Antimicrobial Activity Of Antimicrobial Peptide PaDef And Its Mutants

Plant defensins are an important component of the nonspecific immune system in plants. In recent years, the study found that it not only has broad-spectrum bactericidal activity. It was also found that most plant defensins have no harmful effects on human,animal and plant cells. This means that they have great potential in medical and health care,food and gene breeding applications. Plant Defensin PaDef is a 45 amino acid polypeptide isolated from avocado fruit. In this study, a series of activity studies were conducted using defensin PaDef expressed by Pichia pastoris. On this basis, site-directed mutations were carried out in order to obtain better antimicrobial peptides. The results are as follows:(1) In the optimization of induction time, the increase of protein expression of antibacterial peptide PaDef increased with time in the range of 0-120 h. However, there was no significant difference (P> 0.05) in the supernatant of 72,96,120 h for Staphylococcus aureus, which was 62.5%, 65.6% and 56.2%. Combined time, cost and other factors, the final determination of 72 h for its optimal induction time. In the optimization of induction methanol concentration, the expression was induced by 0.5%, 1%, 1.5% and 2%respectively. When the concentration of methanol was 1.5%, the protein concentration was 79.6 ?g/mL and the inhibitory effect on Staphylococcus aureus was 70.0%. Therefore, 1.5%was the optimal methanol concentration.(2) The antimicrobial peptides PaDef were tested for minimal antimicrobial activity(MIC),and had an antibacterial activity against 11 strains. When the concentration reached 120 ?g / mL (purity> 97%), the inhibitory rate of the tested bacteria was over 90% . In the temperature, PH value, protease stability test, the activity of PaDef is only affected by the pH (pH = 2, 4) environment,alkaline,temperature (4? to 90?),protease (pepsin,papain, protease K, trypsin) did not affect their activity. This shows that PaDef has good stability.(3) In the site-directed mutagenesis test, the mutant PaDef-T3R, PaDef-D29R,PaDef-Q33H, PaDef-K43M were successfully expressed. In the study of MIC activity, in addition to PaDef-D29R on the MIC value of Escherichia coli lower than the wild type, the other mutant activity increased.The effect of PaDef-Q33H was especially obvious. At the concentration of 30 ?g/mL, the inhibitory rates against Staphylococcus aureus(ATCC 25923), Listeria monocytogenes (ATCC 21633), Escherichia coli 0157(ATCC 35150) and Escherichia coli(ATCC 10305)were 97.7%, 95.9%, 99.1%, 65.8%(98.5% at 40 ?g/mL).This is much higher than the wild type.At the same time, in the agarose diffusion test, we observed that the diameter of the zone of PaDef-Q33H and PaDef-K43M was significantly higher than the wild type at 60 ?g/mL.In summar We found that PaDef has broad spectrum bactericidal properties and it to have temperature, pH (except acidity), and protease stability. Mutant PaDef-Q33H activity is superior to wild type.This will not only provide the experience base for further in-depth testing, but also improve the practical application of antibacterial peptide PaDef in food,cosmetics, toxicology and clinical trials.