Multivariate strategy in screening of enzymes to be used for whey protein hydrolysis in an enzymatic membrane reactor

Proteinase activities for Alcalase® 2.4L (EC 3.4.21.62), Flavourzyme® (EC 3.4.11.1), Protease A (EC 3.4.24.39) and Protease N (IUB 3.4.24.28) were determined using 2% whey protein isolate (WPI) and 2% casein. The optimum substrate and enzyme concentrations and temperature were determined by the pH-stat method. Residual enzyme activity, hydrolysate molecular weight and free amino acid (FAA) content were determined. Protease N and Alcalase® 2.4L had the highest proteinase activities on casein and WPI, respectively. Alcalase® 2.4L was more stable in the presence of WPI while Protease N was inhibited by hydrolysates, and like Protease A which released high FAAs, they produced shorter peptides. Flavourzyme® hydrolysed WPI poorly and released the highest FAAs. Short peptides were removed by 5% trichloroacetic acid (TCA) and 3.5% 5-sulphosalicylic acid before FAA analysis by reversed phase high-performance liquid chromatography (RP-HPLC) of Flavourzyme® and Protease A hydrolysates, but were detected in Alcalase® 2.4L and Protease N hydrolysates. The enzyme activities for WPI hydrolysis in an enzymatic membrane reactor were Flavourzyme®