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Increased Enantioselectivity by Engineering Bottleneck Mutants in an Esterase from Pseudomonas fluorescens
Author(s) -
Schließmann Anna,
Hidalgo Aurelio,
Berenguer José,
Bornscheuer Uwe T.
Publication year - 2009
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200900563
Subject(s) - pseudomonas fluorescens , esterase , mutant , enantioselective synthesis , kinetic resolution , chemistry , directed evolution , stereochemistry , protein engineering , substrate (aquarium) , amino acid , active site , pseudomonas , enzyme , biochemistry , catalysis , gene , bacteria , biology , genetics , ecology
Four hydrophobic and bulky amino acid residues (F126, F144, F159, and I225) were identified to form a bottleneck guarding the entrance to the active site of an esterase from Pseudomonas fluorescens (PFE I). Hence, a range of nonpolar amino acids were introduced into PFE I to broaden the substrate range and to increase enantioselectivity while preserving the hydrophobicity of the tunnel. First, single variants were created and then the most enantioselective ones were combined to find cooperative effects. This resulted in several mutants, which showed substantially enhanced enantioselectivity; for instance, in the kinetic resolution of 1‐phenyl‐1‐propyl acetate, with which the wild type only showed E =1.2, two mutants gave E >46. For 1‐phenyl‐1‐ethyl acetate enantioselectivity increased from ∼50 to >100 for all mutants studied. Furthermore, higher conversions could be found at shorter reaction times; this indicates that the mutations not only enhanced selectivity, but that also the entrance into the active site was indeed facilitated by these mutations. The experimental results could be explained by computer modeling.