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Directed evolution of transketolase activity on non-phosphorylated substrates

Paper ID Volume ID Publish Year Pages File Format Full-Text
25001 43551 2007 8 PDF Available
Title
Directed evolution of transketolase activity on non-phosphorylated substrates
Abstract

We have used active-site targeted directed evolution by saturation mutagenesis to improve the activity of E. coli transketolase towards non-phosphorylated substrates. Residues were selected for each set based on either structural proximity to substrate, or on phylogenetic variation. Each library was screened towards the reaction between hydroxypyruvate (HPA) and glycolaldehyde (GA) to form l-erythrulose, and the location of improved mutants related to the natural sequence entropy at each residue. A number of mutants from the phylogenetically defined library were found to outperform the wild-type with up to 3-fold specific activity under biocatalytically relevant conditions, though interestingly with substituted residues that differed from those found in nature. Conserved residues which interact with the phosphate group in natural substrates also yielded mutants with almost 5-fold improved specific activity on the non-phosphorylated substrates. These results suggest that phylogenetically variant active-site residues are useful for modulating activity on natural or structurally-homologous substrates, and that conserved residues which no longer interact with modified target substrates are useful sites to apply saturation mutagenesis for improvement of activity.

Keywords
DSW, deep square well; Ery, erythrulose; GA, glycolaldehyde; HPA, hydroxypyruvate; TK, transketolase; TPP, thiamine pyrophosphateDirected evolution; Biocatalysis; Phylogenetics; Protein engineering; Transketolase
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Directed evolution of transketolase activity on non-phosphorylated substrates
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 131, Issue 4, 30 September 2007, Pages 425–432
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us