Coimmobilization of lipases exhibiting three very different stability ranges. Reuse of the active enzymes and selective discarding of the inactivated ones

• Published in: International journal of biological macromolecules 2022-05, Vol.206, p.580-590
• Main Authors: Carballares, Diego, Rocha-Martin, Javier, Fernandez-Lafuente, Roberto
• Format: Article
• Language: English
• Subjects: Ammonium Sulfate; Candida; Coimmobilized enzyme stability; Enzyme coimmobilization; Enzyme Stability; Enzymes, Immobilized - metabolism; Fungal Proteins - metabolism; Lipase - metabolism; Reuse of coimmobilized stable enzymes; Selective enzyme desorption; Sepharose
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/j.ijbiomac.2022.02.084

Lipase B from Candida antarctica (CALB) and lipases from Candida rugosa (CRL) and Rhizomucor miehei (RML) have been coimmobilized on octyl and octyl-Asp agarose beads. CALB was much more stable than CRL, that was significantly more stable than RML. This forces the user to discard immobilized CALB and CRL when only RML has been inactivated, or immobilized CALB when CRL have been inactivated. To solve this problem, a new strategy has been proposed using three different immobilization protocols. CALB was covalently immobilized on octyl-vinyl sulfone agarose and blocked with Asp. Then, CRL was immobilized via interfacial activation. After coating both immobilized enzymes with polyethylenimine, RML could be immobilized via ion exchange. That way, by incubating in ammonium sulfate solutions, inactivated RML could be released enabling the reuse of coimmobilized CRL and CALB to build a new combi-lipase. Incubating in triton and ammonium sulfate solutions, it was possible to release inactivated CRL and RML, enabling the reuse of immobilized CALB when CRL was inactivated. These cycles could be repeated for 3 full cycles, maintaining the activity of the active and immobilized enzymes. •RML, CALB and CRL immobilized in octyl-Asp agarose presented very different stabilities at pH 7.•CALB could be covalently immobilized on octyl-vinyl sulfone agarose.•CRL could be immobilized by interfacial activation on octyl-VS-CALB-Asp agarose.•Activity/stability of immobilized CRL and CALB increases by PEI coating and it permits to immobilize RML.•The release of RML or CRL/RML from octyl-VS-CALB-Asp-CRL-PEI-RML enabled reusing immobilized CAL/CRL or CALB.