Changing the electron donor improves azoreductase dye degrading activity at neutral pH

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°C). AzoRo is not an efficient biocatalyst 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 2mol nicotinamide coenzyme are needed. AzoRo displayed Methyl Red (MR) reduction activities with NADH and NADPH of 5.49±0.14Umg^-1 and 4.96±0.25Umg^-1, respectively, whereas with BNAH it displayed 17.01±0.74Umg^-1 (following BNAH oxidation) and 7.16±0.06Umg^-1 (following MR reduction). Binding of BNAH to AzoRo was determined with a K_m of 18.75±2.45μM (BNAH oxidation) and 12.45±0.47μM (MR reduction). In order to show applicability of this system an upscaled reaction was performed using 78.6μg of purified AzoRo to convert 2.96μmol of MR (total reaction volume: 40ml) within a 1h reaction.