Prasad, Shreenath and Bocola, Marco and Reetz, Manfred T. (2011) Revisiting the Lipase from Pseudomonas aeruginosa: Directed Evolution of Substrate Acceptance and Enantioselectivity Using Iterative Saturation Mutagenesis. CHEMPHYSCHEM, 12 (8). pp. 1550-1557. ISSN 1439-4235,
Full text not available from this repository. (Request a copy)Abstract
The most thoroughly studied enzyme in directed evolution is the lipase from Pseudomonas aeruginosa (PAL) as a catalyst in the hydrolytic kinetic resolution of 2-methyldecanoic acid p-nitrophenyl ester. Seminal studies utilized epPCR, saturation mutagenesis and DNA shuffling or combinations thereof. With current emphasis on efficacy in laboratory evolution, however, we recently applied our previously developed method, iterative saturation mutagenesis (ISM), to the same catalytic system, discovering that this approach is much more efficient than the original strategies. Herein, we consider PAL once more, this time testing ISM as a means to broaden the substrate scope of this lipase by studying bulky substrates of the type 2-phenylalkanoic acid esters as substrates that are not accepted by the WT. Highly active and enantioselective (E up to 436) mutants were evolved, a process that required only small mutant libraries and thus a minimum of screening effort. A theoretical investigation using molecular dynamics simulations and docking experiments revealed the source of enhanced activity and stereoselectivity.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | ACTIVE-SITE; FORCE-FIELD; LABORATORY EVOLUTION; GENETIC SELECTION; BINDING-SITE; ENZYMES; BIOCATALYSTS; CREATION; SPACE; PARAMETERIZATION; directed evolution; enantioselectivity; enzymes; molecular dynamics; mutagenesis |
| Subjects: | 500 Science > 540 Chemistry & allied sciences |
| Divisions: | Biology, Preclinical Medicine > Institut für Biophysik und physikalische Biochemie |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 09 Jun 2020 07:13 |
| Last Modified: | 09 Jun 2020 07:13 |
| URI: | https://pred.uni-regensburg.de/id/eprint/20673 |
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