THERMAL STABILIZATION OF IMMOBILIZED ALPHA-AMYLASE BY COUPLING WITH PROLINE

alpha-Amylase was immobilized to Sepharose-4B activated by electrophilic method using cyanogen bromide. The alpha-amylase coupled was 77% of the total protein added, Further L-proline was covalently linked to the immobilized alpha-amylase by carbodiimide. Optimum carbodiimide concentration for the coupling of proline to the immobilized alpha-amylase and the suitable proline concentration for the coupling were determined. Activity of immobilized alpha-amylase was not altered after coupling to proline. The thermal stability of soluble alpha-amylase, immobilized alpha-amylase and immobilized alpha-amylase-proline conjugates (samples coupled to two different proline concentrations) were studied at 45 and 60 degrees C. Soluble alpha-amylase lost its total activity in 4 days and 1 day at 45 and 60 degrees C, respectively, Immobilized alpha-amylase lost its total activity on the 30th and 16th days at 45 and 60 degrees C, respectively. Immobilized alpha-amylase-proline conjugate (85.35 mu g proline/g gel) last only 78% activity at 45 degrees C on the 30th day while the same preparation took 20 days at 60 degrees C to lose the total activity. On the other hand the immobilized alpha-amylase-proline conjugate (785.32 mu g proline/g gel) lost only 30% of its original activity at 45 degrees C on the 30th day and took 30 days at 60 degrees C to lose its total activity. These results show that the coupling of proline to immobilized enzymes increases their thermal stability.