Increased Catalyst Productivity in alpha-Hydroxy Acids Resolution by Esterase Mutation and Substrate Modification

Optically pure alpha-hydroxy acids and their derivatives are versatile chiral building blocks in the pharmaceutical industry. In this study, the potential of a recombinant Pseudomonas putida esterase (rPPE01) for the enzymatic resolution of alpha-acetoxy acids was significantly improved by combinatorial engineering of both the biocatalyst and substrate. Semirational design based on homologous modeling and molecular docking provided a single-point variant, W187H, whose K-cat/K-M for sodium 2-acetoxy-2-(2'-chlorophenyl)acetate (Ac-CPA-Na) was increased 100-fold, from 0.0611 to 6.20 mM(-1) s(-1), while retaining its excellent enantioselectivity and broad substrate spectrum. Biocatalyst deactivation under the operating conditions was decreased by using the potassium salt of Ac-CPA instead of Ac-CPA-Na. With 0.5 g L-1 of lyophilized cells containing rPPE01-W187H, 500 mM (R,S)-Ac-CPA-K was selectively deacylated with 49.9% conversion within 15 h, giving satisfactory enantiomeric excesses (ee) for both the S product (>99% cc) and the remaining R substrate (98.7% ee). Consequently, the amount of (S)-2-hydroxy-2-(2'-chlorophenyl)acetate prepared per unit weight of lyophilized cells was improved by a factor of 18.9 compared with the original productivity of the wild-type esterase. Further enzymatic resolution of other important hydroxy acids at the 100 mL scale demonstrated that the rPPE01-W187H-based bioprocess is versatile and practical for the large-scale preparation of chiral alpha-hydroxy acids.