In-flow small angle X-ray scattering (SAXS) was used to probe in real-time (typically every second) the hydrolysis of whey protein isolate (WPI) by bromelain. The WPI concentration was 2.5%, the enzyme to substrate ratio was 1:10, and the enzymatic reaction was followed for 90 min at 50 °C and pH 7. SAXS showed that the average size of WPI molecules was about 20 A and that even at the completion of the enzymatic reaction some intact molecules of similar size remained; these are likely bromelain molecules and trace amount of BSA. SAXS allowed us to monitor the hydrolysis course through the calculation of the power-law exponent (P) and the Guinier scale factor (G) using a theoretical unified model fitting. The fitting exercise also indicated that bromelain hydrolysis transforms the globular WPI molecules into Gaussian polypeptides. The hydrolysis of WPI, which was completed within 40 min of hydrolysis, was confirmed by the degree of hydrolysis and turbidity measurements and by SDS-PAGE, which showed that bromelain has a broad specificity. This study demonstrates that SAXS is a powerful method to monitor in situ and real-time protein hydrolysis and can offer insights into protein structural changes that occur.
