Influence of water availability on the enzymatic hydrolysis of proteins

The overall rate of enzymatic protein hydrolysis decreases with increasing protein concentration (0.1–30% (w/v)) at constant enzyme/substrate ratio. To understand the role of water, the amount of available water was expressed as the ratio between free and bound water and experimentally determined from water activity and T2 relaxation time (NMR) measurements. At low protein concentrations a large excess of water is present (1.5 × 106 water molecules per protein molecule at 0.1% (w/v) whey protein isolate (WPI), but only 3984 at 30% (w/v) WPI. Assuming that 357 molecules of water are needed for full hydration of the protein, these values correspond to a 4280 and 11 times excess of water, showing that at 30% (w/v) WPI the amount of water becomes limited. At the same time, only a small decrease was observed in water activity (1.00–0.997 for 0.1–30% (w/v) WPI), and an increase of bound water measured by NMR (