Improved immobilization and stabilization of lipase from Rhizomucor miehei on octyl-glyoxyl agarose beads by using CaCl2

Abstract A strategy to stabilize octyl (OC)- Rhizomucor miehei lipase (RML) and OC- Candida rugosa lipase (CRL) at pH 10 is necessary to take full advantage of the immobilization of these enzymes on OC-glyoxyl (OCGLX) supports. CaCl 2 and MnCl 2 have been reported to stabilize OC-RM and OC-CRL at pH 5.0 and 7.0. After screening different buffers, 5 mM CaCl 2 was found to be fully soluble in Gly at pH 10. OC-RML was 15 folds stabilized at this pH value by CaCl 2 , while OC-CRL was not. This salt was used in the preparation of octyl-glyoxyl (OCGLX)-RML, permitting to maintain almost unaltered the OC-RML activity that was 3 fold higher than that of the free. This preparation was 30% and 3 folds more stable in thermal or acetonitrile inactivations respectively than the standard OCGLX one. The stabilizing effect of CaCl 2 and MnCl 2 on the OCGLX preparations was studied. These salts stabilized both OCGLX-RML preparations, although the one prepared using Ca 2+ during the covalent attachment was more stabilized than the standard one by the presence of Ca 2+ , even 7-8 folds in the presence of aceonitrile. Thus, this additive permits to recover an OCGLX-RML preparation more stable and active than the standard protocol.