The oxygen-insensitive azoreductase AzoRo originating from Rhodococcus opacus 1CP was found to be most active at low pH (ca. 4) and high temperature (ca. 50 degrees C). AzoRo is not an efficient biodatalyst when used at low pH due to stability problems. To overcome this issue, we discovered that AzoRo accepts an alternative electron donor, 1-benzyl-1,4-dihydronicotinamide (BNAH), which allows fast turnover at neutral pH. In order to screen this nicotinamide coenzyme mimic as a source of electrons, AzoRo-catalysed reactions were run under neutral conditions, under which typically slow rates are observed with NADH. For the reduction of 1 azo bond by azoreductases 2 mol nicotinamide coenzyme are needed. AzoRo displayed Methyl Red (MR) reduction activities with NADH and NADPH of 5.49 +/- 0.14 U mg(-1) and 4.96 +/- 0.25 U mg(-1), respectively, whereas with BNAH it displayed 17.01 +/- 0.74 U mg(-1) (following BNAH oxidation) and 7.16 +/- 0.06 U mg(-1) (following MR reduction). Binding of BNAH to AzoRo was determined with a Km of 18.75 +/- 2.45 mu M (BNAH oxidation) and 12.45 +/- 0.47 mu M (MR reduction). In order to show applicability of this system an upscaled reaction was performed using 78.6 mu g of purified AzoRo to convert 2.96 mu mol of MR (total reaction volume: 40 ml) within a 1 h reaction. (C) 2017 Elsevier Inc. All rights reserved.