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Improved enantioselectivity of thermostable esterase toward (S)-ketoprofen ethyl ester

초록/요약

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.

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Abstract ⅰ
List of tables viii
List of figures ix
Chapter I. General introduction 1
1. Extremozyme 2
2. Esterases 7
3. Nonsteroidal anti-inflammatory drug and Ketoprofen 14
4. Aims of this study 19
Chapter II. Cloning and characterization of a novel thermostable esterase from Bacillus gelatini KACC 12197 21
1. Introduction 22
2. Materials and methods 24
2.1. Strains and growth conditions 24
2.2. Screening of the genomic DNA library for the esterase gene 24
2.3. Subcloning and sequence analysis of the esterase gene 25
2.4. Overexpression and purification of the esterase 26
2.5. Substrate specificity 27
2.6. Enantioselectivity toward (R,S)-ketoprofen ethyl ester 28
2.7. Effects of temperature and pH on the enzymatic activity 28
2.8. Effects of surfactants and organic solvents on the enzymatic activity 29
2.9. The kinetic assay 30
3. Results 31
3.1. Screening and cloning of the esterase 31
3.2. Overexpression and purification of Est-gela 35
3.3. Substrate specificity 37
3.4. Effects of temperature and pH on the enzymatic activity 39
3.5. Effects of surfactants and organic solvents on the enzymatic activity 42
3.6. The kinetic assay 45
4. Discussion 47
Chapter III. Improved enantioselectivity of thermostable esterase from Archaeoglobus fulgidus toward (S)-ketoprofen ethyl ester by directed evolution and characterization of mutant esterases 54
1. Introduction 55
2. Materials and Methods 58
2.1. Chemicals and enzymes 58
2.2. Error-prone PCR and library construction of Est-AF 58
2.3. Screening for improved enantioselectivity 59
2.4. Esterase expression and purification 60
2.5. Enzyme activity assay 61
2.6. Homology modeling assay 62
2.7. Characterization of enzymes 62
2.8. Enzyme kinetic assay 63
3. Results 64
3.1. Mutagenesis of Est-AF 64
3.2. Structural modeling 67
3.3. Effect of temperature and pH on enzyme activity 69
3.4. Effect of various additives and organic solvents on enzyme activity 72
3.5. Kinetic parameters 75
4. Discussion 77
Chapter IV. Strategies for increasing heterologous expression of a thermostable esterase from Archaeoglobus fulgidus in Escherichia coli 81
1. Introduction 82
2. Materials and methods 84
2.1. Strains, plasmids and growth conditions 84
2.2. Construction of expression systems 84
2.3. Expression and purification 85
2.4. Optimization of induction conditions 86
2.5. Analytical procedures 87
3. Results 89
3.1. Codon optimization of the Est-AF gene and construction of expression systems 89
3.2. Comparison of expression systems 93
3.3. Optimization of induction conditions 99
4. Discussion 101
Chapter V. Overall conclusion and further study 108
Reference 112
Abstract in Korean 131

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