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Anna Christina R. Ngo, Fabian Peter Josef Schultes, Artur Maier, Simon Niklas Herman Hadewig, Dirk Tischler

• Azoreductases require NAD(P)H to reduce azo dyes but the high cost of NAD(P)H limits its application. Formate dehydrogenase (FDH) allows \(NAD(P)^{+}\) recycling and therefore, the fusion of these two biocatalysts seems promising. This study investigated the changes to the fusion protein involving azoreductase (AzoRo) of \(\textit {Rhodococcus opacus}\) 1CP and FDH (\(FDH_{C23S}\) and \(FDH_{C23SD195QY196H}\)) of \(\textit {Candida boidinii}\) in different positions with His-tag as the linker. The position affected enzyme activities as AzoRo activity decreased by 20-fold when it is in the \(\it N\)-terminus of the fusion protein. \(FDH_{C23S}\)+ AzoRo was the most active construct and was further characterized. Enzymatic activities of \(FDH_{C23S}\)+ AzoRo decreased compared to parental enzymes but showed improved substrate scope – accepting bulkier dyes. Moreover, pH has an influence on the stability and activity of the fusion protein because at pH 6 (pH that is suboptimal for FDH), the dye reduction decreased to more than 50 % and this could be attributed to the impaired NADH supply for the AzoRo part.