ABSTRACT
We developed a synthetic route for producing 3-amino-2-hydroxy acetophenone (3AHAP) from m-nitroacetophenone (3NAP) using an inâ vitro approach. Various reaction systems were evaluated, and a direct reaction method with crude enzyme and supersaturated substrates for optimal catalytic efficiency was chosen. The reaction system included three enzymes and was enhanced by adjusting enzyme molar ratios and optimizing ribosomal binding sites. We performed substrate docking and alanine scanning to identify key sites in the enzymes nitrobenzene nitroreductase (nbzA) and hydroxylaminobenzene mutase (habA). The optimal mutant was obtained through site-directed mutagenesis, and incorporated into the reaction system, resulting in increased product yield. After optimization, the yield of 3AHAP increased from 75â mg/L to 580â mg/L within 5â hours, the highest reported yield using biosynthesis. This work provides a promising strategy for the efficient and sustainable production of 3AHAP, which has critical applications in the chemical and pharmaceutical industries.