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Improved enantioselectivity of thermostable esterase toward (S)-ketoprofen ethyl ester
- Author(s)
- 김진영
- Advisor
- 유연우
- Department
- 일반대학원 분자과학기술학과
- Publisher
- The Graduate School, Ajou University
- Publication Year
- 2017-02
- Language
- eng
- Keyword
- Esterase; Ketoprofen; Enantioselectivity
- Alternative Abstract
- Esterases are widely used in various biotechnological applications because of their useful properties. In particular, thermostable esterases are used in biotechnological applications requiring high temperature and organic solvent because their structural properties ensure high tolerance of such conditions. We isolated novel thermostable esterase (designated as Est-gela) from the moderate thermophile Bacillus gelatini KACC 12197 and conducted characterization of Est-gela. The open reading frame of this gene (1170 bp) encodes 389 amino acid residues, and the molecular weight of Est-gela is approximately 42 kDa. The Est-gela was overexpressed in Escherichia coli XL1-blue and purified using a His-tag. This showed an enhanced enzymatic activity at 65-75 °C and retained more than 90% of the activity after incubation at 65 °C for 180 min. These results indicated that Est-gela was thermostable. We evaluated the effects of surfactants and organic solvents. Surfactants were more effective at improving the enzymatic activity than were organic solvents. However, Est-gela showed lower thermostability than Est-AF which was isolated from the extremophile Archaeoglobus fulgidus DSM 4304 in previous study. In comparison to other esterases, Est-AF showed high thermostability but low enantioselectivity toward (S)-ketoprofen. The (R)-ketoprofen or (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. The 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 (E=0.7±0.0): V138G (E=3.0±0.1) and V138G/L200R (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. However, further studies of Est-AF were difficult owing to its low expression levels in E. coli. We used various strategies, such as changing the expression vector and host strain, codon optimization, and optimization of induction conditions, to increase the expression of Est-AF. Through codon optimization and by changing the vector and host strain, Est-AF expression was increased from 1.00 ± 0.01 to 1.80 ± 0.01. The optimized expression system consisted of a codon-optimized Est-AF gene in a pET28a(+)-based expression plasmid in E. coli Rosetta cells. The expression level was further increased by optimizing the induction conditions. The optimized conditions were induction with 0.4 mM isopropyl-b-D-1-thiogalactoside (IPTG) at 37 °C for 5 h. Under these conditions, the expression level of Est-AF was increased from 1.00 ± 0.01 to 3.48 ± 0.01.
- Fulltext
- Appears in Collections:
- Graduate School of Ajou University > Department of Molecular Science and Technology > 4. Theses(Ph.D)
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