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A 1-step microplate method for assessing the substrate range of l-α-amino acid aminotransferase

Paper ID Volume ID Publish Year Pages File Format Full-Text
17364 42663 2013 8 PDF Available
Title
A 1-step microplate method for assessing the substrate range of l-α-amino acid aminotransferase
Abstract

Aminotransferase enzymes catalyse the reversible substitution of a keto group for an amino group. While this reaction is highly stereoselective with respect to the amino group, each enzyme can usually catalyse the turnover of a number of different substrates. As the substrate range cannot be inferred from the sequence, it remains an early bottleneck when selecting an enzyme for a biocatalysis application. We have developed a simple first round characterisation method applicable to the broad range of aminotransferases that accept l-glutamate, the central junction of cellular transamination, as one of the amino donors. The assay is based on l-glutamate detection by its highly specific dehydrogenase enzyme in a coupled assay, ending in the reduction of the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-tetrazolium-5-carboxanilide (XTT). While products of most tetrazolium salts are water-insoluble, XTT is reduced to a water soluble colored formazan, allowing direct spectrophotometric detection. The reaction is carried out in microplate format using a single endpoint measurement and is thus suitable for automation.The setup was tested with 7 aminotransferase enzymes: Escherichia coli branched chain amino acid aminotransferase, Pseudomonas aeruginosa and Klebsiella pneumoniae aromatic amino acid AT, Bacillus subtilis histidinol-phosphate AT, and Thermus aquaticus aspartate, serine and histidinol-phosphate AT. In addition to 17 of the 20 proteinogenic amino acids, 32 alternative substrates were tested.

► Simple microplate assay for α-transaminases. ► Substrate range of Thermus, Pseudomonas and Klebsiella α-transaminases. ► Glutamate dehydrogenase detection using colorimetric substrates.

Keywords
AT, aminotransferase; EcoBcAT, Escherichia coli branched chain AT; KpnAroAT, Klebsiella pneumoniae aromatic amino acid AT; PaeAroAT, Pseudomonas aeruginosa aromatic amino acid AT; BsuHispAT, Bacillus subtilis histidinol-phosphate AT; TaqSerAT, Thermus aqu
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A 1-step microplate method for assessing the substrate range of l-α-amino acid aminotransferase
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 52, Issues 4–5, 10 April 2013, Pages 218–225
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