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Engineering of NADPH-dependent aldo-keto reductase from Penicillium citrinum by directed evolution to improve thermostability and enantioselectivity

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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Correspondence to Hiroyuki Asako.

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Asako, H., Shimizu, M. & Itoh, N. Engineering of NADPH-dependent aldo-keto reductase from Penicillium citrinum by directed evolution to improve thermostability and enantioselectivity. Appl Microbiol Biotechnol 80, 805–812 (2008). https://doi.org/10.1007/s00253-008-1594-z

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Keywords

  • Aldo-keto reductase (NADPH)
  • Thermostability
  • Enantioselectivity
  • Directed evolution
  • Mutagenesis
  • Penicillium citrinum