A Novel Antibacterial And Immunopeptide Preparated By Casein Proteolysis And Its Antibacterial Mechanism

The proteolytic solution of bovine casein which has antimicrobial and immunostimulating activity was found and a novel immunopeptide named Tyr-rich polypeptide was separated from the solution by the process of ethanolprecipitation——dialysis——Sephadex G-25 chromatography, the results of A serialexperiments showed that Trpi has antibacterial action to Gram-positive and Gram-negative bacteria, such as E.coli, S.aureus, and enhance lymphocyte cell mediated immunity markedly in vitro.The amino acid composition of the peptide was also tested, and its position in the bovine milk casein was determined with the help of the site-specific character of trypsin. The result of x~2 -test proved the deduce to be correct; The result showed that this immunopeptide was a novel peptide that hadn't been reported comparing with the known antibacterial and immunopeptide sequences. This novel immunopeptide was composed of 53 amino acids, and it was the fragment of the bovine milkκ-CNf11-63. Some of the biochemical properties of the Tyr-rich polypeptide were tested, such as its high thermostablility, high contents of Tyr, pI and aminal acid sequence. On the basis of these works, the elucidation of the mechanisms of the action of Trpi was able to provide a basis for research and development of functional sanitarian food and oral medicament, more efficient methods to prepare immunopeptides are also being tried, so that a scale up method had established.The results of the study showed that the optimum enzyme hydrolysis conditions of enzymolysis of casein catalysed by trypsin were that dissolving casein in water at 80℃to denaturalize the casein moderately first, then trypsinized at 45℃, substrate 9%(w/v), enzyme: substrate ratio [E/S] l:120(U/mg), hydrolyzed for 90 minutes. NKA-8 , which has better adsorption and desorption function, was the most appropriate resin for the purification of Trpi. The concentration of 0.6mg/ml, pH 7.2 and being eluted with 4BV 50% ethanol in 1BV/h speed were the optimal conditions. The result of IEF showed that pI of Trpi is 8.61. Trpi was likely to displace the magnesium ions on the surface of the cellular wall, then either bind tightly to the negatively charged membrane lipopolysaccharide (LPS) or neutralize the charges over an area of the cellular wall, subsequently distorting the cellular wall structure. Once this is accomplished, Trpi can drill through the cellular wall. In addition, the three-dimensional structure of Trpi was studied in this thesis. The three-dimensional structure of Trpi was modeled according as the parameter of amino acid residue, formed Trpi, computed by The Swiss-PdbViewer(v.3.7), such as bonds, nonbonded, electrostatic constraint, torsion and so on.