Purification And Identification Of An Novel Antifungal Protein From Bacillus Subtilis Strain RN-61

An antifungal bacterial strain was isolated from soil samples collected from different agricultural sites in Beijing, China. An novel antifungal protein was also isolated and identified from the bacteria strain. In this study, Fusarium moniliforme、Colletotrichum gloesporioides、 Verticillium dahliae、Fusarium solani、Ralstonia solanacearum、Fusarium vasinfectum、 Pseudoperonospora cubensis、Polvporus hirsutus、Botrytis cinerea、Monilinia fructicola was used as indicators to screen antifungal bacteria. The bacteria were identified, based on their biochemical characteristics, which showed the highest antifungal activity, were selected for further identification by partial sequence analysis of their16S rRNA genes. Because of the effective antifungal activity of the strain RN-61and RN-88, it was investigated.The strain RN-61shared99%similarity with that of Bacillus subtilis subsp. subtilis str, named Bacillus subtilis RN-61, which was exhibited the hightest antifungal activity.The medium and culture conditions of the strain RN-61were optimized by single factor experiments.In the investigation of the culture condition, the major ingredients being investigated included carbon sources and nitrogen sources. The optimal antifungal activity production condition were determined as follows:initial pH6.5,culture temperature at37℃, culture time24h, rotation rate180rpm, soybean peptone10.0g/L, yeast extract5g/L.The antifungal metabolites from the bacteria B.subtilis RN-61were further purified by consecutive chromatographic fractionations including ion exchange chromatography and gel filtration chromatography. The purified fractions were tested for their antifugal effect.the result showed that the fraction B had the best effect with the inhibition of40.94%after96h culture.The purified fraction B was shown to have a relative low molecular weight of32kd by sodium dodecyl sulphate poly-acrylamide gel electrophoresis (SDS-PAGE).Antifungal protein was ideniified with Q-TOF after digested with trypsin. six Peptides were get.six peptides sequenee was INHNIAAL NTLNR；LSSGLR; AGDDAAGLAISEK; NSQDGI SLIQTAEGAL TETHAILQRTEFNGKK: LGAVQNRLEHTINNLS: ASGENLTAAE SRIRDVDMAK EMSEFTKNNI LSQASQAMLA QANQQPQNVLQLLR.These peptides were used to search in MASCOT database:we found the protein was observed related with this antifungal Protein.The protein was flagllin. LC/ESI-MS analysis confirmed the fraction from the metabolites of B.subtilis RN-61which has the strongly inhibition to the indicator that formed as a result of a novel protein belonged to the flagellin family. A flagellin gene (FLA-RN61) was amplified from the B.subtilis RN-61genome by using specifically designed PCR primers. The sequence analysis showed the new cloned gene had a degree of91%identity to a known FLA gene in a new different B.subtilis. The sequence was also expressed in E.coli and showed high antifungal effect. We report here the isolation of a new antifungal bacteria from soils in Beijing China that may be very well adapted to the environmental conditions of this area. We also first report the complete cloning of the new antifungal gene which is belongs to the flagellin family. Due to their high antifungal activity, the bacteria and the novel protein isolated from this work merit further study as a potential biological agents for the organic food production in agriculture.