Hen egg white lysozyme encapsulated in ZIF-8 for performing promiscuous enzymatic Mannich reaction

Hen egg white lysozyme (HEWL) was exploited for the synthesis of β-amino carbonyl compounds through a direct and three-component Mannich reaction in aqueous, confirming high chemoselectivity toward imine. In order to further extend the applications of the enzyme, HEWL was encapsulated using a metal-organic framework (MOF). The reactivity, stereoselectivity, and reusability of the encapsulated enzyme were investigated. The reaction was significantly enhanced as compared to the non-encapsulated enzyme. A mutated version of the enzyme, containing Asp52Ala (D52A), lacking important catalytical residue, has lost the bacterial site activity against Micrococcus luteus (M. luteus) while the D52A variant displayed an increased rate of the Mannich reaction, indicating a different catalytical residue involved in the promiscuous reaction. Based on site-directed mutagenesis, molecular docking, and molecular dynamic studies, it was proposed that π-stacking, H-bond interactions, and the presence of water in the active site may play crucial roles in the mechanism of the reaction.