Purification And Characterization Of An Antimicrobial Peptide From Fraxinus Pennsylvanica Fruits

Plants need to obtain the full spectrum of nutrients from the environment where areextremely rich in microorganisms during the growth of them, but infection remains arare event. Plants form respective antimicrobial path-way during the long-termdevelopment. For example, plants were induced to produce many class ofantimicrobial substance including secondary metabolites and peptides, andantimicrobial peptides play an important role in the protection against infection.Antimicrobial peptides, as their names imply, serve a protective function againstfungal invasion. They are produced by a multitude of organisms including plants,gymnosperms, fungi, bacteria, insects and mammals. They often have a basic pI andare highly resistant to extreme temperature treatment. They can be divided into threegroups according to their target sites and mechanisms: cell wall-acting AMPs,membrane-acting AMPs and intracellular target-acting AMPs. Cell wall-actingAMPs act their inhibitory effect on growth by disturbing the synthesis of chitins andglucans, or catalyzing the degradation of them. Although membrane-acting peptidesare extremely diverse as regards their primary and secondary structure, they share atleast two common features, namely a positive net charge under physiologicalconditions that facilitates interaction with negatively charged microbial surfaces andthey assume amphipathic structures which permit incorporation into microbialmembranes. Antimicrobial peptides entered the cell can interact with a variety ofintracellular targets, such as ribosomes or proteases.In this paper, Sephadex G-15 was used twice to partially purify heat-resistantantibiotics combined with biology test against Bacillus subtilis. Five tubes containingtarget peptide were got following the second isolation with Sephadex G-15. 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 fullyisolated and peak2 was found to have antimicrobial activity. LC/ESI-MS, a powerfultool for rapid identification and sequence determination of peptides was used toidentify these metabolites. From the MS chromatogram, we can conjecture that themolecular weight of this peptide is 927.4Da, and the amino acid sequence is Lys, Asp,Pro, Gly, Ile(Leu), Gly, Glu, Pro, BcZ.The analysis of the antimicrobial activity showed that the antimicrobial peptidefrom Fraxinus pennsylvanica fruits was highly resistant to extreme pH andtemperature treatment. Study on the micro-morphology of the hyphae or cell ofBacillus subtilis by SEM showed that this peptide can inhibit microbes by disturbingtheir membranes.