Engineering of NADPH-dependent aldo-keto reductase from Penicillium citrinum by directed evolution to improve thermostability and enantioselectivity.
Penicillium citrinum beta-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 degrees 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.
Asako
Hiroyuki
H
Organic Synthesis Research Laboratory, Sumitomo Chemical Co., Ltd., Konohana-ku, Osaka, Japan. asako@sc.sumitomo-chem.co.jp
Shimizu
Masatoshi
M
Itoh
Nobuya
N
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000449:Aldehyde Reductase; D000074408:Aldo-Keto Reductases; D019943:Amino Acid Substitution; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016296:Mutagenesis; D010407:Penicillium / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D015202:Protein Engineering; D013696:Temperature
D005656:Fungal Proteins; D000429:Alcohol Oxidoreductases; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase