caa a22 4500 605506647 CHVBK 20210128100628.0 cr unu---uuuuu 210128e20150801xx s 000 0 eng 10.1007/s00253-015-6422-7 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6422-7 Improved enantioselectivity of thermostable esterase from Archaeoglobus fulgidus toward ( S )-ketoprofen ethyl ester by directed evolution and characterization of mutant esterases [Elektronische Daten] [Jinyeong Kim, Seungbum Kim, Sangyoung Yoon, Eunsoo Hong, Yeonwoo Ryu] 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. Springer-Verlag Berlin Heidelberg, 2015 Thermostable esterase nationallicence Ketoprofen nationallicence Enantioselectivity nationallicence Directed evolution nationallicence Error-prone PCR nationallicence Site-saturation mutagenesis nationallicence Kim Jinyeong Department of Molecular Science and Technology, Ajou University, 443-749, Suwon, Korea aut Kim Seungbum Department of Molecular Science and Technology, Ajou University, 443-749, Suwon, Korea aut Yoon Sangyoung Department of Molecular Science and Technology, Ajou University, 443-749, Suwon, Korea aut Hong Eunsoo Department of Molecular Science and Technology, Ajou University, 443-749, Suwon, Korea aut Ryu Yeonwoo Department of Molecular Science and Technology, Ajou University, 443-749, Suwon, Korea aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/15(2015-08-01), 6293-6301 0175-7598 99:15<6293 2015 99 253 https://doi.org/10.1007/s00253-015-6422-7 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6422-7 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Kim Jinyeong Department of Molecular Science and Technology, Ajou University, 443-749, Suwon, Korea aut NATIONALLICENCE 700 1- Kim Seungbum Department of Molecular Science and Technology, Ajou University, 443-749, Suwon, Korea aut NATIONALLICENCE 700 1- Yoon Sangyoung Department of Molecular Science and Technology, Ajou University, 443-749, Suwon, Korea aut NATIONALLICENCE 700 1- Hong Eunsoo Department of Molecular Science and Technology, Ajou University, 443-749, Suwon, Korea aut NATIONALLICENCE 700 1- Ryu Yeonwoo Department of Molecular Science and Technology, Ajou University, 443-749, Suwon, Korea aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/15(2015-08-01), 6293-6301 0175-7598 99:15<6293 2015 99 253