Intensifying conformational dynamics enables HRP catalysis in organic phase

Organic solvents unlock unique opportunities for biocatalysis but also present challenges, as enzymes often suffer from significant activity loss or even complete inactivation in such environments. In this study, we utilized horseradish peroxidase (HRP) in pure toluene for the oxidative degradation of phenol/aniline derivatives and demonstrated that conjugation with Pluronic, a class of amphiphilic polymers, enables HRP to achieve a substrate spectrum comparable to that observed in aqueous systems. Furthermore, the polymer conjugation significantly enhances the enzyme's catalytic activity compared to the free enzyme in toluene. Low-field NMR spectroscopy measurements indicated that polymer conjugation enhances the conformational dynamics of HRP on the millisecond timescale. Molecular dynamics simulations suggested that polymer conjugation improves the flexibility of HRP's active sites and substrate channels, thereby enabling its substrate uptake and catalytic processing in organic solvents. Together, these experimental and theoretical findings revealed a novel mechanism to enhance enzyme activity by amphiphilic polymer conjugation through conformational dynamic enhancement, providing insights for advancing enzyme catalysis in organic solvent systems.