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

Proteinase activities for Alcalase (R) 2.4L (EC 3.4.21.62), Flavourzyme (R) (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 (R) 2.4L had the highest proteinase activities on casein and WPI, respectively. Alcalase (R) 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 (R) 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 (R) and Protease A hydrolysates, but were detected in Alcalase (R) 2.4L and Protease N hydrolysates. The enzyme activities for WPI hydrolysis in an enzymatic membrane reactor were Flavourzyme (R) < Protease A < Alcalase (R) 2.4L <= Protease N. (c) 2006 Elsevier Ltd. All rights reserved.