Construction, expression and characterization of fusion enzyme containing azoreductase and glucose 1-dehydrogenase for dye removal

• An artificial fusion enzyme called GFA was constructed.
• The characterization of GFA has been researched.
• The results indicated GFA was potential for dye removal.

Azoreductase (AZR) has been found to be an effective enzyme for azo dye removal. However, NAD(P)H is required as electron donor for the cleavage of the azo bond by AZR. Due to the high cost of the reduced nicotinamide coenzyme, the large scale application of AZR is impractical. In this study, an artificial fusion enzyme of azoreductase and alkaline-resistant NAD(P)-dependent glucose 1-dehydrogenase (LsGDH) called GFA was constructed for NAD(P)H regeneration and cyclic utilization. The purified fusion enzyme migrated as a single protein band on SDS–PAGE with a molecular weight of ∼55 kDa, exhibiting both azoreductase and glucose 1-dehydrogenase activities. The azoreductase moiety of the fusion enzyme exhibited high enzyme activities towards Methyl Red (160.62 U mg−1), which was slightly higher than parental azoreductase (153.16 U mg−1). Compared with parental enzymes, the azoreductase moiety was characterized by kinetic parameters of decreased Km (0.52-fold) and increased Kcat (1.25-fold). These indicate a 2.41-fold net increase in catalytic efficiency (Kcat/Km) of the azoreductase moiety. The highest enzymatic degradation was achieved at the temperature of 45 °C and pH of 7.0, respectively. Mg2+ and Ca2+ enhanced the fusion enzyme activity, while Cr2+, Mn2+and Zn2+ inhibited the activity to varying extents. The enzyme activity of the fusion enzyme was enhanced by low concentrations of dodecyl trimethyl ammonium bromide (DTAB) and cetylpyridinium chloride (CPC) but inhibited by high concentrations of surfactants.