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Improvement of thermostable aldehyde dehydrogenase by directed evolution for application in Synthetic Cascade Biomanufacturing

Paper ID Volume ID Publish Year Pages File Format Full-Text
17127 42644 2013 8 PDF Available
Title
Improvement of thermostable aldehyde dehydrogenase by directed evolution for application in Synthetic Cascade Biomanufacturing
Abstract

•Soluble production of a thermostable aldehyde dehydrogenase.•Simultaneous improvement of solubility and specific activity.•Enhancement of catalytic efficiency with NAD+.

The aldehyde dehydrogenase from Thermoplasma acidophilum, which was previously implemented as a key enzyme in a synthetic cell-free reaction cascade for the production of alcohols, was optimized by directed evolution. Improvements have been made to enhance reaction velocity and solubility. Using a random approach followed by site-directed and saturation mutagenesis, three beneficial amino acid mutations were found after screening of ca. 20,000 variants. Mutation Y399C enhanced the protein solubility after recombinant expression in Escherichia coli 6-fold. Two further mutations, F34M and S405N, enhanced enzyme activity with the cofactor NAD+ by a factor of eight. Impacts on enzyme stability and substrate specificity were negligible.Modeling of the enzyme structure did not reveal any direct interactions between the amino acid substitutions and residues of the active site or the enzyme's substrates. Thus, a directed evolution approach allowed for the generation of improved enzyme variants which were unlikely to be found by rational or semi-rational strategies.

Keywords
Biocatalysis; Directed evolution; Enzyme engineering; Thermostable dehydrogenase; Screening; Synthetic Cascade BiomanufacturingAlDH, aldehyde dehydrogenase; TaAlDH, aldehyde dehydrogenase from Thermoplasma acidophilum
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 53, Issue 5, 10 October 2013, Pages 307–314
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