Immobilization of Potato Acid Phosphatase on Succinamidopropyl Glass Beads for the Dephosphorylation of Bovine Whole Casein

Potato acid phosphatase was immobilized on succinamidopropyl-derivatized glass beads for use in removing phosphate groups from bovine whole casein. Controlled pore glass beads (with and without glyceryl coating) were silanized and succinylated. The enzyme was covalently bound to derivatized glass beads through carbodiimide coupling. The pH optima for the immobilized enzyme remained at pH 5.5 for p-nitrophenyl phosphate and pH 6.0 to 6.5 for casein substrates. Activities of acid phosphatase (pH 5.5, p-nitrophenyl phosphate substrate) immobilized on the plain and glyceryl-coated controlled pore glass beads were 14 and 4%, respectively, of the free enzyme. When whole casein was used as substrate, the 15 to 20min enzyme activity of free enzyme was 16% of its activity on p-nitrophenyl phosphate and after 4h, 14% of its original rate on casein. After 18h of incubation, the activity of the immobilized enzyme was 36 to 40% of its rate at 4h. A 70% dephosphorylation of whole casein was attained using a concentration of 2.6mg enzyme controlled pore glass beads complex per milligram of whole casein at pH 6.5, 37°C, for 18h.