Data Mining Of A New Nitroreductase And Its Application In Highly Selective And Controllable Synthesis Of N-Arylhydroxylamines

N-Arylhyroxylamines are important intermediates in organic chemistry, where they have been widely used for antioxidants, pesticides, pharmaceuticals, cosmetics and electronic industry products, in addition to having physiological activities themselves. However, the conventional chemical reduction methods have been challenged due to serious pollution, expensive catalyst, hydroxylamines rapidly reduced to corresponding amine. In this study we developed a new method to synthesize arylhydroxylamines by using bacterial nitroreductase. This method represents a green, efficient and controllable method for the synthesis of arylhydroxylamines.By using genomic data mining method, we discovered a new nitroreductase from Bacillus amyloliquefaciens (BaNTR1). It was purified by Ni-NTA column and its catalytic properties were studied. The purified enzyme has a molecular mass of25.7kDa as confirmed by SDS-PAGE. It exhibited the highest activity at35℃and0.1M KPB (pH7.0), the half-life of purified enzyme at40℃,50℃and60℃was64h,20h and0.39h. Metal ions Cu2+and Ag+significantly inhibit the enzyme activity. Kinetic parameters of the purified enzyme were measured as Vmax=77.64μmol min-1mg-1Protein, Km=0.162mM. BaNTR1exhibited abroad substrates specificity with nitroarenes including nitrobenzene and TNT.In a10-mL reactor, BmGDH (glucose dehydrogenase from Bacillus megaterium) was used for coenzyme regeneration. By adding0.3mM NADP+,10mg BarNTR1can completely converted100mM p-cyanonitrobenzene to corresponding arylhydroxylamine in1h (>99%selectivity). It is worthwhile to note that when the reaction time was extended to24h, the selectivity was still remained>99%. In a100-mL reaction, Ba/NTR1exhibited excellent performance with nitroarenes bearing an electron-withdrawing group (e.g.,-cyano,-hydroxy,-carbonyl,-carbamoyl,-nitro) or without any other substitution group (e.g., nitrobenzene,>90%conversion and>98.3%selectivity). Although the conversion of nitroarenes bearing an electron-donating group was>96.7%, but the corresponding hydroxylamines was not detected at all.At last,BaNTR1and BmGDH genes were coexpressed in E. coli BL21for cell self-regeneration of the coenzyme. In a1-L reaction, p-cyanonitrobenzene was converted to arylhydroxylamine in1h with100%conversion and>99%selectivity by10g/L coexpressed cells without any external addition of NADP+. A two-phase system was applied to increase the substrate loading. In toluene/water (v/v,1/1),30g/L coespressed cells,300mM p-cyanonitrobenzene was efficiently converted to hydroxylamine in2.5h with100%conversion and>99%selectivity. The above results indicate that the nitroreductase BaNTR1is promising for the synthesis of various arylhydroxylamines.