Purification And Characterization Of An Antimicrobial Peptide From Paris Polyphylla Var. Chinensis

Plant endophyte is a group of resource microorganism with broud perspective, which have ability of nitrogen fixation, could encourage plant growth and reinforce antireversion force of the plant . It can be separated from the inner part of plant by histology or disinfection. Paris L is a class of plant with variety of medicine value ,which have ability of detoxication, analgesia, antibiosis, anticancer et al. It is the major material of many prepared Chinese medicines. However, with the exploitation of medicine industrial, we are facing the crisis of it's gradually drying up.Base on the research results of medicine plants, it's endophyte can synthetize the same active ingredient, some endophyte can encourage plant to synthetize active ingredient.We are study on endophyte in Paris polyphylla var.chinensis Hara which could produce active ingredient in order to resolve the crisis of wild paridis lacking.Through surface sterilization ,82 strains endophytes were isolated and purified from the fresh hypogeal stems of Paris polyphylla var.chinensis . Many endophytes showed antimicrobial activity through preliminary antimicrobial screening against pathogenic bacterias and further antimicrobial screening against pathogenic fungi. The strain PCE45 with best antimicrobial activity was selected to the next series of studies.Based on the characteristics of morphology,physiology,and biochemistry as well as 16SrDNA analysis, the strain PCE45was classified into Bacillus subtilis.We made the crude extract of PCE45 after the steps of ammonium sulfate salting-out, dialysis and acetone precipitation. Through the method of Oxford Plate,we find the best sulfate saturation is 50%~70% .After SephadexG75,DE52 and SephadexG25 column chromatography, we separated an antimicrobial peptide PCP-1 from the strain PCE45.The stability against high temperature and proteinase, and antimicrobial activity were also analyzed. The antimicrobial peptide PCP-1 was stable to proteinase and tolerated high temperature, strong acid and strong base. PCP-1 caused deformation of the hyphae of Pyricularia oryzae and prohibited the spore germination. It also inhibited fungi such as Curvularia lunata and bacteria such as Escherichia coli. Mass spectrogram measurement revealed its molecular weight of 1058.3 Da. The amino acid composition of the peptide composed of 7 amino acids. Ninhydrin reaction showed negative trait whereas after acid hydrolysis with positive ninhydrin reaction and biuret reaction. The ninhydrin reaction and biuret reaction imply that the peptide PCP-1 is a cyclic lipeptide.