Preparation and characterization of cross linked enzyme aggregates (CLEAs) of Bacillus amyloliquefaciens alpha amylase

Stabilization of enzymes is one of the major challenges in biocatalytic processes. Alpha amylase from Bacillus amyloliquefaciens was immobilized as cross-linked enzyme aggregates (CLEAs). Alpha amylase was aggregated using ammonium sulfate. The resultant aggregates on cross-linking with glutaraldehyde produced insoluble catalytically active cross-linked enzyme aggregates. The effects of precipitation and cross-linking were studied and immobilized alpha amylase was characterized. Seventy percent ammonium sulfate saturation, 2% (v/v) glutaraldehyde, were used; 6 h cross-linking reaction at room temperature was performed and 100% activity recovery was achieved in CLEAs with enhanced thermal and acidic condition stabilities. The cross-linked enzyme aggregates exhibited pH optima of 6.0 and higher temperature optima of 60°C. Although after immobilization maximum velocity of enzyme reaction did not change, substrate affinity of the enzyme increased. Alpha amylase CLEAs retained 65% activity after 4 reuses with 30 min of each reaction time. The Scanning electron microscopy analysis showed that morphology of CLEAs substantially changed after 4 reuses.