title: Directed evolution of an aminoalcohol dehydrogenase for efficient production of double chiral aminoalcohols.
abstract: The aminoalcohol dehydrogenase (AADH) of Rhodococcus erythropolis MAK154, which can be used as a catalyst for the stereoselective reduction of (S)-1-phenyl-1-keto-2-methylaminopropane to d-pseudoephedrine (dPE), is inhibited by the accumulation of dPE in the reaction mixture, limiting the yield of dPE. To improve this weak point of the enzyme, random mutations were introduced into aadh, and a mutant enzyme library was constructed. The mutant library was screened with a color detectable high-throughput screening method to obtain the evolved enzymes showing the activity in the presence of a high concentration of dPE. Two mutant enzymes showed higher tolerability to dPE than the wild type enzyme. Each of these enzymes had a single amino acid substitution in a different position (G73S and S214R), and a third mutant enzyme carrying both of these amino acid substitutions was constructed. Escherichia coli transformant cells, which express mutant AADHs, showed activity in the presence of 100mg/ml dPE. A kinetic parameter analysis of the wild type and mutant enzymes was carried out. As compared with the wild type enzyme, the mutant enzymes carrying the S214R amino acid substitution or both the S214R and G73S substitutions showed higher k(cat) values, and the mutant enzymes carrying the G73S amino acid substitution or both the G73S and S214R substitutions showed higher K(m) values. These results suggest that the Ser214 residue plays an important role in enzyme activity, and that the Gly73 residue participates in enzyme-substrate binding.
authors.affiliation: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Urano
authors.forename: Satoko
authors.initials: S
authors.lastname: Fukui
authors.forename: Shoko
authors.initials: S
authors.lastname: Kumashiro
authors.forename: Takeru
authors.initials: T
authors.lastname: Ishige
authors.forename: Shinji
authors.initials: S
authors.lastname: Kita
authors.forename: Keiji
authors.initials: K
authors.lastname: Sakamoto
authors.forename: Michihiko
authors.initials: M
authors.lastname: Kataoka
authors.forename: Sakayu
authors.initials: S
authors.lastname: Shimizu
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics*; D019943:Amino Acid Substitution; D000605:Amino Alcohols / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D016296:Mutagenesis; D011485:Protein Binding; D012240:Rhodococcus / Q000201:enzymology* / Q000235:genetics; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
chemical_list: D000605:Amino Alcohols; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D000429:Alcohol Oxidoreductases