Primary Structure Identification And Heterologous Expression Of A Novel Bacteriocin BtCspB From Bacillus Thuringiensis

Bacillus thuringiensis(Bt)not only could produce a variety of toxin proteins had poison effect to agricultural pests,it also could synthesize and secrete proteins or peptides with antibacterial or bactericidal activity,called Bacteriocins.Until now,Bt preparation mainly use mixture of spore and crystal,the fermentation supernatant was almost useless,so use the Bt supernatant to extract bacteriocin can improve the efficiency of Bt production.This study used a food-borne pathogen Bacillus cereus(Bc)0938 as an indicator strain to acquire a bacteriocin BtCspB,which was purified from the supernatants of\Bt BRC-ZYR2.The results showed that the bacterioncin was purified to homogeneity from the supernatants of 22 h broth culture by 100%ammonium sulfate precipitation,two steps of Sephadex G-50 gel filtration chromatography and cellulose DEAE-52 anion-exchange chromatography.The tricine-SDS-PAGE analysis of the purified bacteriocin BtcspB showed one band by protein staining.The molecular weight of BtCspB was 7366.2935 Da by MALDI-TOF-TOF determination.The complete amino acid sequence of BtCspB was further derived from its N-terminal amino acid sequencing,its liquid chromatography and tandem mass spectrometry(LC-MS/MS)analysis,and its draft genome sequence.Sequence alignment showed that this bacteriocin has a high similarity with cold-shock protein B(CspB).The physical and chemical properties of pure BtCspB were studied.BtCspB activity was completely lost after enzymatic treatment with proteinase K.Compared with the CK,the activities exhibitd a significant difference after treated with catalase,α-amylase,α-chymotrypsin Ⅶ,lipase Ⅶ and lipase Ⅱ.However,trypsin,α-chymotrypsin Ⅱ and RNase A did not influence the activity.The circular dichroism(CD)spectra of BtCspB did not have the representative patterns of α-helical-rich andβ-strand-rich proteins or disordered proteins,but the CD spectra of lipase Ⅱ-treated thuricin were close to the representative pattern of α-helical-rich proteins.BtCspB was active after treated at 60℃ for 30 min,and was stable in the pH range of 5-7,demonstrating its thermostable property and pH resistance.The results of CD and differential scanning calorimetry showed that the temperature did not have significant effects on secondary structure of BtCspB.But the pH could affect the secondary structure of BtCspB and influenced its activity.Among 32 species of potential food-borne bacteria,BtCspB was only active against Bc 0938 and ATCC 10987 with MIC values of 3.125 μg/mL and 0.781 μg/mL,respectively.The MBC values of BtCspB against Bc 0938 and ATCC 10987 were determined to be 12.5 μg/mL and 6.25 μg/mL,respectively.BtCspB did not detectably inhibit any other strains,exhibiting an extremely narrow antibacterial spectrum and high specificity.A recombinant plasmid with BtCspB gene was transformed into Escherichia coli BL21(DE3).The fusion protein could be expressed in supernatants,which was completely coincided with the BtCspB.The expressed peptide was further purified by Ni-NTA affinity chromatography.This method not only improved the production but also simplified the purification method.