Directed evolution of an enantioselective lipase with broad substrate scope for hydrolysis of alpha-substituted esters.
A variant of Candida antarctica lipase A (CalA) was developed for the hydrolysis of alpha-substituted p-nitrophenyl esters by directed evolution. The E values of this variant for 7 different esters was 45-276, which is a large improvement compared to 2-20 for the wild type. The broad substrate scope of this enzyme variant is of synthetic use, and hydrolysis of the tested substrates proceeded with an enantiomeric excess between 95-99%. A 30-fold increase in activity was also observed for most substrates. The developed enzyme variant shows (R)-selectivity, which is reversed compared to the wild type that is (S)-selective for most substrates.
Engström
Karin
K
Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, SE-106 91 Stockholm, Sweden.
Nyhlén
Jonas
J
Sandström
Anders G
AG
Bäckvall
Jan-E
JE
D002175:Candida / Q000201:enzymology; D020134:Catalytic Domain; D019020:Directed Molecular Evolution / Q000379:methods*; D004952:Esters / Q000737:chemistry* / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D010666:Phenylpropionates / Q000737:chemistry / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity
D004952:Esters; D010666:Phenylpropionates; D008049:Lipase