Overcoming flavin-driven inactivation of alcohol dehydrogenases through enzyme immobilization

Abstract

Flavins are organocatalysts that can be employed as photocatalysts, but also as cofactor regeneration systems when coupled with alcohol dehydrogenases (ADHs) for alcohol oxidation reactions. In previous work, we developed a confined (organo)enzymatic system where ADH catalyzes NAD⁺-dependent regioselective diol oxidation, while FMN enables ground-state NAD⁺ regeneration using molecular oxygen as the electron acceptor. Despite co-immobilization on the same support, the flavin induces premature ADH inactivation during discontinuous use of the heterogeneous hybrid catalyst. Through experimental and computational methods, we herein investigate how FMN interacts with ADHs, triggering their inactivation. We found that the isoalloxazine ring and the ribityl moiety of flavins disrupt ADH's quaternary structure via nonspecific binding at subunit interfaces, creating an oxidative microenvironment that oxidizes surface-exposed cysteine residues. By immobilizing and compartmentalizing flavins and ADHs through a cationic polymer layer, we enhanced the operational stability of this hybrid heterogeneous catalyst, maintaining its maximum oxidative performance for three consecutive reaction cycles, outperforming flavin-dependent oxidation systems previously reported. Collectively, this work shows that microscopic catalyst compartmentalization via rational immobilization overcomes flavin–dehydrogenase incompatibilities in cascade reactions, enabling a robust integration of chemo- and biocatalysts for concurrent reaction routes.