초록
<P>A method for determining lipase enantioselectivity in the transacylation of <I>sec</I>-alcohols in organic solvent was developed. The method was applied to a model library of <I>Candida antarctica</I> lipase A (CalA) variants for improved enantioselectivity (<I>E</I> values) in the kinetic resolution of 1-phenylethanol in isooctane. A focused combinatorial gene library simultaneously targeting seven positions in the enzyme active site was designed. Enzyme variants were immobilized on nickel-coated 96-well microtiter plates through a histidine tag (His<SUB>6</SUB>-tag), screened for transacylation of 1-phenylethanol in isooctane, and analyzed by GC. The highest enantioselectivity was shown by the double mutant Y93L/L367I. This enzyme variant gave an <I>E</I> value of 100 (<I>R</I>), which is a dramatic improvement on the wild-type CalA (<I>E</I>=3). This variant also showed high to excellent enantioselectivity for other secondary alcohols tested.</P>