Purification, Characterization And Antibiotic Property Of Natural Antifungal Peptide APS

The emergency of drags-resistant fungi makes the systemic fungal infections represent a challenging problem for clinicians. The era of the classical antibiotics may be over because of their deficiencies. Recently, antimicrobial peptides have received increasing attention as their fundamental role in host innate immunity defense mechanism. Natural antifungal peptide is a potential candidate for the new antibiotics. APS, found by Prof. Pei Yan, et al, is a novel antifungal peptide isolated from Bacillus cereus S-l. It has been reported that in the presence of APS, hypha growth and spore germination of several phytopathogenic fungi in agriculture were strongly inhibited. This paper elucidated the process of isolation and purification, the characterization, the antibiotic property to some phytopathogenic fungi in clinic and possible antifungal mechanism of APS.On the base of having been purified by Sephadex G100 previously, the process of combining subsection elution and linear gradient elution with RPC Sephasil Peptide C8 column was established. And a class of functional components were obtained, designated APS1, APS2, APS3, APS4 and APS5. ESI-MS analyses indicated single molecule species of 1042, 1056, 1056,1070 and 1070 Da, respectively. The UV scaning spectrum showed APSs had the absorbance peak at 280nm. The ninhydrin reaction was negative, but when the preparation was hydrolyzed with 12N HC1 at 110℃ for 24h, it became positive. APSs showed high resistance to heat and some proteases, including thermolysin and chymotrypsin. The amino acid analyzer couldn't detect amino acid sequence with Edman degradation, which meant APSs hadthe blocked N-terminus. The primary structure was given by ESI-MS/MS. The amino acid sequence showed APSs were high homologous, and APS4 sharing the same one with APS5. According to the amino acid sequences of APS1 and APS4, some peptides were synthesized, but no antifungal avtivity was found. The minimal inhibitory concentration(MIC) of APSs to seven kinds of fungi were detected by microdilution test, including Saccharomyces cerevisiae, Candida albicans, Candida albicans CMCC(F)98001, Cryptococcus neoformans, Cryptococcus neoformans(Sanf.)Vvuill. and Trichophyton verrucosum. Compared with fluconazole, APSs had lower MIC to seven fungi tested. Furthermore, the growth of Escherichia coli TG1 and Staphyloccus aureur ATCC25923 were not under the influence of APSs. The switching pulse induced a perceptible sustained transmembrane current on the planar lipid bilayer after unpurified APS added.The optimized purification process of APS was founded. From the results, it could be concluded that APS was a class of cyclic peptides. Its mechanism of action is most likely to cause membrane permeation and the death of fungi. Its strong activity of inhibition to different phytopathogenic fungi, showed the great potential to be used as new antifungal drug in future.