Purification And Characterization Of Novel Antimicrobial Peptides From Xenorhabdus Budapestensis NMC-10

Antimicrobial peptides (AMP) are small peptides with antibacterial, antifungal and other bioactivities and exist in all classes of organisms. These peptides are demonstrated to be potential and broad spectrum antibiotics and do not cause target microorganism resistances. Gram negative bacteria belonging to the genera Xenorhabdus play a dual role in the process of nematode infecting susceptible host insect larvae, where they are mutually symbiotic with nematode and pathogenic to the insect. Novel antibacterial substances from these symbiotic bacteria showed a wide utilization prospect in agricultural and medical field.In this thesis, the Xenorhabdus budapestensis strain NMC-10 was selected as the test microorganism and two novel antimicrobial peptides was isolated and purified from metabolites of NMC-10. The two peptides exhibited broad-spectrum and significant antibiotic activity against a variety of plant pathogens. This study provides a potential application prospect in plant diseases protection and novel therapeutics design. The major results are as follows:1. Strain NMC-10 with notable inhibition activity against Bacillus subtilis and Phytophthora capsici was screened out from 15 strains of Xenorhabdus and Photohabdus. NMC-10 was classified to Xenorhabdus budapestensis according to phylogenetic tree based on 16S RNA sequence homology (99%).2. Phytophthora capsici is screened out from 5 plant pathogens as the sensitive indication microorganism, with 99.45% mycelium growth inhibition by the method of medium mixed with crude cultures of NMC-10.3. The major bioactive components of the NMC-10 cultures consist of different chemical substances, and are thermal stable, insensitive to protease digestion, insensitive to pH, soluble in organic solvent.4. Two peptides are isolated, purified by ammonium sulfate precipitation, ion exchange chromatography and reversed phase chromatography. The purity is over 90%. Two peptides are thermal stable and insensitive to pH.5,The amino acid sequences of two peptides was obtained by ESI-MS-MS and De novo of the peptides and molecular weights are 1658.93 Da and 1787.96 Da respectively. We designated the two peptides as GP-19 and EP-20, respectively according to the number and type of the amino acids.6,Sequence alignment using BLAST demonstrated that both are novel peptides with low homology of 40.9% in database. Bioinformatics analysis showed that both two peptides are extracellular products with no signal peptide or transmembrane domain. Secondary structures consist of coils and strands. 3D models of GP-19 and EP-20 are also obtained by homology modeling.7. GP-19 and EP-20 were synthesized by solid phase synthesis method and purified by LC-MS, with the purity of over 98%. Both natural and synthetic peptides are tested against a variety of bacteria and fungus. The results demonstrated GP-19 and EP-20 are broad spectrum antimicrobial peptides.