Lumping Kinetic Model For Protein Enzymatic Hydrolysis Reaction

Enzymatic hydrolysis of protein is a kind of biochemical process to yield small molecular products such as peptones and peptides. But it is difficult to investigate the kinetic model of this process because of the diversity of hydrolysates and the complexity of reaction. This thesis tried to introduce the lumping method, which has been successfully used in the kinetic model of complicated system in petrochemical industry, into biochemical reaction field. Then a universal lumping model for protein enzymatic hydrolysis was established, and was applied to BSA-trypsin reaction. Investigation on the operating parameters: The rise of temperature can increase the reaction rate at the beginning, but the rate decreases faster with time due to the inactivation of enzyme. The hydrolysis rate is the fastest between pH8 to pH9. High concentration of substrate will result in substrate inhibition. The reaction rate increases when the concentration of enzyme rises.Analysis of hydrolysis with HPSEC: The tryptic hydrolysis of BSA was analyzed with HPSEC. The mass concentration of polypeptides corresponding to the peak whose molecular weight is the biggest declined, while those of other peptides rose. Comparing the molecular weight of theoretical peptides with that from experiments, the possible cleavage position of BSA can be deduced roughly.Establishment of universal lumping kinetic model: On the basis of hydrolysis mechanism, hypotheses about the hydrolysis process were made, and the intrinsic kinetic equation was deduced. Then the lumping reaction network was built up and the microcosmic reactions were described by intrinsic kinetic equations. Thus the universal lumping model for protein enzymatic hydrolysis was established.Four lumps kinetic model for BSA-trypsin system: The components of BSA-trypsin reaction system were grouped into four lumps according to the data of HPSEC, and the network for reaction was built up. The six reaction rate constants were estimated by Marquardt method in two steps from lower level to upper level after the intrinsic kinetic constants of each lump had been measured. The ability of prediction and extrapolation of the kinetic model was tested by comparing the computed values with the experimental values. BSA-trypsin virtual first-order reaction lumping kinetic model: The intrinsic kinetic equation was simplified as a virtual first-order equation, and a simple lumping kinetic model was obtained. The deviation between computed values and observed values is similar to that in the mechanism model. In addition, this model has the advantage of simple form and small workload.In sum, lumping method is an effective way to study the complicated reaction of protein enzymatic hydrolysis. It can be applied to the investigation of similar complicated reaction systems such as polysaccharides, lipids or nucleic acids. But there are some problems to be solved by further research in this method.