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Control of Lipase Enantioselectivity by Engineering the Substrate Binding Site and Access Channel

Abstract : [a] ((Dedication, optional)) Lipase from Burkholderia cepacia (BCL) has proven to be a very useful biocatalyst for the resolution of 2-substituted racemic acid derivatives which are important chiral building blocks. Our previous work showed that enantioselectivity of the wild-type BCL could be improved by chemical engineering of the substrate molecular structure. From this earlier study, three amino acids (L17, V266 and L287) were proposed as targets for mutagenesis aiming at tailoring enzyme enantioselectivity. In the present work, a small library of 57 BCL single mutants targeted on these three residues was constructed and screened for their enantioselectivity towards (R,S)-2-chloro ethyl 2-bromophenylacetate. This led to the fast isolation of three single mutants with a remarkable 10 times enhanced or reversed enantioselectivity. Analysis of substrate docking and trajectories in the active site was then performed. From this analysis, the construction of 13 double-mutants was proposed. Among them, an outstanding improved mutant of BCL was isolated that showed an E-value of 178 and a 15 times enhanced specific activity compared to the parental enzyme, thus demonstrating the efficiency of the semi-rational engineering strategy.
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https://hal.laas.fr/hal-01986285
Déposant : Thierry Simeon <>
Soumis le : vendredi 18 janvier 2019 - 16:27:28
Dernière modification le : lundi 23 novembre 2020 - 12:56:03

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Vincent Lafaquière, Sophie Barbe, Sophie Puech-Guenot, David Guieysse, Juan Cortés, et al.. Control of Lipase Enantioselectivity by Engineering the Substrate Binding Site and Access Channel. ChemBioChem, Wiley-VCH Verlag, 2009, 10 (17), pp.2760-2771. ⟨10.1002/cbic.200900439⟩. ⟨hal-01986285⟩

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