Improved enzyme screening by automated fast protein liquid chromatography.
The assay for NADH-dependent dehydrogenases in crude extracts is often interfered with non-specific reactions. Therefore a screening for such enzymes is hampered by high blank values. To overcome such problems we chromatographed crude extracts on a fast protein liquid chromatography system during part of an enzyme screening for 2-hydroxyisocaproate dehydrogenases and lactate dehydrogenases. The automated chromatography procedure presented consists of a combination of gel filtration and ion-exchange chromatography. The total time needed to perform one cycle of the two-column purification, including the equilibration and regeneration steps, is about 35 min. The procedure described separates the desired enzyme, 2-hydroxyisocaproate dehydrogenase, totally from any interfering activity such as NADH-oxidase and also from the second enzyme of interest, the lactate dehydrogenase. Besides the elimination of the side reactions the desired enzymes are purified up to 20-fold.
D000429:Alcohol Oxidoreductases / Q000032:analysis*; D001322:Autoanalysis; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods; D007202:Indicators and Reagents; D007770:L-Lactate Dehydrogenase / Q000032:analysis*; D007778:Lactobacillus / Q000201:enzymology*; D007934:Leuconostoc / Q000201:enzymology*; D013045:Species Specificity
D007202:Indicators and Reagents; D000429:Alcohol Oxidoreductases; C047961:2-hydroxyisocaproate dehydrogenase; D007770:L-Lactate Dehydrogenase