Kinetic studies on soluble and immobilized alpha amylase and glucoamylase

Alpha amylase and glucoamylase were coupled separately to Sepharose- 4B which was activated by an electrophilic method (by using cyanogen bromide). The kinetic properties of these enzymes in immobilized form were compared with those of their soluble enzymes. The Km values of soluble and immobilized alpha amylase for starch were 0.89% (WIV) and 1.33% (WM respectively at pH 6.9 and at 40⁰ C and the Km values of soluble and immobilized glucoamylase for starch were 0.25% (W/V) and 0.72% (W/V) respectively at pH 4.8 and at 50⁰ C. The optimum pH for soluble alpha amylase compared with that of the immobilized enzyme shifted from 6.9 to 6.5 in.0.02M phosphate buffer while in the case of ghcoamylase the pH optimum in 0.01M acetate buffer increased from 4.8 to 5.2 on immobilization. There was no shift in optimum phosphate concentration .(0.02M) for both soluble and immobilized alpha amylase at pH.6.9 and at 40⁰ C and likewise no shift in optimum acetate concentration (0.01M) for soluble and immobilized glucoamylase at pH 4.8 and at 50⁰ C. The optimum temperature for the activities of soluble alpha amylase compared with the immobilized enzyme assayed in 0.02M phosphate buffer (pH 6.9) shifted from 45⁰ C to 50⁰C and that of glucoamylase assayed in 0.0lM acetate buffer (pH 4.8) shifted from 55⁰ C to 58⁰ C.