A simple approach to a vastly improved acetylcholinesterase activity and stability at elevated temperatures using magnetic microbeads and poly(N-(3-aminopropyl methacrylamide)) hydrogel supports

Abstract The thermal stabilization of enzymes is a critical factor in the development and reliability of enzyme-based processes and functional materials. Using a simple amine coupling approach for enzyme immobilization onto magnetic microbeads, followed by encasement of the beads in a hydrogel, we demonstrate that the thermal stability of the enzyme acetylcholinesterase can be increased dramatically. For example, when free and microbead-immobilized enzyme (“EM Conjugate”) are incubated overnight in a dry state at 63 °C (140 °F), the catalytic efficiency (k cat /K m ) of the latter is higher than the former by six orders of magnitude (a factor of 2.16 × 10 6 ). This effect arises mostly through a ∼29,700-fold decrease in K m experienced by the EM Conjugate, relative to that of the free enzyme. Encapsulation of the EM Conjugate in a hydrogel based on poly(N-(3-aminopropyl methacrylamide)), which contains a primary amine, affords the enzyme additional stability when incubated overnight at 63 °C in an aqueous state. For example, its catalytic efficiency is four orders of magnitude higher than that of both the free enzyme (a factor of 4.34 × 10 4 ) and that of the EM Conjugate alone (a factor of 1.78 × 10 4 ) after all are incubated overnight at 63 °C. The presence of the hydrogel also caused the Michaelis constant to decrease by 1.38 × 10 4 relative to that of the EM Conjugate, reaching a value of 2.18 × 10 −3  M. Thus the hydrogel enables the AChE substrate binding site to retain a significant amount of its natural affinity for the substrate, after heating. This effect may occur via ion-pairing by the primary amines in the hydrogel polymer repeat unit, which are protonated and positively-charged at the assay pH. To the best of our knowledge, this simple method for enzyme thermal stabilization is novel and has not yet been investigated.