TAILORING LIPASE SPECIFICITY BY SOLVENT AND SUBSTRATE CHEMISTRIES

An acyl binding structural model has been developed to explain the observed catalytic efficiencies and enantioselectivities of Candida rugosa lipase-catalyzed (trans)esterification reactions involving 2-hydroxy acids and vinyl esters, respectively, and acylation reactions involving both cyclic and acyclic alcohols. A clear minimum was observed for (trans)esterification of six-carbon acyl moieties. Morever, the stereoselectivity of 2-hydroxy acid esterification in a number of hydrophilic and hydrophobic solvents was dependent on the acyl chain length: S-isomers of 2-hydroxy acids were acylated for acyl chain lengths of six or fewer, whereas the R-isomers were preferentially esterified for acyl chain lengths of eight or more. These results suggest that CRL contains both large and small acyl binding regions or pockets with high catalysis 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 observed with vinyl caproate (C6) as acyl donor. This phenomenon may reflect the greater degree of steric hinderance in the acyl enzyme intermediate caused by the caproate group. A mechanistic and thermodynamic rationale was proposed for the effects of solvent and substrate chemistries on CRL catalysis in organic solvents.