72377-05-0Relevant articles and documents
Iterative saturation mutagenesis accelerates laboratory evolution of enzyme stereoselectivity: Rigorous comparison with traditional methods
Reetz, Manfred T.,Prasad, Shreenath,Carballeira, Jose D.,Gumulya, Yosephine,Bocola, Marco
experimental part, p. 9144 - 9152 (2010/08/21)
Efficacy in laboratory evolution of enzymes is currently a pressing issue, making comparative studies of different methods and strategies mandatory. Recent reports indicate that iterative saturation mutagenesis (ISM) provides a means to accelerate directed evolution of stereoselectivity and thermostability, but statistically meaningful comparisons with other methods have not been documented to date. In the present study, the efficacy of ISM has been rigorously tested by applying it to the previously most systematically studied enzyme in directed evolution, the lipase from Pseudomonas aeruginosa as a catalyst in the stereoselective hydrolytic kinetic resolution of a chiral ester. Upon screening only 10 000 transformants, unprecedented enantioselectivity was achieved (E = 594). ISM proves to be considerably more efficient than all previous systematic efforts utilizing error-prone polymerase chain reaction at different mutation rates, saturation mutagenesis at hot spots, and/or DNA shuffling, pronounced positive epistatic effects being the underlying reason.
Directed evolution of an enantioselective lipase with broad substrate scope for hydrolysis of α-substituted esters
Engstroem, Karin,Nyhlen, Jonas,Sandstroem, Anders G.,Baeckvall, Jan-E.
supporting information; experimental part, p. 7038 - 7042 (2010/07/05)
A variant of Candida antarctica lipase A (CalA) was developed for the hydrolysis of α-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.