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Enhancement of stability of l-tryptophan dehydrogenase from Nostoc punctiforme ATCC29133 and its application to l-tryptophan assay

Paper ID Volume ID Publish Year Pages File Format Full-Text
22963 43403 2015 6 PDF Available
Title
Enhancement of stability of l-tryptophan dehydrogenase from Nostoc punctiforme ATCC29133 and its application to l-tryptophan assay
Abstract

•TrpDH variant was obtained by random mutagenesis and complementation in a Trp auxotroph.•The specific activity and stability of the variant were higher than those of the wild type enzyme.•The variant was available for determination of Trp in human plasma.

Microbial NAD+-dependent l-tryptophan dehydrogenase (TrpDH, EC1.4.1.19), which catalyzes the reversible oxidative deamination and the reductive amination between l-tryptophan and indole-3-pyruvic acid, was found in the scytonemin biosynthetic pathway of Nostoc punctiforme ATCC29133. The TrpDH exhibited high specificity toward l-tryptophan, but its instability was a drawback for l-tryptophan determination. The mutant enzyme TrpDH L59F/D168G/A234D/I296N with thermal stability was obtained by screening of Escherichia coli transformants harboring various mutant genes, which were generated by error-prone PCR using complementation in an l-tryptophan auxotroph of E. coli. The specific activity and stability of this mutant enzyme were higher than those of the wild type enzyme. We also revealed here that in these four mutation points, the two amino acid residues Asp168 and Ile296 contributed to increase the enzyme stability, and the Leu59, Ala234 residues to increase its specific activity. Growth of the strain harboring the gene of above 4 point mutated enzyme was accelerated by the enhanced performance. In the present study, we demonstrated that TrpDH L59F/D168G/A234D/I296N was available for determination of l-tryptophan in human plasma.

Keywords
l-Tryptophan dehydrogenase; Random mutagenesis; l-Tryptophan auxotroph; Directed evolution; l-Amino acid determination
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Enhancement of stability of l-tryptophan dehydrogenase from Nostoc punctiforme ATCC29133 and its application to l-tryptophan assay
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volumes 196–197, 20 February–10 March 2015, Pages 27–32
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