Directed evolution of an aminoalcohol dehydrogenase for efficient production of double chiral aminoalcohols

作者：Urano Nobuyuki; Fukui Satoko; Kumashiro Shoko; Ishige Takeru; Kita Shinji; Sakamoto Keiji; Kataoka Michihiko^*; Shimizu Sakayu

来源：Journal of Bioscience and Bioengineering, 2011, 111(3): 266-271.

DOI：10.1016/j.jbiosc.2010.11.005

• Aminoalcohol dehydrogenase
• Short-chain alcohol dehydrogenase
• Directed enzyme evolution
• Amino acid substitution
• Enzyme screening

The aminoalcohol dehydrogenase (AADH) of Rhodococcus erythropolis MAK154, which can be used as a catalyst for the stereoselective reduction of (5)-1-phenyl-l-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 (G735 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 100 mg/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 kat values, and the mutant enzymes carrying the G73S amino acid substitution or both the G73S and S214R substitutions showed higher K, 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.

• 出版日期2011-3