Improving Biocatalytic Properties of an Azoreductase via the N‐Terminal Fusion of Formate Dehydrogenase

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 Rhodococcus opacus 1CP and FDH (FDHC23S and FDHC23SD195QY196H) of 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 N‐terminus of the fusion protein. FDHC23S+AzoRo was the most active construct and was further characterized. Enzymatic activities of FDHC23S+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. A fusion protein comprised of formate dehydrogenase and azoreductase was constructed to allow the simultaneous activity of dye reduction and NAD+ regeneration. The fusion protein showed improved substrate scope, but it only showed partial degradation for the bulkier azo dyes.