Purification And Tandem Mass Spectrum Analysis Of Bacillus Thuringiensis BRC-ZYR2 Bacteriocins

Bacteriocins synthesized and secreted by bacterial ribosomes are proteins or peptides with antibacterial or bactericidal activity.Since bacteriocins have many advantages such as thermostability,safety,free of affecting product flavor and no drug resistance,they have good application prospects in the fields of food,fodder,medicine,biological control.The purpose of this study were to(1)monitor the bacterioncins production during the growth of Bacillus thuringiensis(Bt)BRC-ZYR2;(2)preliminary determine the molecular weight of BRC-ZYR2 bacterioncins;(3)purify BRC-ZYR2 bacterioncins with salt fractionation and column chromatography;(4)preliminary analysis of BRC-ZYR2 bacterioncins by MALDI-TOF MS analysis and NCBInr database database searching.The detailed experimental results are as follows:Bacillus cereus(Be)0938 was used as an indicator strain to evaluate the activities of BRC-ZYR2 bacteriocin by a modified agar-well diffusion method.In tryptic soy broth(TSB),BRC-ZYR2 bacteriocin was first detected at 18 h after incubation,and reached maximum level at 22 h after incubation when Bt BRC-ZYR2 was in sporangium period.To preliminary determine the molecular weight of the bacteriocin,the bacteriocin activity was detected on a SDS-PAGE gel.The inhibition zone indicated the molecular weight of BRC-ZYR2 bacterioncins was slightly larger than 10 kDa.Pure BRC-ZYR2 bacterioncins were extracted by three-step chromatographic processes.Bt BRC-ZYR2 was grown for 22 h and showed strongest antibacterial activities.The bacteriocin-Iike inhabitation substrances(BLIS)were precipitated with 100%ammonium sulfate and stocked at 4oC overnight.The resulting pellets,re-suspended with 0.01 mol/L Tris-Cl buffer(pH 7.4),were loaded onto a Sephadex G-50 gel filtration column(φ10x600 mm)and eluted with 0.01 mol/L Tris-Cl buffer(pH 7.4).There are two absorption peaks and the fractions between 78 min and 108 min in the peakⅡ,positive for antimicrobial activity,were pooled,dialysed overnight.Then they were loaded into a DEAE-52 Sepharose column(φ16x300 mm)and eluted with 0.01 mol/L Tris-Cl buffer(pH 7.4)containing 0,0.1,0.6 mol/L NaCl,respectively.Four absorption peaks appeared and the active eluents of 167-188 min in the peak Ⅳ were pooled,dialysed overnight.They were loaded into the DEAE-52 Sepharose column(φ16x300 mm)and eluted with 0.01 mol/L Tris-Cl buffer(pH 7.4)containing 0,0.15,0.3,0.6 mol/L NaCl,respectively.Of the five absorption peaks,the active eluents of 64-94 min in the peak Ⅱ were pooled and dialysed overnight.The purified BRC-ZYR2 bacterioncins were analyzed by SDS一PAGE,revealing a single band which was slightly larger than 10 kDa.The pure BRC-ZYR2 bacterioncin was 35.6 μg/mL and 235.7 U in a 2.5 mL solution.The recovery rate was 38.8%.Based on MALDI-TOF mass spectrometry,the first-order diagram and second-order diagram of BRC-ZYR2 bacteriocins were obtained and compared wtih NCBInr database through Mascot software.But no matching protein can be detected.Thus BRC-ZYR2 bacterioncins might harbor novel structures.Taken together,the purification method of Bt BRC-ZYR2 bacterioncinS was established and the pure BRC-ZYR2 bacterioncins might have novel structures.This study provide theoretical basis for mass production of Bt bacterioncins.