Physical And Chemical Mutation In Bacillus Subtilis NJA-9and The Structure Identification Of Subtilosina

Recently, an increasing number of people are focusing on investigation in order to find the kind of natural substance which is safer and more stable with a higher efficiency.Bacillus subtilis is a mesophilic, aerobic or facultative anaerobic, Gram-positive rod-shaped bacteria which is able to produce endogenous spores.It can produce a variety of antimicrobial substances,including lipopeptide, phospholipids, protein, polyene etc. These antimicrobial substances can inhibit the normal growth of fungi, bacteria, viruses, and bacteria of the original body, and have a wide range of practical applications.Bacillus subtilis nja-9isolated from compost have strong antibacterial on positive and gram-negative. In this paper, the strain is mutagenized by physical and chemical methods in order to get a high activity and stability strain,and thus it reserach the separation, purification and stability properties of the antibacterium substances,the last, the antibacterium substances of structure is identified by LC-MS、IRand UV.The main research findings are as follows:1. Study of physical and chenmical mutation in Bacillus subtilis nja-9:The fermentation time is24h by measuring the growth curve of Bacillus subtilis nja-9and dynamic curve of its antimicrobial substances.The strain was mutagenized by NTQ EMS, LiCl and the implantation of low energy ion beam of N+. The most optimized experimental conditions of NTG, EMS, LiCl and implantation time of low energy N+are0.25mg/mL,0.6mol/mL,4%and10s under lOkev energy repectively. Last, after several rounds of screening, we got a strain named NO. N133which have better antibacterial activity and genetic stability from1250bacterias.Because the diameter of inhibition zone which antibacterial substances of N133acted on Pseudomonas fluorescens was23.23mm,which increased by6.9%compared wild strain,it was the starting strain for subsequent experimements.LB will be the fermentation medium of next experiments through determination of antibacterial effect of the fermentation different medium.2. The study of purification and property of antibacterial substances produced by Bacillus subtilis N133:The acid precipitation regard as a means of crude extract lastly after comparing acid precipitation, organic solvent extraction and ethanol precipitation.A group of active ingredient which preliminary considered surfactin Homologues were obtained by SephadexLH-20chromatography purification of methanol crude extracts.The results of stability studies on the fermentation broth of strain N133showed that it was very stable at acidiccondition, but became unstabile as pH ascended. The antimicrobial activity decreased after100℃treatment for2hours and inhibition zone diameter compared with the control is reduced about25.55%after120℃for20minutes, so it show good thermal and UV irradiation stability.3. The structue identification of antibacterial substances produced by Bacillus subtilis N133:The maximum UV absorption wavelength is210nm after scanning of Methanol crude extraction broth,so210nm is the detection wavelength in HPLC and LC-MS. LC-MS showed that molecular weight of a group are994Da,1008Da,1022Da and1036Da respectively and its HPLC chromatogram compared with HPLC chromatogram of standard Surfactin.It concluded that the N133antibacterial substances contain Surfactin Homologues.In addition,it was that another broad-spectrum antimicrobial substance was successfully isolated from the fermentation broth of strain N133by aseries of separation and purification steps which included acid precipitation, methanol extraction,semi-preparative HPLC collection. UV scan showed that its contains amino acid groups. MALDI-TOF MS showed that its molecular weight is3401Da and consistent molecular weight of Subtiliosin reported.The results of IR indicated that the substance consist of element of C, H, O, N, and contains functional groups of-CH3,-CH2,-CO-NH-, and benzene rings. Through the full scan MS/MS of the antibacteria substance, on fragment peaks were observed at m/z3022.197、2447.522、2245.937、1507.724、1049.769、765.244、262.053etc.We determined that the molecular structure of the activity peak3was SubtilosinA.By testing the broad-spectrum of activity peak3and comparing with Nisin, the activity peak3has better antibacterial activity under the same concentration.Among them, activity peak3displayed strong antibacterial activity on Pseudomonas fluorescens, Pseudomonas aeruginosa, Micrococcus luteus, Bacillus pumilus, Bacillus cereus and general antibacterial activity on E. coli and Staphylococcus aureus.