Immobilization and Stabilization of Lipase (CaLB) through Hierarchical Interfacial Assembly

Abstract

Nanostructure-enabled hierarchical assembly holds promise for efficient biocatalyst immobilization for improved stability in bioprocessing. In this work we demonstrate the use of a hierarchical assembly immobilization strategy to enhance the physicochemical properties and stability of lipase B from Candida antarctica (CaLB). CaLB was complexed with iron oxide nanoparticles followed by interfacial assembly at the surface of an oil-in-water emulsion. Subsequent ring opening polymerization of the oil provided cross-linked microparticles that displayed an increase in catalytic efficiency when compared to the native enzyme and Novozym 435. The hierarchical immobilized enzyme assembly showed no leakage from the support in 50% acetonitrile and could be magnetically recovered across five cycles. Immobilized lipase exhibited enhanced thermal and pH stability, providing 72% activity retention after 24 h at 50 °C (pH 7.0) and 62% activity retention after 24 h at pH 3.0 (30 °C); conditions resulting in complete deactivation of the native lipase.

Influence of Hierarchical Interfacial Assembly on Lipase Stability and Performance in Deep Eutectic Solvent

“…33,34 Dopamine capped iron oxide nanoparticles were prepared via a one-pot procedure as described previously. seconds, 3.8 cm tube height), washed three times with nanopure water, and stored in 10 mM MES buffer, pH 7, at 4 ºC for further testing.…”

“…Activity measurements were performed using the synthetic substrate resorufin butyrate (10 µΜ in 10 mM MES buffer, pH 7). 33 7…”

“…32 Commercially available lipase is adsorbed on acrylic resin beads (Novozym 435), permitting recovery, but is also susceptible to protein leaching. 33 Therefore, immobilization techniques incorporating both macroscale and nanoscale features, may improve the commercial feasibility of immobilized enzyme systems.…”

“…33,34 This technique harnessed the magnetic and interfacial properties of the nanoparticles and the facile recovery of the macroscale core. 33,35,36 These hierarchically assembled microparticles exhibited stability against protein leaching, and displayed enhanced stability to pH and temperature extremes, compared to both native lipase and Novozym 435.…”

“…33,34 This technique harnessed the magnetic and interfacial properties of the nanoparticles and the facile recovery of the macroscale core. 33,35,36 These hierarchically assembled microparticles exhibited stability against protein leaching, and displayed enhanced stability to pH and temperature extremes, compared to both native lipase and Novozym 435. 33 There remains a need to demonstrate the performance of these hierarchical structures under commercially relevant conditions; specifically, limited 3 research exists on the use of deep eutectic solvents for the production of sugar esters.…”