fulltext.study @t Gmail

A high-throughput colorimetric assay to measure the activity of glutamate decarboxylase

Paper ID Volume ID Publish Year Pages File Format Full-Text
17484 42672 2011 5 PDF Available
Title
A high-throughput colorimetric assay to measure the activity of glutamate decarboxylase
Abstract

A pH-sensitive colorimetric assay has been established to quantitatively measure glutamate decarboxylase (GAD) activity in bacterial cell extracts using a microplate format. GAD catalyzes the irreversible α-decarboxylation of l-glutamate to γ-aminobutyrate. The assay is based on the color change of bromocresol green due to an increase in pH as protons are consumed during the enzyme-catalyzed reaction. Bromocresol green was chosen as the indicator because it has a similar pKa to the acetate buffer used. The corresponding absorbance change at 620 nm was recorded with a microplate reader as the reaction proceeded. A difference in the enzyme preparation pH and optimal pH for GAD activity of 2.5 did not prevent this method from successfully allowing the determination of reaction kinetic parameters and the detection of improvements in enzymatic activity with a low coefficient of variance. Our assay is simple, rapid, requires minimal sample concentration and can be carried out in robotic high-throughput devices used as standard in directed evolution experiments. In addition, it is also applicable to other reactions that involve a change in pH.

► A high-throughput colorimetric assay to measure the activity of GAD. ► We monitor the absorbance change of bromocresol green at 620 nm. ► A pH difference of 2.5 units does not prevent this method from application. ► This method allows the determination of reaction kinetic parameters. ► Improvements in enzyme activity can be detected with a low coefficient of variance.

Keywords
GAD, glutamate decarboxylase; GABA, γ-aminobutyrate; l-MSG, monosodium l-glutamate; PLP, pyridoxal 5′-phosphate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresisGlutamate decarboxylase; pH indicator; Enzyme catalysis; Colorimetric assay
First Page Preview
A high-throughput colorimetric assay to measure the activity of glutamate decarboxylase
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
Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 49, Issue 3, 10 August 2011, Pages 272–276
Authors
, , , ,
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