Semi-rational design of the nitroreductase EcNTR for the reduction of m-nitroacetophenone

To enhance the catalytic efficacy of nitroreductase and optimize the utilization of m-nitroacetophenone substrate, a novel nitroreductase, EcNTR, was isolated from Enterobacter cloacae ZJUT16 (CGMCC NO:28,310). The gene encoding EcNTR was successfully cloned and heterologously expressed. Through alanine scanning and site-saturation mutagenesis, two mutants, EcNTR-G20N/I54N and EcNTR-G20N/Y154D, were identified, exhibiting 5.01-fold and 4.69-fold higher specific activities, respectively, compared to wild-type EcNTR. At a substrate concentration of 80 mM, these mutants achieved a conversion rate of 98.52 %. Further optimization of the whole-cell catalytic conditions for mutant EcNTR-G20N/Y154D—specifically, a reaction time of 12 h, temperature of 30 °C, cell concentration of 100 g /L, use of DMSO as co-solvent, and 80 mM substrate—resulted in a yield of 99.2 %. These findings indicate that conformational changes in the active site region improved enzyme-substrate binding efficacy, thereby enhancing enzyme activity and catalytic ability. This study lays a foundation for the further development of nitroreductases for the efficient degradation of nitro-containing compounds.