Abstract: To establish a method to determine the activity of phospholipase Lecitase?Ultra using oil-free phospholipids as the substrate, three reaction parameters including time, temperature and pH were optimized by response surface methodology based on one-factor-at-a-time experiments. A quadratic regression model for phospholipase activity was proposed. The results showed all these parameters had an extremely significant effect on phospholipase activity. The optimal conditions for determining phospholipase activity required substrate concentration of 4 g/100 mL, final pH 9.0, enzyme dilution factor of 100, reaction pH 5.1, reaction temperature of 53 ℃ and reaction time of 8 min.

Key words: phospholipase Lecitase?Ultra, enzyme activity determination, acid-base titration, response surface methodology