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 pK(a) 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.
authors: Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, China.
authors: Kai
authors: K
authors: Yu
authors: Sheng
authors: S
authors: Hu
authors: Jun
authors: J
authors: Huang
authors: Le-He
authors: LH
authors: Mei
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D001961:Bromcresol Green; D002021:Buffers; D003124:Colorimetry / Q000379:methods*; D004926:Escherichia coli; D005968:Glutamate Decarboxylase / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007700:Kinetics; D052196:Levilactobacillus brevis / Q000201:enzymology* / Q000235:genetics; D008832:Microchemistry / Q000379:methods; D011522:Protons; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D012371:Robotics; D012680:Sensitivity and Specificity; D013053:Spectrophotometry
chemical_list: D001426:Bacterial Proteins; D002021:Buffers; D007202:Indicators and Reagents; D011522:Protons; D011993:Recombinant Fusion Proteins; D018698:Glutamic Acid; D001961:Bromcresol Green; D005968:Glutamate Decarboxylase