Monitoring of enzymes during chromatographic separations.
An on-line enzyme assay is presented based on flow injection techniques combined with fluorimetric detection. It allows to monitor NAD-dependent oxidoreductases during the purification of microbial crude extracts or partially purified enzymes by fast protein liquid chromatography (FPLC) in a near real-time mode. The arrangement is simple and can be easily integrated in the chromatographic system avoiding dead volumes. A high measuring frequency (up to 180 samples h-1) and a short response time (10-30 s) are achieved. The method has a low limit of detection (approximately 0.01 U ml-1), and a good reproducibility (1-4%), the injected sample volume is only 2 microliters.
Institute of Enzyme Technology, Heinrich-Heine University Düsseldorf, Jülich, F.R.G.
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WW
Stamm
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MR
Kula
D050788:Alanine Dehydrogenase; D000445:Aldehyde Oxidoreductases / Q000032:analysis*; D000594:Amino Acid Oxidoreductases / Q000032:analysis*; D002845:Chromatography; D005560:Formate Dehydrogenases / Q000032:analysis*; D006863:Hydrogen-Ion Concentration
D005560:Formate Dehydrogenases; D000445:Aldehyde Oxidoreductases; D000594:Amino Acid Oxidoreductases; D050788:Alanine Dehydrogenase; C022342:phenylalanine oxidase