FLOW INJECTION ANALYSIS FOR THE ON-LINE DETECTION OF LIPASE; A TOOL FOR THE AUTOMATIZATION OF LIQUID CHROMATOGRAPHY

Abstract

The application of a FIA-FPLC unit for the post-column on-line detection of lipase activity is presented. As the developmentof techniques such as Fast Protein Liquid Chromatography (FPLC) and High Performance Liquid Chromatography (HPLC) has greatly reduced the time required for protein purification, fast identification of enzyme-containingfractions is desired. Rapid detection of enzymeactivity can be achieved by coupling a flow injection analysis (FIA) system to the liquid chromatography unit. Usually lipase activity is detected in the presence of emulsified substrates, whose evendistribution in the FIA system causes problems. In orderto solve these problems, the solubilized substrates S,O,O’-tributyryl-1-thiogycerol (TBTG) and 1,2-O-dilauryl-rac-glycero-3- glutaric-resorufinester (BM) were applied to determine the relationship between lipase concentration and FIA response. Lipase and its substrate were injected simultaneously into two carrier streams which were mixed together before passing a thermostated reaction coil. The cleaved productof the lipase substrate was detected photometrically. The BM-substrate turned out to be most suitable for FIA applications. The FPLC-FIA unit using the BM-substrate has successfully been applied for post-column on-line monitoring of lipase activity during different lipase purification steps. FIA response showsa linear correlation to lipase activity up to lipase concentrations of 120 U/ml.