Characterisation of an oxygen-tolerant recombinant nitroreductase for development of an aryl amine biosynthesis platform using cofactor-regenerating crude extract system.

Biocatalytic reduction of nitroaromatic compounds (NACs) to aryl amines offers a route for integrating pollutant transformation with the synthesis of important chemical intermediates under mild conditions, providing an alternative to energy- and resource-intensive conventional chemical reductions. However, practical deployment of nitroreductases is often limited by oxygen sensitivity, high cofactor cost, and inefficient progression beyond hydroxylamine intermediates. Here, we report the characterisation of a novel oxygen-tolerant nitroreductase (C_Ntr) from a Cupriavidus strain a3. Phylogenetic analysis placed C_Ntr within a distinct Cupriavidus clade, sharing 97.89% identity with C. oxalaticus nitroreductase. It is a homodimeric flavoprotein with a mixed α/β fold, and exhibits optimal activity at pH 8.0 and 30 °C, with broad pH (6.0-9.0) tolerance and thermal stability (Tₘ = 58.9 °C). Gene expression analysis demonstrated significant induction of C_Ntr expression in response to NACs exposure. It displayed broad substrate specificity with catalytic efficiencies ranging from 0.57 to 15.3 × 10⁴ M⁻¹ s⁻¹ and catalysed full six-electron reduction of several NACs (nitrobenzene, 3-nitrotoluene, and 2,4-dinitrophenol) to corresponding amines. Mechanistic analysis revealed strong NADPH stoichiometric dependence and enhanced terminal amine formation under oxic relative to anoxic conditions. To address the NADPH cofactor cost, a crude extract-based system was developed, enabling in situ NADPH regeneration using citrate as economical electron donor, achieving 82% conversion of nitrobenzene to aniline without the need for enzyme purification or NADPH/ NADP⁺ supplementation. This work establishes a laboratory-scale platform and provides a practical framework for future evaluation of crude extract-based nitroreduction strategy under environmentally relevant conditions.