A colorimetric assay to quantify dehydrogenase activity in crude cell lysates.
Nitroblue tetrazolium (NBT) in the presence of phenazine methosulfate (PMS) reacts with the NADPH produced by dehydrogenases to produce an insoluble blue-purple formazan. Endpoint assays taking advantage of this reaction have been successfully used to detect the activity of several dehydrogenases. Here we present a version of this assay suitable for determining the kinetics of 6-phosphogluconate dehydrogenase catalysis in crude lysates of bacterial cells prepared in 96-well plates. Using the assay to screen a small library of variant 6-phosphogluconate dehydrogenases generated by error-prone polymerase chain reaction, we were able to identify three variants with improved activity and thermostability over the parent enzyme. These enzymes were partially purified and shown to be expressed at higher levels than the parent (leading to the increase in activity), and all three variants were indeed more thermostable than the parent (temperature midpoints 4-7 degrees C higher) after purification. Thus the NBT-PMS assay appears suitable for screening libraries of variant dehydrogenases.
Mayer
Kimberly M
KM
California Institute of Technology, Pasadena, California 91125, USA.
Arnold
Frances H
FH
D002384:Catalysis; D002942:Circular Dichroism; D003124:Colorimetry / Q000379:methods*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D015723:Gene Library; D007700:Kinetics; D008773:Methylphenazonium Methosulfate / Q000494:pharmacology*; D008956:Models, Chemical; D009249:NADP / Q000378:metabolism; D009580:Nitroblue Tetrazolium / Q000494:pharmacology*; D010088:Oxidoreductases / Q000378:metabolism*; D010734:Phosphogluconate Dehydrogenase / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D013696:Temperature; D013997:Time Factors
D009580:Nitroblue Tetrazolium; D008773:Methylphenazonium Methosulfate; D009249:NADP; D010088:Oxidoreductases; D010734:Phosphogluconate Dehydrogenase