Immobilization of thermostable α-amylase from Bacillus licheniformis by cross-linked enzyme aggregates method using calcium and sodium ions as additives

Cross-linked enzyme aggregates (CLEAs) of thermostable α-amylase from Bacillus licheniformis have been prepared for starch liquefaction. Among the six different precipitants, tert-butanol performed the best with an aggregation efficiency of 99.54% for starch hydrolysis. The optimal conditions for the immobilization process required 5 mM glutaraldehyde, 0.96 mg/mL enzyme, 1:2 ratio (0.5 ratio) of enzyme/bovine serum albumin (BSA), and 12 h crosslinking at 2–3 °C. Starch was used as the main substrate in enzyme assay. Immobilization did not affect pH (5.5) and temperature (95 °C) optima of free α-amylase thus, these values remained constant for produced CLEAs. Different concentrations of calcium and sodium ions were added to the enzyme during the aggregation process. It was observed that simultaneous addition of both calcium and sodium (1200 ppm of calcium and 400 ppm of sodium ions, in a ratio of 3:1) significantly improved the catalytic efficiency (kcat/Km), of CLEAs–BSA–CN from 3.91 × 105 to 4.57 × 105 (1.2-folds) compared with free enzyme. Although immobilization did not significantly affect Vmax (5.35–5.25), substrate affinity of the enzyme increased (1.34–1.12) after addition of mixed ions to the prepared CLEAs. Moreover, compared with free α-amylase, enzyme half-life (t1/2) of CLEAs–BSA–CN increased from 43.31 to 115 min (about 3-folds) at 110 °C. The CLEAs–BSA–CN retained 76% residual activity even after 10 cycles of reuse.