Cross-linked lipase particles with improved activity; application of a non-toxic linker for cross-linking

The preparation of cross-linked enzymes is often performed through enzyme aggregation or crystallization prior to cross-linking by glutaraldehyde as the most common linker. Isocyanide-based multi-component reactions (ICMRs) provide a one-step process to prepare cross-linked preparations of enzymes directly from their soluble forms. ICMRs accept a variety of functional groups to be involved in the coupling reaction. We here introduce cysteamine as a novel non-toxic alternative for glutaraldehyde to be used as a cross-linker in cross-linking reactions implemented by ICMRs. The cross-linked enzymes (CLEs) of Rhizomucor miehei lipase (RML) and Thermomyces lanuginosa lipase (TLL) were prepared with high immobilization yields (up to 97%). The results showed the maximum activities of 43.7 U/mg and 213.35 U/mg for the immobilized derivatives of RML and TLL, respectively, presenting an 11-fold and 10-fold improvement compared to the activity of the corresponding soluble lipases. The immobilized derivatives showed improved thermal and co-solvent stability. Additionally, the CLEs of TLL showed a selectivity of 19.9 in the selective hydrolysis of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from fish oil. The used linker provides an alternative strategy for CLEs production, particularly where the traditional cross-linking method has the possible risk of reaction contamination by weakly bonded glutaraldehyde molecules.