Study Of Adsorption And Separation Of Musca Domestica Antibacterial Peptides By Macroporous Adsorption Resin Chromatography

Insect antibacterial peptides are the important components of insect immune systems, and have advantages of small molecule weight, heat-stability and broad antibacterial activity. Musca domestica has the strong disease- resistance and the unique immunity mechanism,distributes widely in China, is easy to culture, and low-cost, thus the housefly is a good resource deserving to be explored. Study on the antibacterial peptide from the housefly is both theoretically and practically important.Antibacterial Peptides from Musca domestica larvae was separated and purified by macroporous adsorption resin chromatography, of which antibacterial activity has particular relationship with its hydrophobic property. The optimized parameters for the processing of the active compounds in antibacterial peptides separated by maroporous adsorption resin chromatography were known by test. The more active peptide obtained was purified by RP-HPLC further, and the most antibacterial peptides was acquired.Adsorbed ability of macroporous adsorption resin chromatography was recognized. The adsorbing quantity of D101 macroporous adsorption resin chromatography for the Antibacterial Peptides reached 217.18 mg/g. Ethanol was a variety of optimized eluted solution. Increased with concentration of ethanol, the eluted efficiency was elevated. Eluted efficiency of Antibacterial Peptides with D101 macroporous adsorption resin was 75.70%, when eluted by 75% concentration ethanol. The best loading parameters for peptides on D101 macroporous adsorption resin was 15mg/mL sample concentration with 0.5 BV/h (BV, Bed Volume) loading rate. The 15%,35% and 55% ethanol solution was used to elute on D101 macroporous adsorption resin. The protein content of compounds separated by D101 macroporous adsorption resin with 15%,35% and 55% ethanol solution were 39.40%, 71.72% and 72.74% respectively, while the protein content of the origin Antibacterial Peptides was 12.79%, indicated that the protein content was increased after eluting. At the same time, hydrophobic value of compounds eluted by the three different concentration ethanol was 4.30,4.43 and 4.75 kJ/mol, 0.13~0.64 kJ/mol increased than the origin Antibacterial Peptides, which suggested that the antibacterial activity of Antibacterial Peptides was higher with hydrophobic value increased. The compounds eluted by 55% ethanol owned the best antibacterial activity.The adsorbed capacity decreased dramaticly after separation and purify, but the traditional methods would not recover the adsorbed capacity of D101 macroporous adsorption resin. The optimized condition for reuse method with acid and cedrol was investigated as following: with the static reuse, the concentration of HCL was 0.1mol/L, the concentration and quantity of ethanol was 85% and 50 mL/g dry resin at 30℃for six hours; with dynamic reuse, the rate of reuse solution was 1 BV/h and the quantity of ethanol was 66.7 mL/g dry resin, and the other parameters was same as the static reuse .The optimized condition for reuse method with base and cedrol was investigated as following: with the static reuse, the concentration of NaOH was 0.1 mol/L, the concentration and quantity of ethanol was 85% and 66.7 mL/g dry resin at 30℃for six hours; with dynamic reuse, rate of reuse solution was 1 BV/h and the other parameters was same as the static reuse. Both reuse method with acid and cedrol and with base and cedrol used to reuse D101 macroporous adsorption resin, the adsorbed capacity reduced by 1.5% after employing 20 times.RP-HPLC was applied to purify the compound obtained by D101 macroporous adsorption resin with 55% ethanol, the peak of F12 was the highest antibacterial activity among the peaks with Antibacterial activity. After the second RP-HPLC and SDS-PAGE electrophoresis test, chromatographically and electrophoretically pure ingredient was detected with molar weight of 4842. Analysis of amino acid showed that F12 was rich in basic amino acid and hydrophobic amino acid and hydrophobic value was 5.82 kJ/mol, indicated that it was a basic antibacterial peptide with more hydrophoby. Compared with the compound separated by D101 macroporous adsorption resin, the hydrophobicity and antibacterial activity of F12 was enhanced. The MIC for test organism of F12 was the lowest among each ingredients, which was 30μg/mL. The ingredient of F12 was insensitive to either high or zero temperature and acid or basic envirment, with the most antibacterial activity at pH 9.