Immobilization of aldehyde dehydrogenase on montmorillonite using polyethyleneimine as a stabilization and bridging agent

In the following study the tetrameric enzyme, aldehyde dehydrogenase (ALDH - 228 kDa), was immobilized on montmorillonite using polyethyleneimine (PEI) as a bridging agent. Previous studies have shown that small enzymes such as laccases and peroxidases exhibit increased stability and enhanced activity over time when immobilized on the surface of clay minerals. However, low-cost immobilization methods for high MW, multimeric enzymes, are lacking because these enzymes are prone to disassociation and deactivation upon adsorption. Herein, ALDH was successfully immobilized by pre-stabilization with a polyelectrolyte prior to adsorption on the surface of montmorillonite. The resulting complex was characterized in terms of enzyme conformation and loading using zeta potential, SEM, FTIR, and TGA measurements. Aldehyde transformation activity was measured by following benzaldehyde transformation to benzoic acid and NADH formation. An optimal ALDH:PEI ratio of 4:1 (w/w) was identified, yielding the highest activity at the adsorbed state due to conformational stability. The adsorption isotherm best fit the dual Langmuir-Freundlich model and revealed a significantly high loading capacity of 6.2 mg protein per mg montmorillonite. Rate experiments showed that although activity was maintained at the adsorbed state, the immobilization slowed aldehyde transformation, probably due to diffusion limitations caused by aggregation and heterogeneity of the material. Overall, the results suggest that this method has the potential to efficiently immobilize large multimeric proteins. This in turn can help solve separation issues and advance the design of efficient platforms for bio-catalysis and bioremediation technologies. [Display omitted] •A tetrameric enzyme was immobilized on montmorillonite for aldehyde transformation.•An optimal enzyme: polymer ratio of 4:1 resulted in high activity at the adsorbed state.•Polyethyleneimine stabilized the enzyme and conserved conformation upon adsorption.•A high loading capacity of 6.2 mg protein per mg montmorillonite was achieved.•Aldehyde transformation activity was maintained at the adsorbed state.