Screening Of Antagonistic Bacullis And Its Antimicrobial Peptides' Purification And Analysis

Antibacterial peptides are a kind of protein antibiotics widely found in nature.Almost all species of organisms can produce a certain degree of antimicrobial peptides.Because of these substances with broad-spectrum antibacterial effect on some bacteria,fungi and others,they had been got much attention in various fields.In recent years,traditional sense antibiotics course of some problems in practical use,looking for some new antibiotics with different antibacterial mechanisms as a supplement to traditional antibiotics in practical applications has become one of the main tasks of some areas.Because of unique antimicrobial properties of the antimicrobial peptide,thus it has been a hot spot in recent years.In addition,some pharmacological effects of antimicrobial peptides?such as antiviral,anti-tumor,etc.?were detected.So that the study of such substances quickly to develop in depth.In this study,the antimicrobial peptides were successfully isolated from Bacillus licheniformis B32 and the optimum conditions were determined.At the same time,the isolation and purification conditions of antimicrobial peptides were explored,and their stability,structure,antibacterial properties were studied in depth The above results can provide a reference for the application of the antagonistic Bacillus in certain fields and the study of the substance.The main results of this paper are as follows:?1?With Staphylococcus aureus as an indicator of bacteria,successfully selected from the soil of a significant antagonistic indicator of the strains,named B32.The strain was identified as Bacillus licheniformis by colony identification and molecular biology identification.?2?The optimum conditions for the production of high antibacterial peptides from Bacillus licheniformis were determined by optimizing the culture condition and the medium components,such as carbon source,nitrogen source,inorganic salt and growth factor.The results showed that the optimum medium was 1.5%soluble starch,3.5%of mixed nitrogen source?soybean meal powder:ammonium sulfate = 5:1?,0.1%manganese sulfate,0.2%calcium carbonate and 0.025%methionine.The optimum culture conditions were as follows:cultureat 35? and pH 6.0,inoculation amount 2%.Under this condition,the fermentation broth with Antibacterial potency up to 82.57 U/mL?ampicillin sodium?was obtained for 36 h.?3?The extraction conditions of the antibacterial substances in the fermentation broth were optimized by using the organic solvent.The extraction conditions were determined by using n-butanol as the organic extractant.The volume ratio of organic solvent to fermentation broth was 2:1,and the operating temperature was 45?,The optimum extraction efficiency is about 44.51%.At the same time,the main factors influencing the effect of the reversed extraction process were the operating temperature and the operations times.At the operating temperature of 45?,the volume ratio of organic solvent topure water was 1:1,the single effectiveness is 53.80%,and the secondary operating is close to 61.28%.The samples prepared by extraction were separated by high performance liquid chromatography?mobile phase:acetonitrile/ultrapure water?.Antibacterial peptides with purity of 90%were successfully obtained.?4?The stability and antimicrobial activity of the antimicrobial peptides were measured,and it was found that it had strong stability,high temperature?more than 100??,acid and alkali changes and other factors can not make its antibacterial titer decreased significantly.The antimicrobial peptides were treated with certain proteases?pepsin,trypsin,protease K?,andit can be concluded that the effect of protease on the antimicrobial peptides is not consistent.Trypsin can destroy the material structure.At the same time by the antibacterial results can be obtained that the antimicrobial peptides for some Gram-positive pathogens?such as Staphylococcus aureus?has a good antagonistic,while some fungi?such as Trichoderma reesei,etc.?also have a certain inhibition effect.?5?The amino acid sequence of antimicrobial peptides was identified by mass spectrometry,and it was compared and analyzed in the genomic protein library of Bacillus licheniformis?NC₀06270.3?.The gene was cloned by molecular biology method,and finally the antibacterial The sequence of the peptide MAVKQKKEYLIRQLAKPGRLDNLKDLSSHKLDEVRQHQL.Bioinformatics analysis showed that the antimicrobial peptide was C203H345N63O58S1,the relative molecular weight was 4628.42 Da,and the theoretical isoelectric point was 9.87,which belonged to soluble protein.