کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
21477 | 43224 | 2011 | 6 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: Directed evolution of an aminoalcohol dehydrogenase for efficient production of double chiral aminoalcohols Directed evolution of an aminoalcohol dehydrogenase for efficient production of double chiral aminoalcohols](/preview/png/21477.png)
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 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 kcat values, and the mutant enzymes carrying the G73S amino acid substitution or both the G73S and S214R substitutions showed higher Km 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.
Journal: Journal of Bioscience and Bioengineering - Volume 111, Issue 3, March 2011, Pages 266–271