Purification And Characterization Of An Antimicrobial Peptide From Fraxinus Pennsylvanica Fruits

Antimicrobial peptides are produced by a multitude of organisms including plants, bacteria, insects and mammals. Antimicrobial peptide was found in 1972 from Hyatophora cecropia. They often have a basic pI and are highly resistant to extreme temperature treatment. They can be divided into four groups according to their origin: 1. the AMPs originate from the insect. These AMPs include Cecropins, Insect defensins, Prolinerich peptides, Glycine- rich peptides, Anti- fungal peptide; 2. the AMPs originate from the Vertebrate. These AMPs include Magainins, Defensins, Cathelicidins;3. the AMPs originate from the microbial. These AMPs include Nisin, Killer toxins, Peptaibols;4. the AMPs originate from the plant. These AMPs include oligopeptides, cyclopeptide, cyclopeptide alkaloids. Along with separation appraisal technology development and multi-peptides research thoroughly, the people already separated more than 100 kinds of antimicrobial multi- peptides from plant. Although people had found many important multi-peptides from animals, The mechanisms of AMPs still is not unified. More classical has the following several kind of patterns: Barrel-Stavemodel,Carpetmodel,toroidal (wormhole) pore model.This article used water for extraction solvent to screen 50 kinds of seeds(fruit). We found that Cornus officinasis, Brassica oleraces, Illicium verum has strong inhibitory effect to gram-positive microorganisms, but has weaker inhibitory effect to gram-negative microorgansms. Melia azedarach only has inhibitory effect to Bacillus subtilis and Staphylococcus aureus. Sinapia alba also has inhibitory effect to Bacillus subtilis. Lysimachia pentapetala has inhibitory to all fungal except Saccharomyces cerevisiae.In this paper, Sephadex G-15 was used twice to partially purify heat-resistant antibiotics combined with biology test against Bacillus subtilis. Followed by the second isolation with Sephadex G-15, five tubes containing target peptide were got. Then, analytical HPLC was used to analyze samples in these tubes. Three peaks named P1, P2 and P3 with the respective retention time (2.8min, 5.6min and 14.4min) were fully isolated and peak3 was found to have antimicrobial activity. LC/ESI-MS, a powerful tool for rapid identification and sequence determination of peptides, was used to identify these metabolites. From the MS chromatogram, we hypothesize that the molecular weight of this peptide is 765.3Da, and the amino acid sequence is N-Methy-Arginine, Pyroglutamate, phe, pro, Cystine.The analysis of the antimicrobial activity shows that the antimicrobial peptide from Fraxinus pennsylvanica fruits was highly resistant to extreme pH and temperature treatment. Simultaneously this antibacterial peptide also may resistant high ion concentration. And this antibacterial peptide also keeps stability to some organic solvents and the surface active agent.