TAILORING LIPASE SPECIFICITY BY SOLVENT AND SUBSTRATE CHEMISTRIES

An acyl binding structural model has been developed to explain the obs erved catalytic efficiencies and enantioselectivities of Candida rugos a lipase-catalyzed (trans)esterification reactions involving 2-hydroxy acids and vinyl esters, respectively, and acylation reactions involvi ng both cyclic and acyclic alcohols. A clear minimum was observed for (trans)esterification of six-carbon acyl moieties. Morever, the stereo selectivity of 2-hydroxy acid esterification in a number of hydrophili c and hydrophobic solvents was dependent on the acyl chain length: S-i somers of 2-hydroxy acids were acylated for acyl chain lengths of six or fewer, whereas the R-isomers were preferentially esterified for acy l chain lengths of eight or more. These results suggest that CRL conta ins both large and small acyl binding regions or pockets with high cat alysis observed for proper fitting substrates into either pocket. CRL is also highly selective and reactive on secondary cyclic alcohols. In particular, the R isomers of menthol and sec-phenethanol are acylated efficiently by straight-chain vinyl esters. The catalytic efficiency of acylation (i.e., V(max)/K(m) for the secondary alcohol) is strongly dependent on the acyl chain length. Once again, a clear minimum is ob served with vinyl caproate (C6) as acyl donor. This phenomenon may ref lect the greater degree of steric hinderance in the acyl enzyme interm ediate caused by the caproate group. A mechanistic and thermodynamic r ationale was proposed for the effects of solvent and substrate chemist ries on CRL catalysis in organic solvents.