Control of Lipase Enantioselectivity by Engineering the Substrate Binding Site and Access Channel
2009
Lafaquière, Vincent | Barbe, Sophie | Puech-Guenot, Sophie | Guieysse, David | Cortés, Juan | Monsan, Pierre | Simeon, Thierry | André, Isabelle | Remaud Simeon, Magali | Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP) ; Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse) ; Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS) | Équipe Robotique et InteractionS (LAAS-RIS) ; Laboratoire d'analyse et d'architecture des systèmes (LAAS) ; Université Toulouse Capitole (UT Capitole) ; Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse) ; Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université Toulouse - Jean Jaurès (UT2J) ; Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3) ; Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP) ; Université de Toulouse (UT)-Université Toulouse Capitole (UT Capitole) ; Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse) ; Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université Toulouse - Jean Jaurès (UT2J) ; Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3) ; Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP) ; Université de Toulouse (UT) | Institut des Technologies Avancees du Vivant (ITAV) ; Toulouse Canceropole campus
International audience
اظهر المزيد [+] اقل [-]إنجليزي. [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.
اظهر المزيد [+] اقل [-]الكلمات المفتاحية الخاصة بالمكنز الزراعي (أجروفوك)
المعلومات البيبليوغرافية
تم تزويد هذا السجل من قبل Institut national de la recherche agronomique