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 (FDH _C23S and FDH _{C23SD195QY196H} ) 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. 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.
(© 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH.)