Mutagenesis-independent, 1000-fold stabilization of class B Flavin monooxygenases in operation

Abstract

In this study we describe the stabilization of f flavin-dependent monooxygenases under reaction conditions, using an engineered formulation of additives (the natural cofactors NADPH, FAD, and superoxide dismutase and catalase as catalytic antixodans, Figure 1). This way, a 10^3 to 10^4-fold increase of the half-life under reaction conditions was reached without resource-intensive directed evolution or structure-dependent protein engineering methods. The stabilized enzymes are highly valued for their synthetic potential in biotechnology and medicinal chemistry (enantioselective sulfur, nitrogen and Baeyer-Villiger oxidations; oxidative human metabolism) but widespread application was so far hindered by their notorious fragility. Our technology immediately enables their use, does not require structural knowledge of the biocatalyst, and creates a strong basis for the targeted development of improved variants by mutagenesis.