Improved enantioselectivity of a lipase by rational protein engineering

A model based on two different binding modes for alcohol enantiomers in the active site of a lipase allowed rational redesign of its enantioselectivit y, 1-Halo-2-octanols were poorly resolved by Candida antarctica lipase B. I nteractions between the substrates and the lipase were investigated with mo lecular modeling. Unfavorable interactions were found between the halogen m oiety of the fast-reacting S enantiomer and a region situated at the bottom of the active site (stereoselectivity pocket). The lipase was virtually mu tated in this region and energy contour maps of some variants displayed bet ter interactions for the target substrates. Four selected variants of the l ipase were produced and kinetic resolution experiments were undertaken with these mutants. Single point mutants gave rise to one variant with doubled enantioselectivity as well as one variant with annihilated enantioselectivi ty towards the target halohydrins. An increased volume of the stereoselecti vity pocket caused a decrease in enantioselectivity, while changes in elect rostatic potential increased enantioselectivity, The enantioselectivity of these new lipase variants towards other types of alcohols was also investig ated. The changes in enantioselectivity caused by the mutations were well i n agreement with the proposed model concerning the chiral recognition of al cohol enantiomers by this lipase.