the immobilizied enzyme was carried out. The results were compared with the free enzyme. Globe artichoke leaves were homogenized with 100 mM phosphate (pH 6,5) buffer. Ultrafiltration fraction of pullulanase-active homogenate was obtained and the retantate was applied to DEAE-cellulose column and pullulanase was purified 21,53 fold. The enzyme activity was assayed with Miller?s reducing sugar method and the protein content was determined by using Bradford and Warburg-Christian methods. After SDS-PAGE electrophoresis which was commited due to determine the molecular weight and the purity of the globe artichoke pullulanase, it was found that the purified pullulanase showed one activity band and had a moleculer weight of 141,37 kDa. The affinity of the purified enzyme against pullulan, dextrin, amylopectin and starch was investigated. It was found out that the highest affinity of the enzyme was to dextrin having an optimum pH of 5,0 and an optimum temperature of 45°C. Km values were 5,1382X10-5 mg/mL, 5,9093X10-4 mg/mL, 2,0986X10-3 mg/mL and 3,88726X10-3 mg/mL and Vmax values were 0,8037 U/mL, 0,9848 U/mL, 0,8089 U/mL ve 0,7892 U/mL for the same substrates respectively. Storage stability and thermal stability of globe artichoke pullulanase were also determined. The effects of several organic and inorganic compounds, organic solvents, various acids and bases, detergents were investigated. The immobilization of globe artichoke pullulanase on chitosan, PEG-chitosan and amberlite XAD-16 by adsorption was held. Properties of the immobilizied enzymes such as optimum pH, temperature, thermal and storage stability and effects of vaious compounds were also examined. The results were compared to the free enzyme.