By using a isocyanide-based multi-component reaction for the immobilization of the soluble forms of Rhizomucor miehei lipase (RML) and Thermomyces lanuginosa lipase (TLL), the first step of enzyme aggregation or crystallization in the traditional methods of cross-linking was bypassed. High immobilization yields and specific activities were achieved for both lipases.

We describe here a novel and simple method for making bioconjugation and immobilization of Rhizomucor miehei lipase (RML) on carboxylated multiwall carbon nanotubes (MWCNT-COOH) and carboxylated graphene nanosheets (Gr-COOH) by using an isocyanide-based four-component reaction. In this approach, the enzyme supplies amino groups, the support supplies carboxylic acid groups, and the missing components (isocyanide and aldehyde) are added to the reaction medium. This coupling reaction was carried out in water at 25 °C, in which rapid and high enzyme loading were achieved. The maximum loading capacity of 530 mg and 680 mg was obtained for Gr-COOH and MWCNT-COOH, respectively. A variety of techniques including FTIR, Raman spectroscopy, XRD, SEM, and TGA were employed to characterize the immobilized derivatives of RML. The immobilized preparations showed significantly increased thermal stability and co-solvent stability as compared to the soluble enzyme. Kinetic parameters and optimum pH activity of RML and its immobilized preparations were also determined. The K_m values of 0.44, 0.23, and 0.18 mM and the maximum reaction rates (V_max) of 0.09, 0.1, and 0.08 mM min⁻¹ were obtained for MWCNTs-RML, Gr-RML, and free RML, respectively. This approach may provide a general and efficient method to attach biomolecules on a variety of carboxylated solid surfaces at ambient conditions.