Abstract
Combined cross-linked enzyme aggregates (combi-CLEAs) are a novel prospect for immobilization of a mixture of enzymes and the present study addresses the preparation, characterization and application of pectinases-cellulases combi-CLEAs for grape juice clarification. Initially, 8 enzymatic preparations were tested for turbidity reduction in grape juice and Rohapect 10L provided the best results (around 50% in 1 h) being selected for CLEA preparation. The optimization of combi-CLEAs, was performed using a central composite design (CCD) and response surface methodology (RSM) varying the glutaraldehyde concentration and reaction time, using isopropanol as the precipitant agent. The best conditions for the Rohapect 10L CLEA preparation was 110 mM of glutaraldehyde and 2 h. Bovine serum albumin (BSA) was used as a feeder and improved the volumetric activity, recovered activity and thermal stability. Combi-CLEAs-BSA prepared using 0.4 mg mL(-1) of the enzyme mixture and 2.4 mg mL(-1) of BSA presented an activity of 14 U mL(-1), 18% of recovered activity and 3-times more thermal stability compared to soluble enzymes. The combi-CLEAs and combi-CLEAs-BSA were tested in repeated batches, being reused for 4 and 6 cycles, respectively, keeping 100% of the initial activity. The combi-CLEAs and combi-CLEAs-BSA appear to be suitable alternatives of immobilized biocatalyst for the clarification of grape juices.