초록
<P>Optically pure α-hydroxy acids and their derivatives are versatile chiral building blocks in the pharmaceutical industry. In this study, the potential of a recombinant <I>Pseudomonas putida</I> esterase (rPPE01) for the enzymatic resolution of α-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 <I>k</I><SUB>cat</SUB>/<I>K</I><SUB>M</SUB> for sodium 2-acetoxy-2-(2′-chlorophenyl)acetate (Ac-CPA-Na) was increased 100-fold, from 0.0611 to 6.20 mM<SUP>–1</SUP> s<SUP>–1</SUP>, 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<SUP>–1</SUP> of lyophilized cells containing rPPE01-W187H, 500 mM (<I>R</I>,<I>S</I>)-Ac-CPA-K was selectively deacylated with 49.9% conversion within 15 h, giving satisfactory enantiomeric excesses (ee) for both the <I>S</I> product (>99% ee) and the remaining <I>R</I> substrate (98.7% ee). Consequently, the amount of (<I>S</I>)-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 α-hydroxy acids.</P><P><B>Graphic Abstract</B><BR><IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/accacs/2014/accacs.2014.4.issue-3/cs401183e/production/images/medium/cs-2013-01183e_0006.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/cs401183e'>ACS Electronic Supporting Info</A></P>