Peptide Sequence, Kinetic Model And Structural Analysis Of Protein Enzymatic Hydrolysis For Preparing Active Peptides

Enzymatic hydrolysis of protein is a kind of biochemical process to produce small molecular products such as peptones and peptides. This process has the characters such as variety of products and complexity of the reaction. In this thesis, the process of protein enzymatic hydrolysis has been researched in detail by using HPLC-ESI-MS/MS and the Dynamic Light Scattering.Determination of the HPLC analysis conditions: The HPLC separation conditions (such as the acentonitrile gradient condition and the content of the TFA in the mobile phase) were optimized for BSA-trypsin reaction system. The MS analysis of the sequences for the BSA tryptic hydrolysis peptides: The complex hydrolysates were analyzed by using RP-HPLC/ESI-MS/MS. According to the mass spectrum data, the sequence of total 33 peptides could be obtained by Bioworks software.Prediction model of retention time for enzymatic hydrolysis peptides in RP-HPLC: Using ridge regression method, the chromatographic behavior of hydrolysis products was investigated, and an appropriate prediction model of RP-HPLC retention time was established. Compared with several other models, it demonstrated that the model established by ridge regression was more suitable for the enzymatic hydrolysis peptides.Establishment of hydrolysis conditions– hydrolysis degree model: The degree of hydrolysis is affected by the hydrolysis conditions such as concentration of substrate and enzyme, temperature and pH of the reaction, and the reaction time. Based on the experimental data, artificial neural network was introduced to simulate the complex relationship between the hydrolysis conditions and the hydrolysis degree. Establishment of release kinetic model for the enzymatic hydrolysis peptides: Based on the eletrospray ionization mass spectrum, the relative concentration of each peptide could be calculated, and then the proper equations were selected to model the kinetic behavior of each peptide during the whole process of the reaction via data fitting.Elementary studies of structural change for the tryptic hydrolysis of casein by dynamic light scattering: The structure of protein is associated with the physiologic function of peptides. In present work, casein was selected as an example, and the effects of temperature, ionic strength and enzymatic hydrolysis on the average hydrodynamic radius of casein micelles in phosphate buffer were studied by using...