Thermal Stabilization of Immobilized α-Amylase by Coupling with Proline

Abstract

α-Amylase was immobilized to Sepharose-4B activated by electrophilic method using cyanogen bromide. The α-amylase coupled was 77% of the total protein added. Further l -proline was covalently linked to the immobilized α-amylase by carbodiimide. Optimum carbodiimide concentration for the coupling of proline to the immobilized α-amylase and the suitable proline concentration for the coupling were determined. Activity of immobilized α-amylase was not altered after coupling to proline. The thermal stability of soluble α-amylase, immobilized α-amylase and immobilized α-amylse-proline conjugates (samples coupled to two different proline concentrations) were studied at 45 and 60°C. Soluble α-amylase lost its total activity in 4 days and 1 day at 45 and 60°C, respectively. Immobilized α-amylase lost its total activity on the 30th and 16th days at 45 and 60°C, respectively. Immobilized α-amylase-proline conjugate (85·35 μg proline/g gel) lost only 78% activity at 45°C on the 30th day while the same preparation took 20 days at 60°C to lose the total activity. On the other hand the immobilized α-amylase-proline conjugate (785·32 μg proline/g gel) lost only 30% of its original activity at 45°C on the 30th day and took 30 days at 60°C to lose its total activity. These results show that the coupling of proline to immobilized enzymes increases their thermal stability.