Purification And Characterization Of Antimicrobial Protein From Bacillus Sp.MD

The contamination of food spoilage and food-borne pathogens have been thought as the main Food safety problem on a global scale,such as Escherichia coli,Salmonella enterica,Pseudomonas,Clostridium botulinum,Listeria monocytogenes,Staphylococcus aureus and Streptococcus suis.The need of the drugs to fight bacterial have became more and more inflated whether in the field of food or in the pharmaceutical field.But the increasing use of chemical food preservatives to inhibit the bacteria has imposed a large negative effect on people's health.The natural antibacterial material has attracted people's attentions because of its widely-distributed and unique mechanism of action and provided important information about the research of new anti bacterial active substances.Besides,with the rate of discovering new antimicrobial substances from land-base is on the decline,it is imperative for scientists to turn their eyes to the ocean to explore new resources.The sea provides a special ecological environment,which may led to the development of new metabolic pathways of marine microorganisms and be more likely to produce new antimicrobial substances.These antimicrobial substances which usually have a novel structure will play a huge role in the control of the growth of food spoilage and food-borne pathogens.So,exploring marine microorganisms to find antimicrobial peptides has an important academic significance.The secondary metabolites of Bacillus are an important source of antimicrobial protein,enzyme and active compounds.Therefore,the selections of active strains in my study aimed at Bacillus sp.of marine origin.In order to screen the bacteria of deep-sea mud samples and water samples from the Arctic and oceans,three culture mediums(M1,NA and 2216E)were used as selection mediums.There were 610 strains of bacteria were screened by gradient dilution method and most of which were identified as bacillus.The antimicrobial activities of fermentation supernatants were determined by agar well diffusion methods as Staphylococcus aureus,Escherichia coli and Samomella enteritidis were used as indicator strains.There were one hundred twenty strains of S.aureus resistant,90 strains of E.coli resistant,forty-two strains of S.enteritidis resistant respectively.Thirty-four strains can inhibit three indicators at the same time.For further study in the detailed characterization of bacteriocin,a strain,which had a antimicrobial activity against S.aureus,was fermented in LB medium and purificated for antimicrobial substances.The strain was identified and named as Bacillus sp.MD-5.Firstly,the crude protein was isolated by the method of ammonium sulfate precipitation,combined with centrifugation,dialysis and freeze drying.Then,the pure antibacterial protein was isolated from crude protein using ion exchange chromatography and ultrafiltration concentration technology.The protein was called as MD antibacterial protein.The antimicrobial experiments showed that the protein had a good antibacterial effect on the positive bacteria such as S.aureus and L.monocytogenes.The molecular weight was about 4.6 kDa and had high purity by SDS-PAGE electrophoresis and silver staining.MALDI-TOF-TOF mass spectrometry and LC-MS were used to identify MD protein.Compared with the protein sequence predicted by genomic draft of Bacillus sp.MD-5,the deduced protein contained ninety-three amno acids was considered as the MD protein sequence.Homology analysis results showed that it might be an unknown antibacterial protein.The MD protein has a good inhibitory effect on the positive bacteria,it is similar to the antimicrobial spectrum of nisin which was widely used in food.Therefore,the minimal inhibitory concentration(MIC)and some physical and chemical properties of the protein were studied by comparing with nisin.The MIC against S.aureus of MD protein and nisin were detected by the microdilution technique in 96-well plates.The MD protein and nisin showed strong antimicrobial activities against S.aureus with MIC of 7.33 and 2.34 ?g/mL respectively.The physical and chemical properties were determined by pH,temperature,metal ions,protease and surfactants.The activity of the MD protein mainly did not decrease while incubated in three surfactants.But it is sensitive to the high temperature and the protease K.In addition,0.2M Mg2+,0.05M Ba2+and 0.01 M Al3+had a certain decrease on the activity of MD protein.It maintained the basic stability for MD protein under the condition of weak acidic,neutral and alkaline,but nisin only in acidic and weakly acidic conditions.Perhaps,it will have an obvious advantage over nisin applied in food industry.