Comprehensive study of Candida antarctica lipase B enantioselectivity : using experimental and molecular modeling approaches

• Main Author: Chaput, Ludovic
• Other Authors: LIttoral ENvironnement et Sociétés - UMRi 7266 (LIENSs), Université de La Rochelle (ULR)-Centre National de la Recherche Scientifique (CNRS), Université de La Rochelle, Marianne Graber, Huu Vinh Tran
• Format: Doctoral or Postdoctoral Thesis
• Language: French
• Published: HAL CCSD 2012
• Subjects: Biocatalysis; Lipase B from Candida antarctica; Enantioselectivity; Molecular modeling; Tetrahedral intermediate; Free energy perturbation; Imprinting effect; Mutant; T42V; S47A; T42V/S47A; Lipase B de Candida antarctica; Énantiosélectivité; Biocatalyse; Modélisation moléculaire; Intermédiaire tétraédrique; Perturbation de l’énergie libre; Effet d’empreinte; [SDV.SA]Life Sciences [q-bio]/Agricultural sciences; Antarc*; Antarctica
• Online Access: https://tel.archives-ouvertes.fr/tel-00825876; https://tel.archives-ouvertes.fr/tel-00825876/document; https://tel.archives-ouvertes.fr/tel-00825876/file/2012Chaput30539.pdf

The lipase B from Candida antarctica is an enzyme displaying enantioselective properties which are interesting to obtain enantio pure compounds by kinetic resolution of racemic mixtures, which are used as pharmaceutical intermediates and fine chemicals. Indeed, for most of the drugs, only one of the two chiral formsis efficient as bioactive compound, whereas the other chiral form may display deleterious effects. Present work concerns the understanding of the origin of Candida antarctica lipase B enantioselectivity, and more especially in case of the resolution of secondary alcohols by transesterification. We used, for the first time, the free energy perturbation method to evaluate the free energy difference between tetrahedral intermediates with R and S alcohol enantiomers for a series of secondary alcohols in order to predict in silico enantiomeric ratio of CALB-catalyzed reactions. The apparent kinetic parameters were experimentally determined for two enantio pure substrates and allow to evalute the relative contribution of both Km and kcat for R and S enantiomers in the enantiomeric ratio of CALB-catalyzed reactions. Experimental study of imprinting effect hypothesis by co-substrate molecules was done. Molecular modeling studies of imprinting effect hypothesis were performed, in which the first substrate ester of the reaction could mould the active site. At least, the third part of this thesis concerns the study of wild-type CALB and three different variants (T42V and S47A which allow to increase enantioselectivity and T42/S47A) of CALB by molecular modeling. A detailed study of the conformation of the stereo specificity pocket in the active site is presented, based on molecular dynamics simulations. La lipase B de Candida antarctica (CALB) est un enzyme présentant des propriétés énantiosélectives très intéressantes pour l’obtention de molécules énantio pures par dédoublement cinétique de mélanges racémiques,molécules utilisées comme synthons dans l’industrie pharmaceutique. En effet, le principe actif de ...