Preparation and characterization of cross-linked enzyme aggregates (CLEAs) of Brassica rapa peroxidase

Abstract

Peroxidase from white turnip (Brassica rapa) was immobilized as cross-linked enzyme aggregate (CLEA) via solvent precipitation and glutaraldehyde cross-linking. The nature of the precipitation solvent, enzyme and glutaraldehyde concentrations affected the formation and the activity of the enzyme aggregate. The aggregates resulting from cross-linking with glutaraldehyde produced insoluble catalytically active cross-linked enzyme aggregates. Acetone was selected as the more efficient solvent, giving the highest activity recovery (28%) and aggregation yield (78%). Immobilization was conducted for 3 h under agitation at 4 °C. Optimal conditions were determined as 2% (v/v) glutaraldehyde and 9:1 (v/v) enzyme to glutaraldehyde ratio. The highest activity recovery and aggregation yield were 42.5% and 82% respectively. CLEAs with enhanced thermal and acidic condition stabilities were formed when compared to the free enzyme. The cross-linked enzyme aggregates exhibited optimal pH and temperature in the range 6–7 °C and 40 °C, respectively. CLEAs of Brassica rapa peroxidase exhibited good stability in nonpolar organic solvents and they could be stored in pH 7 buffer with no activity loss at 4 °C for more than three months.