Enhanced Laccase Activity and Stability as Crosslinked Enzyme Aggregates on Magnetic Copper Ferrite Nanoparticles for Biotechnological Processes

Abstract

Highly stable and reusable magnetic crosslinked enzyme aggregates (m-CLEAS) of laccase are synthesized with simultaneous improved enzymatic activity. Magnetic copper ferrite nanoparticles (CFNPs) were synthesized by solvothermal procedure with an average size of ~8 nm. The nanometric m-CLEAS were formed by co-aggregation of enzyme with CFNPs and crosslinked using glutaraldehyde. Different mass ratios of CFNPs:Laccase were assayed (1 : 2, 1 : 3, and 1 : 6), where 1 : 6 resulted in the highest activity recovery (97 %). The m-CLEAS showed an average size of ~239 nm, ~24 % enzyme immobilization efficiency, and loading as high as 1.75 g of protein per g of support. As expected, m-CLEAS oxidized the substrate with a higher transformation rate (kcat) and catalytic efficiency (kcat/Km) than the free enzyme. m-CLEAS showed superior storage and thermostability compared to free enzyme and non-magnetic CLEAS. In particular, the m-CLEAS showed ~150 % and ~100 % residual activity after 30 days of storage at 4 °C and room temperature, respectively. Furthermore, m-CLEAS showed good recyclability, retaining ~78 % and ~54 % laccase activity after 5 and 10 cycles of reuse, respectively. This work highlights the facile and cost-effective synthesis of nanometric m-CLEAS with exceptional storage stability and simultaneously improved laccase activity, making them suitable for a range of green industrial processes.