Immobilization of glucoamylase on biotin-avidin system

The biotin-avidin system was used as carriers to immobilize glucoamylase. First, the optimum conditions of biotin marking glucoamylase were determined by the orthogonal experiments: 4mg/mL enzyme liquid 2mL, the reaction temperature was 20°C, the reactive pH was 4.6, stirring time was 2.5h. Second, the optimum preparation conditions of avidin-magnetic agarose microspheres were determined by the single factor-orthogonal experiments: avidin load was 3 μg/ mg carrier, reaction time was 20min, pH was 6.6. Third, the conditions of oriented-immobilization enzyme with biotin-avidin system were optimized by the single factor-orthogonal experiments: biotin marking glucoamylase load was 40 μL/mg carrier, the reaction time was 20min, the reaction temperature was 20°C, pH was 6.1. Under these conditions, the activity of the immobilized enzyme reached 1629.7 U/g. Activity recovery and relative activity of the immobilized enzyme were 50.3% and 64.7% respectively. Finally, the enzymatic properties of the immobilized glucoamylase were investigated. The results showed that the optimum temperature was 65°C, the optimum pH was 5.1. The Km was 2.28 mg/mL that less than free glucoamylase's. It mains that immobilized glucoamylase has a stronger affinity for substrate than free glucoamylase. And the activity of immobilized enzyme was still maintained 71.8% after used for five times. The half-life of the immobilized enzyme was 84 d.