Hierarchical systems that integrate nano- and macroscale structural elements can offer enhanced enzyme stability over traditional immobilization methods. Microparticles were synthesized using interfacial assembly of lipase B from Candida antarctica with (CLMP-N) and without (CLMP) nanoparticles around a cross-linked polymeric core, to characterize the influence of the hierarchical assembly on lipase stability in extreme environments. Kinetic analysis revealed that the turnover rate (kcat) significantly increased after immobilization. The macrostructure stabilized lipase at neutral and basic pH values, while the nanoparticles influenced stability under acidic pH conditions. Performance of CLMPs was demonstrated by production of sugar ester surfactants in a greener, deep eutectic solvent system (choline chloride and urea). Turnover rate (kcat) and catalytic efficiency (kcat/Km) of the CLMPs decreased following solvent exposure but retained over 60% and 20% activity after 48 h storage at 50 and 60 °C, respectively. CLMP and CLMP-N outperformed the commercially available lipase per unit protein in the production of sugar esters. Improving enzyme performance in greener solvent systems via hierarchical assembly can improve processing efficiency and sustainability for the production of value-added agricultural products.
Keywords: bioprocessing; deep eutectic solvent; enzyme immobilization; hierarchical assembly; lipase; nanoparticle.