Improved enantioselectivity of thermostable esterase from Archaeoglobus fulgidus toward ( S )-ketoprofen ethyl ester by directed evolution and characterization of mutant esterases
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| 024 | 7 | 0 | |a 10.1007/s00253-015-6422-7 |2 doi |
| 035 | |a (NATIONALLICENCE)springer-10.1007/s00253-015-6422-7 | ||
| 245 | 0 | 0 | |a Improved enantioselectivity of thermostable esterase from Archaeoglobus fulgidus toward ( S )-ketoprofen ethyl ester by directed evolution and characterization of mutant esterases |h [Elektronische Daten] |c [Jinyeong Kim, Seungbum Kim, Sangyoung Yoon, Eunsoo Hong, Yeonwoo Ryu] |
| 520 | 3 | |a 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. | |
| 540 | |a Springer-Verlag Berlin Heidelberg, 2015 | ||
| 690 | 7 | |a Thermostable esterase |2 nationallicence | |
| 690 | 7 | |a Ketoprofen |2 nationallicence | |
| 690 | 7 | |a Enantioselectivity |2 nationallicence | |
| 690 | 7 | |a Directed evolution |2 nationallicence | |
| 690 | 7 | |a Error-prone PCR |2 nationallicence | |
| 690 | 7 | |a Site-saturation mutagenesis |2 nationallicence | |
| 700 | 1 | |a Kim |D Jinyeong |u Department of Molecular Science and Technology, Ajou University, 443-749, Suwon, Korea |4 aut | |
| 700 | 1 | |a Kim |D Seungbum |u Department of Molecular Science and Technology, Ajou University, 443-749, Suwon, Korea |4 aut | |
| 700 | 1 | |a Yoon |D Sangyoung |u Department of Molecular Science and Technology, Ajou University, 443-749, Suwon, Korea |4 aut | |
| 700 | 1 | |a Hong |D Eunsoo |u Department of Molecular Science and Technology, Ajou University, 443-749, Suwon, Korea |4 aut | |
| 700 | 1 | |a Ryu |D Yeonwoo |u Department of Molecular Science and Technology, Ajou University, 443-749, Suwon, Korea |4 aut | |
| 773 | 0 | |t Applied Microbiology and Biotechnology |d Springer Berlin Heidelberg |g 99/15(2015-08-01), 6293-6301 |x 0175-7598 |q 99:15<6293 |1 2015 |2 99 |o 253 | |
| 856 | 4 | 0 | |u https://doi.org/10.1007/s00253-015-6422-7 |q text/html |z Onlinezugriff via DOI |
| 898 | |a BK010053 |b XK010053 |c XK010000 | ||
| 900 | 7 | |a Metadata rights reserved |b Springer special CC-BY-NC licence |2 nationallicence | |
| 908 | |D 1 |a research-article |2 jats | ||
| 949 | |B NATIONALLICENCE |F NATIONALLICENCE |b NL-springer | ||
| 950 | |B NATIONALLICENCE |P 856 |E 40 |u https://doi.org/10.1007/s00253-015-6422-7 |q text/html |z Onlinezugriff via DOI | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Kim |D Jinyeong |u Department of Molecular Science and Technology, Ajou University, 443-749, Suwon, Korea |4 aut | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Kim |D Seungbum |u Department of Molecular Science and Technology, Ajou University, 443-749, Suwon, Korea |4 aut | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Yoon |D Sangyoung |u Department of Molecular Science and Technology, Ajou University, 443-749, Suwon, Korea |4 aut | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Hong |D Eunsoo |u Department of Molecular Science and Technology, Ajou University, 443-749, Suwon, Korea |4 aut | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Ryu |D Yeonwoo |u Department of Molecular Science and Technology, Ajou University, 443-749, Suwon, Korea |4 aut | ||
| 950 | |B NATIONALLICENCE |P 773 |E 0- |t Applied Microbiology and Biotechnology |d Springer Berlin Heidelberg |g 99/15(2015-08-01), 6293-6301 |x 0175-7598 |q 99:15<6293 |1 2015 |2 99 |o 253 | ||