ON-LINE DETECTION OF LIPASE ACTIVITY BY FLOW INJECTION ZYMOGRAPHY

Abstract

The application of a FIA-FPLC unit for the post-column on-line detection of lipase activity is presented. As the development of 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-containing fractions is desired. Rapid detection of enzymeactivity can be achieved by coupling a flow injection analysis (FIA) system to the liquid chromatographyunit. Usually lipase activity is detected in the presence of emulsified substrates, whose even distribution in the FIA system causes problems. In order to solve these problems, the solubilized substrates S,0,0’-tributyryl-1-thiogycerol (TBTG) and 1,2-O-dilauryl-rac-glycero-3-glutaric-resorufinester (BM) were applied to determinethe 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 differentlipase purification steps. FIA response showsa linearcorrelation to lipase activity up to lipase concentrations of 140 U/ml.