Purification, Antimicrobial Activity And Mechanism Of A Natural Antimicrobial Peptide From Bamboo Leaf

In China, the functions and effects of Bamboo leaves'therapy and healthcare have been well known for a long time, and Bamboo leaves have also been used as food, as well as medicine. Therefore, their research and utilization is very significant and beneficial. In this paper, in order to study the novel antimicrobial peptide in Bamboo leaves, the proteins and peptides from Bamboo leaves were extracted by solvent, gradient salting-out and dialysis. The partially purified proteins were purified further by the molecular sieve and weak anionic exchange column chromatography. The molecular weight and purity of these purified peptides were determined by SDS polyacrylamide gelatin electrophoresis, the antibacterial activity and characteristics of the antimicrobial peptide is investigated by their diameter of inhibition zone, and the antibacterial mechanism was also researched. In our research, a novel antimicrobial peptide from Bamboo leaves is obtained, which has very well antibacterial activity against bacteria (Staphylococcus aureus, Escherichia coli, Baciplus subtilis and Bacillus cereus) and stability. Furthermore, the novel antimicrobial peptide is proved to be a trypsin inhibitor, which elucidates its antimicrobial mechanism.In the thesis, firstly, bamboo leaf protein was extracted in PBS buffer, salted out by 30%, 60% and 80% ammonium sulfate respectively, and dialyzed with cut-off molecular weight of 3000Da. By disk diffusion method and SDS–PAGE, the extracted peptides in Bamboo leaves show very well antibacterial activity, especially against bcteria. The antibacterial activities of the peptides from 60% and 80% ammonium sulfate are stronger than that of the peptide from 30%.Secondly, these extracted peptides were separated and purified further by gel filtration chromatography and ion exchange chromatography, respectively. The purified antibacterial peptides show 2 bands by SDS-PAGE, and their molecular weights are 99776Da and 45604Da. The result suggests that the antibacterial peptide in Bamboo leaves consists of 2 defferent peptides, or a dimer of a peptide.Thirdly, the antibacterial spectrum and characteristics of the antibacterial peptide were also studied. The results show that the Bamboo leaf peptide has broad antibacterial spectrum, especially against bcteria, even very low concentration. The peptide also shows some antibacterial effect against Saccharomyces cerevisiae, but little inhibition against mould. The antibacterial peptide exhibits very well stability from pH3.0 to pH11, and is stable at temperature of 120℃for 10min. It also shows good stability against different enzymes and organic solvent as well. Finally, the probable antibacterial mechanism of the purified peptides from bamboo leaves was explored by BAPNA method. It demonstrates that this antimicrobial peptide from bamboo leaves has very well trypsin inhibitory activity, which suggests its antimicrobial mechanism of the novel antimicrobial peptide.