Expanding the substrate scope of enzymes: combining mutations obtained by CASTing.
In a previous paper, the combinatorial active-site saturation test (CAST) was introduced as an effective strategy for the directed evolution of enzymes toward broader substrate acceptance. CASTing comprises the systematic design and screening of focused libraries around the complete binding pocket, but it is only the first step of an evolutionary process because only the initial libraries of mutants are considered. In the present study, a simple method is presented for further optimization of initial hits by combining the mutational changes obtained from two different libraries. Combined lipase mutants were screened for hydrolytic activity against six notoriously difficult substrates (bulky carboxylic acid esters) and improved mutants showing significantly higher activity were identified. The enantioselectivity of the mutants in the hydrolytic kinetic resolution of two substrates was also studied, with the best mutant-substrate combination resulting in a selectivity factor of E=49. Finally, the catalytic profile of the evolved mutants in the hydrolysis of simple nonbranched carboxylic acid esters, ranging from acetate to palmitate, was studied for theoretical reasons.
Reetz
Manfred T
MT
Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim/Ruhr, Germany. reetz@mpi-muelheim.mpg.de
Carballeira
José Daniel
JD
Peyralans
Jérôme
J
Höbenreich
Horst
H
Maichele
Andrea
A
Vogel
Andreas
A
D002384:Catalysis; D004952:Esters / Q000737:chemistry; D006868:Hydrolysis; D008049:Lipase / Q000737:chemistry* / Q000235:genetics*; D015394:Molecular Structure; D009154:Mutation; D019151:Peptide Library; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D013237:Stereoisomerism
D004952:Esters; D019151:Peptide Library; D008049:Lipase