Pluronic‐Conjugated Enzyme Cascade for In Situ Oxidation in Biphasic Media

Abstract

AbstractA Pluronic‐conjugated enzyme cascade was synthesized for catalyzing oxidation reactions using hydrogen peroxide (H2O2) as the oxidant. The synthesis started with encapsulating enzymes in the micelles of aldehyde‐functionalized Pluronic (PL) containing bovine serum albumin (BSA). Schiff‐base bonds formed between the amine groups of proteins and the aldehyde groups of the PL, which were reduced to a more stable form, resulting in enzyme–PL conjugates. The effects of operation parameters on the conjugate yield were examined using glucose oxidase (GOx) and horseradish peroxidase (HRP) as examples. The co‐localization of GOx and HRP was confirmed by Förster resonance energy transfer (FRET), from which the average distance between GOx and HRP was interpreted as 6.1 nm. The proximity effect was confirmed by adding a high concentration of catalase (CAT) to a solution of GOx–HRP–PL to catalyze the oxidation of ABTS. The GOx–HRP–PL conjugate was readily solubilized in organic solvents such as dimethyl sulfoxide, methanol or chloroform. Phenol oxidation with GOx–HRP–PL in dimethyl sulfoxide/water biphasic media was conducted using glucose as the substrate to generate H2O2 in situ. The multiple enzyme conjugate provided a 1.3‐fold higher removal rate compared to their native counterparts in free form. The GOx–HRP–PL conjugate could be readily recovered by centrifugation at 4 °C and 90 % of the initial activity remained after 10 cycles.