Directed evolution of an esterase from Pseudomonas fluorescens yields a mutant with excellent enantioselectivity and activity for the kinetic resolution of a chiral building block.
A triple mutant of an esterase from Pseudomonas fluorescens (PFE) that was created by directed evolution exhibited high enantioselectivity (E=89) in a kinetic resolution and yielded the building block (S)-but-3-yn-2-ol. Surprisingly, a mutation close to the active site caused the formation of inclusion bodies, but remote mutations were found to be responsible for the high selectivity. Back mutations gave a variant (double mutant PFE Ile76Val/Val175Ala) that showed excellent selectivity (E=96) and activity (20 min for 50% conversion, which corresponds to 1.25 U per mg of protein).
Institute of Biochemistry, Department of Biotechnology & Enzyme Catalysis, Greifswald University, Soldmannstrasse 16, 17487 Greifswald, Germany.
Marlen
M
Schmidt
Daniel
D
Hasenpusch
Markus
M
Kähler
Ulrike
U
Kirchner
Kerstin
K
Wiggenhorn
Walter
W
Langel
Uwe T
UT
Bornscheuer
D000480:Alkynes / Q000737:chemistry* / Q000494:pharmacology; D001665:Binding Sites / Q000235:genetics; D000440:Butanols / Q000737:chemistry* / Q000494:pharmacology; D019020:Directed Molecular Evolution; D004950:Esterases / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D008968:Molecular Conformation; D009154:Mutation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011551:Pseudomonas fluorescens / Q000201:enzymology* / Q000235:genetics; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000235:genetics; D013997:Time Factors
D000480:Alkynes; D000440:Butanols; D004950:Esterases