Abstract
► CALB did not crosslink in the final step of CLEA preparation. ► Amination of CALB adsorbed on octyl agarose is simple. ► Aminated CALB yields CLEA properly crosslinked. ► The new CLEAs are more stable than BSA/CALB coCLEAs.
Lipase B from Candida antarctica (CALB) is not very adequate to prepare crosslinked enzyme aggregates (CLEAs). Although the precipitation step is easy using different precipitants, the crosslinking step becomes a problem due to the low amount of Lys residues in this enzyme. In this paper, we have enriched the enzyme in amino groups by chemical amination of the enzyme using ethylenediamine and carbodiimide. The modification was performed using a solid phase strategy modifying the enzyme adsorbed on octyl-Sepharose. After desorption from the support, the enzyme was more active at pH 7.0 than the unmodified enzyme. This modified enzyme showed to be suitable to produce CLEAs. Using this modified enzyme, precipitation is also effective but the crosslinking step did not fail in giving an intense intermolecular crosslinking. This way, the CLEA did not release enzyme molecules even if boiled in SDS. Stability of this CLEA was higher in both thermal and cosolvent inactivation experiments than that of the coCLEA produced by coagregation of BSA and CALB; another alternative to produce a CLEA of this interesting enzyme.
The strategy may be of high interest for many other enzymes as a way to both permit the production of CLEAs and to improve enzyme stability during CLEA production.