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.

Cloning and characterization of a novel thermostable esterase from Bacillus gelatini KACC 12197

“…In addition, Est-gela hydrolyzes (R,S)-ketoprofen ethyl ester preferably to (S)-ketoprofen than to (R)-ketoprofen. An ideal esterase, however, can produce only (S)-ketoprofen with high thermostability, and many studies on improvement of the thermostability and enantioselectivity of esterases have been conducted [58][59][60]. Accordingly, we are also trying to improve the thermostability and enantioselectivity of Est-gela using various mutagenesis methods (work in progress).…”

Improving the ‘tool box’ for robust industrial enzymes

“…Some esterases and a lipase were earlier characterised from this organism including AFEST [47] which is a member of a hormone-sensitive lipase family and can be used for the biocatalytic conversion of poly δ-valerolactone being thermostable up to 90 °C. The first esterase Est-AF [16] and directed mutants of this enzyme have been designed for the industrial production of the ( S )-enantiomer of ketoprofen that acts as an anti-inflammatory drug [37, 38]. …”

Screening, Isolation and Identification of Thermophilic Esterase Enzyme Isolated from Rhodococcus SP: LKE-021

“…Though, nearby some obstructions still to be there for the large scale utilization of esterases in industries, for instance low yield of production, thermal stability, limited pH, and deprived performance in organic solvents 11,12 . There is still need of enhanced their catalytic efficiency on the basis of rational protein design through functional modification of the enzymes in addition to substrate specificity, thermostability and enantioselectivityas required by industries [13][14][15][16] . Better understanding of structure and functionalities will also ease rational improvement in purification, isolation and source of microbes.…”