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 re-actions implemented by ICMRs. The cross-linked enzymes (CLEs) of Rhizomucor miehei lipase (RML) and Ther-momyces 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 doco-sahexaenoic 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.

Automated summary

The preparation of cross-linked enzymes can be done through enzyme aggregation or crystallization followed by cross-linking using glutaraldehyde as a common linker. Isocyanide-based multi-component reactions (ICMRs) provide a one-step process to directly prepare cross-linked enzymes from their soluble forms. Cysteamine is introduced as a non-toxic alternative cross-linker in ICMR-implemented cross-linking reactions, demonstrating improved immobilization yields and enzyme activities.