title: Improved enantioselectivity of thermostable esterase from Archaeoglobus fulgidus toward (S)-ketoprofen ethyl ester by directed evolution and characterization of mutant esterases.
abstract: Thermostable esterases have potential applications in various biotechnology industries because of their resistance to high temperature and organic solvents. In a previous study, we isolated an esterase from Archaeoglobus fulgidus DSM 4304 (Est-AF), which showed high thermostability but low enantioselectivity toward (S)-ketoprofen ethyl ester. (R)-ketoprofenor (S)-ketoprofenis produced by esterase hydrolysis of the ester bond of (R,S)-ketoprofen ethyl ester and (S)-ketoprofen has better pharmaceutical activity and lower side effects than (R)-ketoprofen. Therefore, we have generated mutants of Est-AF that retained high thermostability whilst improving enantioselectivity. A library of Est-AF mutants was created by error-prone polymerase chain reaction, and mutants with improved enantioselectivity were isolated by site-saturation mutagenesis. The regions of Est-AF containing amino acid mutations were analyzed by homology modeling of its three-dimensional structure, and structure-based explanations for the changes in enantioselectivity are proposed. Finally, we isolated two mutants showing improved enantioselectivity over Est-AF (ee% = -16.2 ± 0.2 and E = 0.7 ± 0.0): V138G (ee% = 35.9 ± 1.0 and E = 3.0 ± 0.1) and V138G/L200R (ee% = 89.2 ± 0.2 and E = 19.5 ± 0.5). We also investigated various characteristics of these mutants and found that the mutants showed similar thermostability and resistance to additives or organic solvents to Est-AF, without a significant trade-off between activity and stability.
authors: Department of Molecular Science and Technology, Ajou University, Suwon, 443-749, Korea.
authors: Jinyeong
authors: J
authors: Kim
authors: Seungbum
authors: S
authors: Kim
authors: Sangyoung
authors: S
authors: Yoon
authors: Eunsoo
authors: E
authors: Hong
authors: Yeonwoo
authors: Y
authors: Ryu
mesh_terms: D019611:Archaeoglobus fulgidus / Q000201:enzymology* / Q000235:genetics; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D004950:Esterases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004952:Esters / Q000378:metabolism*; D006358:Hot Temperature; D007660:Ketoprofen / Q000378:metabolism*; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D004952:Esters; D050505:Mutant Proteins; D007660:Ketoprofen; D004950:Esterases