Purification And Characteristic Of Antifungal Protein From Bacillus Amyloliquefaciens MG-3

Bacillus amyloliquefacciens MG-3 was isolated from leaf or stem of mango and identified,which can secreted kinds of active sub stances to against pathogens from fruits.In this study,purification and characteristic of antifungal protein was researched from bacillus amyloliquefaciensMG-3.Bacillus amyloliquefaciensMG-3 was cultured for getting fermentation broth,which the crude protein was obtained by precipitating with ammonium sulfate adding according to 20%,40%,60%saturation,then DEAE-650C.ion change chromatography and Sephacryl S-200 Gel chomatography was used on purified the crude protain as protocol for geting pure protein.Lasiodiplodia theobromaeof Longan as tested pathogenic,checking the antifungal protein has or not antifungal activity,and using SDS-PAGE analyzed indicated the antifungal protein was only one band on the Gel which molecular is about 45KDa.The antifungal protein was identified by MALDI-TOF/TOF with the result showed that the protein is likely serine protease and the molecular is approxicately 48KDa.For reseaeching effect of antifungal protein with stable and activity,the result of experiment showed that the protein has siginificant stability,whatever how change of temperature,pH,ultraviolet radiation,the relative activity of the protein remaining above 70%.Among these condition,the activity ofprotein barely effect disposed with protein K,with 100? dealt with the protein for 30min,the activity of the antifungal protein has 70%active.Studying measure effect of the protein with content against Lasiodiplodia theobromaeof Longan,mode of action against pathogen,and antimicrobial spectrum,the result showed that half maximal inhibitory concentration(IC50)is 40.58ug/mL,manimal inhibitory concentration(MIC)is 80.60ug/mL,and minimum bactericidal concentration(MBC)is 119.8ug/mL;the antifungal protein not only against pathogens,but also inhibited germinating of spore;the protein has a broad antimicrobial spectrum including Lasiodiplodia theobromae,Colletotrichum acutatumand so on.The antifungal can against pathogenic fungi on the surface of loquat,which has longer storing.On the basis of result of identification by MALDI-TOF/TOFand blast analyzed with NCBI database,designing the primer with restriction enzyme sites,target gene was amplified by PCR from genomic of bacillus amyloliquefaciensMG-3 as template,which the target gene was linked with PMD18-T vector transformed into Escherichia Coliand sequencing.The PMD18-T contained target gene was double digestion as same as PET-28a vector,then target gene connected to PET-28a vector by digested for building the expressed vector transforming intoEscherichia Coli.The target gene expressing the target protein induced by IPTG in Escherichia Coli that centrifuging and broken can obtained substances.The recombination antifungal protein was purified by Ni column chromatography,which was tested with antifungal activity by Lasiodiplodia theobromaesis tested strain.