Optimized preparation of CALB-CLEAs by response surface methodology: The necessity to employ a feeder to have an effective crosslinking

Lipase B from Candida antarctica (CALB) has been immobilized using the CLEA technique. Due to the low content of surface Lys on the enzyme and the purity of the commercial preparation, CALB crosslinking did not work properly, and it was always possible to find some CALB (as molecules or soluble aggregates) when analyzing the CLEA using SDS-PAGE. To improve the crosslinking, bovine serum albumin was used as a feeder, and after optimization using response surface methodology, the glutaraldehyde crosslinking step was effective, and permitted to greatly stabilize the enzyme (no activity decrease was observed after a time where the free enzyme was almost fully inactivated). After two experimental designs, the best conditions for preparation of CALB–BSA-CLEA were: protein concentration (3 mg/mL), tert-butylalcohol as precipitant, precipitation for 60 min; precipitant concentration, 50% v/v; and glutaraldehyde concentration (1.5% w/v).