In Vitro Studies Of Papain Hydrolysis Of β-casein Film

Objective: The aim of this study was to in situ and real-time monitor the dynamic hydrolysis process ofβ-casein(β-CN) film catalyzed by papain using a quartz crystal microbalance with dissipation (QCM-D) and to compare the hydrolysis rate of papain at different concentrations. To inspect the property and topography ofβ-CN film before and after catalyzed by contact angle meter and atomic force microscope. Methods: The shift of frequency, film thickness and mass were monitored by QCM-D whenβ-CN adsorbed onto the SAM surfaces. According to the correlation coefficient of Langmuir and Fruendlich adsorption model, to decide which was fit to describe the adsorption ofβ-CN on SAM. A model film ofβ-CN was build-up with QCM-D. The changes in frequency and thickness during incubation at various enzymatic concentrations were monitored. The experiment data were well fitted by Boltzmann equation. Changes of contact angle on different surface as Au(a), 11-MUA(b), EDC-NHS(c),β-CN(d),β-CN/papain(0.5μg/ml)(e),β-CN/papain(12.5μg/ml)(f) were inspected by contact angle meter, and the topography and roughness of surfaces (c, d, e, f) were studied by atomic force microscope. The hydrolysis parameters, contact angle data and root square roughness were compared by analyses of one-way ANOVA and SNK-q test. The level of significance was defined asα= 0.05. Result: Film mass and thickness increased followed the rising concentration ofβ-CN within critical micelle concentration. The Langmuir isotherm was fit to describe the adsorption isotherm ofβ-CN onto the SAM. Increased enzyme concentration generally had a great effect on increasing frequency and hydrolysis rate and decreasing the V50 and the thickness ofβ-casein film (P <0.05). The changed order of contact angle was c > b > d > e > f > a (P <0.05) and root square roughness was d > e > f > c (P <0.05). Protein particles on Au chip extinguished gradually with the increasing concentration of enzyme concentration. Conclusion:β-CN formed a monolayer on the SAM within critical micelle concentration. The hydrolysis rate increased with increasing the enzyme concentration within a certain range. The target of papain onβ-CN molecule located at the C-end of peptide. The protein film can be hydrolyzed evenly by high concentration of enzyme.