Kvantitativno određivanje lipaze pomoću fluoresceina

An easy and fast gel diffusion assay for detecting and monitoring lipase activity by quantification of fluorescein is described. By measuring the intensity of fluorescein, it is possible to obtain a calibration curve with a regression coefficient better than by using the radius of fluorescent haloes. Through the quantification of fluorescence intensity of fluorescein released after the hydrolysis of a fluorescent ester, fluorescein dibutyrate, used as substrate in agar plates, commercial and skimmed milk lipase activity were studied. Moreover, with this method, lipase activity can be monitored in reaction medium that contains compounds which are affected by turbidity or cause measurement interference for UV-spectrophotometer and fluorimeter. In this experiment, boiled skimmed milk was dispersed in the agar gel with fluorescein dibutyrate, and it was used as a reaction medium to mimic natural conditions. The development of such an assay has a potential for applications in industries ranging from pharmaceuticals to food production and monitoring.U radu je opisana jednostavna i brza metoda određivanja i praćenja aktivnosti lipaze pomoću fluoresceina. Mjerenjem intenziteta fluorescencije fluoresceina može se dobiti kalibracijska krivulja s koeficijentom regresije boljim od onog dobivenog mjerenjem promjera fluorescentne zone. Kvantitativnim određivanjem intenziteta fluorescencije fluoresceina, oslobođenog hidrolizom estera fluorescein dibutirata na podlozi s agarom, ispitana je aktivnost komercijalne lipaze, te one izolirane iz obranog mlijeka. Ovom se metodom može pratiti aktivnost lipaze u reakcijskoj smjesi koja sadržava spojeve što uzrokuju zamućenje ili interferiraju s uzorkom pri mjerenju UV-spektrofotometrom i fluorimetrom. U istraživanju je upotrijebljena reakcijska smjesa od prokuhanog obranog mlijeka raspršenog na agar gelu s fluorescein dibutiratom. Daljnim bi se razvojem ova metoda mogla primijeniti u farmaceutskoj i prehrambenoj industriji, zaštiti okoliša i sl