Isolation,Purification And Characterization Of Antimicrobial Proteins From Bacillus Velezensis NT35

This research group isolated and obtained a bacterium NT35 which has a good inhibitory effect on the Cylindrocarpon destructans.This strain has the antagonistic ability to a variety of pathogenic fungi,and shows different degree of antibacterial activity.The purpose of this experiment is to isolate and purify the antibacterial substances of NT35 strain,to explore the characteristics of the antibacterial substances,in order to improve the biological control effect of the strain and lay a foundation for the application of biological control strains to ginseng diseases.The main research results are as follows:1.Identification of strain,determination of antibacterial activity,field testNT35 showed different degree of antibacterial activity against a variety of pathogenic fungi.Field control experiments showed that NT35 fermentation broth?1.5×10⁷cfu/m L?had up to 68.79%effect on the prevention and treatment of Cylindrocarpon destructans.Through the observation of the morphological characteristics,physiological and biochemical tests and 16S r DNA sequencing analysis of bacterial strain NT35,it was identified as Bacillus velezensis.2.Preparation of fermentation broth and preparation of crude proteinPreparation of seed solution:two bacterial dishes were prepared on NT35 plate with dense lines,and then the culture mediumwas inserted into the medium at 28?,160rpm,and cultured for 12h.Preparation of fermentation broth:the inoculation amount of 6%was inserted into the fermentation medium.The temperature was 34?,the rotational speed was 180rpm,the liquid content was 50/250,and the flask was shaken for 72h.The fermentation broth was precipitated with 30%?NH₄?₂SO₄,then desalted by dialysis bag?3500?for 24h,and finally 7.946g of crude protein was obtained.3.Determination of antibacterial propertiesThe crude protein is resistant to high temperature and has antibacterial activity at20-100?.Resistant to acid and alkali,with antibacterial activity under ph4-10condition.Under the irradiation of 30W ultraviolet lamp,it has antibacterial activity.However,protease K?final concentration 100 g/m L?and chloroform were inactivated.4.Separation and purificationUsing chromatography column of Sephadex G-25?DEAE-sepharose Fast Flow?Q Sepharose HP?Superdex75.We obtained antagonistic natural proteins 1-4-2F and1-5-1F.The molecular weights were 6.5 k Da and 3.5 k Da.5.Mass spectrometry identificationThe identification results of 1-4-2F mass spectrometry:through comparison and selectionin the database,the comprehensive score was the highest,the protein registration number:ATX84098.1,the molecular weight was 10.176KDa,the isoelectric point was 9.08,the coverage rate reached 49%,the data was highly reliable.However,functional proteins were not annotated.1-5-1F mass spectrometry identification:through comprehensive analysis,too few protein data were found.6.Effect of antagonistic proteins of 1-4-2F on mycelium growthThe results showed that the antagonistic proteins?1-4-2F?had a significant effect on the pathogen filaments,and the mycelium was disorderly,with cell distortion,vesicle,expansion and then fracture.The cell wall is destroyed,the mycelium protoplasm is leaked from the dissolved cell wall,and the mycelium breaks into segments and then melts.This experiment isolated and purified a kind of anti-ginseng rust pathogen related protein,which provided a theoretical basis for the biocontrol application of bacillus beles,laid a foundation for the discovery of disease-related genes and the study of disease-resistance mechanism,and contributed to the transformation of biocontrol bacteria and the development of drug diversity.