Engineering of NADPH-dependent aldo-keto reductase from Penicillium citrinum by directed evolution to improve thermostability and enantioselectivity |
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Authors: | Hiroyuki Asako Masatoshi Shimizu Nobuya Itoh |
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Affiliation: | (1) Organic Synthesis Research Laboratory, Sumitomo Chemical Co., Ltd., 1-98, Kasugade-naka 3-chome, Konohana-ku, Osaka 554-8558, Japan;(2) Department of Biotechnology (Biotechnology Research Center), Faculty of Engineering, Toyama Prefectural University, Kurokawa 5180, Imizu Toyama, 939-0398, Japan |
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Abstract: | Penicillium citrinum β-keto ester reductase (KER) can catalyze the reduction of methyl 4-bromo-3-oxobutyrate (BAM) to methyl (S)-4-bromo-3-hydroxybutyrate with high optical purity. To improve the thermostability of KER, protein engineering was performed using error-prone polymerase chain reaction-based random mutagenesis. Variants with the highest levels of thermostability contained the single amino acid substitutions L54Q, K245R, and N271D. The engineered L54Q variant of KER retained 62% of its initial activity after heat treatment at 30°C for 6 h, whereas wild-type KER showed only 15% activity. The L54Q substitution also conferred improved enantioselectivity by KER. An Escherichia coli cell biocatalyst that overproduced the L54Q mutant of KER and glucose dehydrogenase as a cofactor regeneration enzyme showed the highest level of BAM reduction in a water/butyl acetate two-phase system. |
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Keywords: | Aldo-keto reductase (NADPH) Thermostability Enantioselectivity Directed evolution Mutagenesis Penicillium citrinum |
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