Enzymatic Production of Ascorbic Acid-2-Phosphate by Engineered Pseudomonas aeruginosa Acid Phosphatase

l-Ascorbic acid-2-phosphate (AsA-2P) is stable in aqueous solution and at high temperatures and is widely used in foods, pharmaceuticals, cosmetics, and fodders; however, practical application of enzymatic synthesis methods to promote industrial-scale production of AsA-2P remains a major challenge. In this study, we enhanced the phosphorylation efficiency of Pseudomonas aeruginosa acid phosphatase (APase; EC 3.1.3.2) for AsA-2P production via protein engineering. Among the mutants obtained, we selected the most efficient mutant (Var₅; G125E/D135T/S136N), which exhibited an increased k_cat of 18.6 s^-1 and a K_m for AsA of 223.9 mM. In addition, Var₅ exhibited a maximum enzyme activity of 2080.4 U/L after 10 h of fermentation, which was 80% higher than the wild-type enzyme. Furthermore, under optimal conditions, Var₅ showed a maximal conversion of 48.6% and achieved a final AsA-2P titer of 61.5 g/L at 8 h, which is considerably higher than that reported for other similar biocatalytic approaches. These findings demonstrate the potential of this method for the large-scale production of AsA-2P.