The catalytic performance of Candida antarctica lipase B (CALB) immobilized on silica-coated magnetic nanoparticles was evaluated for biodiesel production via methanolysis of rapeseed oil. Two different covalent immobilization approaches were compared to assess the effect of immobilization protocols on lipase efficiency. The first approach involved immobilization of CALB on amine-functionalized magnetic nanoparticles (MNPs), which targeted the Lys-rich regions of the enzyme. The second used epoxy-functionalized MNPs, enabling broader nucleophilic groups on the enzyme surface to participate in the coupling reaction. Immobilization of 20 mg of CALB on 1 g of each support resulted in 82 % and 86 % protein loading on the amine- and epoxy-functionalized MNPs, respectively, after 24 h of incubation. Response surface methodology (RSM) was applied to optimize biodiesel production by analyzing the effects of parameters such as reaction temperature, time, t-butanol concentration, biocatalyst loading, and molecular sieve quantity on the yield of fatty acid methyl esters (FAME). Out of 45 designed experiments, the maximum FAME yields were 92 % and 84 % for the epoxy- and amine-functionalized MNPs, respectively.
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