Abstract
•CALB could be immobilized on vinyl sulfone (VS) activated support while RML could not.•Co-immobilization of CALB and RML was not possible using vinyl sulfone activated support.•RML could be immobilized on ethylendiamine blocked VS support via anion exchange.•CALB was covalently immobilized on VS, blocked with ethylediamine, and then RML was co-immobilized.•Immobilized CALB was much more stable than immobilized RML.•RML could be released from the support, enabling the reuse of immobilized CALB.
The coimmobilization of lipases from Rhizomucor miehei (RML) and Candida antarctica (CALB) has been intended using agarose beads activated with divinyl sulfone. CALB could be immobilized on this support, while RML was not. However, RML was ionically exchanged on this support blocked with ethylendiamine. Therefore, both enzymes could be coimmobilized on the same particle, CALB covalently using the vinyl sulfone groups, and RML via anionic exchange on the aminated blocked support. However, immobilized RML was far less stable than immobilized CALB. To avoid the discarding of CALB (that maintained 90% of the initial activity after RML inactivation), a strategy was developed. Inactivated RML was desorbed from the support using ammonium sulfate and 1% Triton X-100 at pH 7.0. That way, 5 cycles of RML thermal inactivation, discharge of the inactivated enzyme and re-immobilization of a fresh sample of RML could be performed. In the last cycle, immobilized CALB activity was still over 90% of the initial one. Thus, the strategy permits that enzymes can be coimmobilized on vinyl sulfone supports even if one of them cannot be immobilized on it, and also permits the reuse of the most stable enzyme (if it is irreversibly attached to the support).