| Tyrosine-rich Peptide of Immunostimulating (Trpi) was separated from the products of enzymolysis of casein catalysed by trypsin in our laboratory. Trpi could possess antimicrobial activity against Gram-negative and Gram-positive bacteria, such as E.coli, S.aureus, and enhance cell mediated immunity markedly.In this paper, the preparative technics of Trpi was optimized by adjusting the parameter of enzymolysis of casein, the purification of Trpi with resin was studied , the pI of Trpi was determined, The spatial structure of Trpi was conjectured, and the mechanism of Trpi was also studied by determining the changes of ions before and after the action of peptide with E.coli in the liquid culture medium. 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 10%(w/v), enzyme: substrate ratio [E/S] 1:100(w/w) for 90 minutes. NKA-9, 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 the elution with 4BV 50% ethanol in 1BV/h speed were the optimal conditions. The pI of Trpi was 8.61, which was determined by IEF-PAGE. The spatial structure of Trpi, determined according as the parameter of amino acid residues, formed Trpi, computed by The Swiss-PdbViewer(v.3.7), such as bonds, nonbonded, electrostatic constraint, torsion and so on, was likely to have four puckers, centralized charge area, and the tendency of distributing symmetrically of polar amino acid and non polar amino acid. The changes of the K+ and Ca2+ were induced by Trpi after 15 min, and that of the Mg2+ was after 10min. The effect of Trpi was induced rapidly. Based on these, the mechanism of Trpi was conjectured as follows: Trpi was likely to displace the magnesium ions on the surface of the outer membrane, then bind tightly to the negatively charged membrane lipopolysaccharide (LPS) or neutralize the charge over an area of the outer membrane, subsequently distorting the outer membrane structure. Once this is accomplished, Trpi can drill through the outer membrane, combine the cytoplasmic membrane by the polar sidechains, then reorient perpendicular to the membrane and align in a manner in which the hydrophobic sidechains face outwards the lipid environment while the polar sidechains align inward to form transmembrane pores, leading to leakage of ions and metabolites, ensuing depolarization, loss of membrane-coupled respiration and biopolymer synthesis, and ultimately cell death. 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. |
