Isolation Of Endophytic Bacillus Amyloliquefaciens ES-2 And Studies On Its Antimicrobial Substances

Endophytes existing in plants have a wide range of antimicrobial strains, which are theimportant potential sources of antimicrobial substances. Some endophytes could excrete theantimicrobial compounds that may be involved in a symbiotic association with a host plant.Meanwhile, many biologically active substances that endophytes excreted were relativelynewly to us. In addition, the antibiotics made by endophytes may reduce cell toxicitytowards higher organisms because the plant itself serves as a natural selection system.Therefore, it is a huge potential to screen novel, highly active and low poisonousantimicrobial substances from endophytes. Therefore, the objectives of this work were: (1)to isolate, screen and identify endophytes with novel, highly active and low poisonousantimicrobial substances from endophytes existing in plants; (2) to isolate and identify theantimicrobial substances from culture filtrate of strain ES-2 by the HPLC and the massspectrum; and (3) to study physics and chemistry nature, the culture medium andfermentation dynamics of antibacterial material from strain ES-2. The results weredescribed as follows:23 endophytes with antimicrobial activity were isolated from 16 kinds of plantsmaterials. After duplicate sieves, 11 strains have obviously inhibited Aspergillus niger. Themajority of strains exhibited antagonism against Gram-positive bacteria. 2-3 strains of themstrongly inhibited Gram-positive and Gram-negative bacteria and Aspergillus niger. Theantibacterial matter produced from the majority of isolates has the certain thermal stability.The antibacterial matter from tested strain has the resistance or the partial resistances to thetrypsin and the pepsin. The strain ES-2 from Chinese medicinal plant Scutellariabaicalensis Georgi exhibited broad-spectrum antibacterial and antifungal activities.Secondary metabolite produced by ES-2 can be resistance to treatment of 121℃20 minheat and the proteinase. Finally, the strain ES-2 was determined for the next step ofexperimental strain.The strain ES-2 with strong antimicrobial activity was identified with molecularmethod. The sequence fragment of 1423bp 16S ribosomal RNA gene is amplified fromtotal DNA of strain ES-2 by PCR. The sequence analysis is down by the application of BLAST on the websites of NCBI and the phylogenetic tree is drawn by Bioedit 7.0^T andTreedrawing^T. The results showed that ES-2 shared 99.72% 16SrRNA gene sequencehomology with Bacillus amyloliquefaciens (AY620954). In phylogenetic framework ofbacteria classification, strain ES-2 belongs to Bacillus amyloliquefaciens.After purification and analyzed on the sephadex LH-20 and HPLC, The methanolextraction of culture filtrate of ES-2 strain exhibited three series of antimicrobial activitypeaks. A HPLC-MS analysis showed three series of ion peaks from the culture filtrate. Afurther electrospray ionization/collision-induced dissociation spectra revealed that the threeseries ion peaks represented different fengycin homologues and surfactin homologues anditurin homologues respectively. The first series of ion peaks represented 6 kinds of fengycinA homologues with C14～C18 fatty acids carbon chains and 5 kinds of fengycin Bhomologues with C14～C18 fatty acids carbon chains and 3 kinds of novel linearlipopepetide; The second series of ion peaks represented C11, C13 and C14 [Leu7]surfactin, as well as C13 [Val7] surfactin and one kind of C-13 and the C-14 [Leu 7+Val7]mixed-type surfactin. By comparing their mass data with those obtained in previous studies,the third series of ion peaks may belong to C18 iturin A or C17 bacillomycin F and C12bacillomycin L of iturins group.The UV spectrum, HPLC chromatogram and posotive ion spectra of LC-MS ofantimicrobial lipopeptide produced by strain ES-2 was described in this paper. The resultsshowed that antimicrobial lipopeptide produced by strain ES-2 is composed of fengycins,surfactins and iturins, in which fengycins is the principal constituent. The methanolextraction of culture filtrate is diffluent in the water, below 90% methanol and 70% alcohol;is dissoluble or slightly soluble in n-butanol, isoamyl alcohol, chloroform, acetone, ether,ethyl acetate; It will precipitate in the pH 2 below watery solution, antimicrobial activity ofantimicrobial lipopeptide is stable under the pH 2-12 condition. The antimicrobiallipopeptide can be resistance to treatment of heat and proteinase. As well as, it displayedbroad-spectrum antibacterial and antifungal activities.The medium screening experiment indicated that the ES-2 could obtain high yield ofantimicrobial peptides in the Landy medium. Through further carbon and nitrogen sourcescreening tests, one kind of improvement Landy synthesis medium was determined for theantimicrobial lipopeptide production.Influence of carbon and nitrogen source of medium on antimicrobial lipopeptidecomponent was examined by HPLC. The result showed that glucose is good carbon source for production of fengycin and surfactin, specially, which obviously promoted surfactinproduction; the sucrose and the lactose also were good carbon source for production ofsurfactin. Therefore different sugars in medium have the certain influence to production oftwo kind of lipopeptide composition. The L-glutanic acid and the L-asparagine are goodnitrogen source for production of the antimicrobial lipopeptide. But the different amino acidcoordination is certainly disadvantageous to the antimicrobial lipopeptide production. Theexperimental result also showed that 6 kinds of fatty acids inhibited antimicrobiallipopeptide production.The submerged fermentation conditions for antimicrobial peptides produced by ES-2were optimized. On the base of single factor experiment, the methodology of Plackett-Burman design was undertaken to screen the key factors rapidly from the related 12 factors.By analyzing the statistical regression and the prediction profiler, the temperature and theconcentrations of KCL and FeSO₄ were found to be the most important factors for theproduction of antimicrobial peptides. Based on the results of previous Plackett-Burmandesign, a three-level three-factor Box-Behnken design was applied to optimize theproduction of antimicrobial lipopeptide. The critical factors selected for the investigationwere mentioned above. By analysis of the 3-D plots and their corresponding plots, theoptimum values of the temperature and the concentrations of KCL and FeSO₄ for obtainingthe most production of antimicrobial lipopeptide were 27.22℃, 0.78g/L, and 0.05mg/L,respectively. The optimized cultivation conditions allowed antimicrobial lipopeptideproduction to be increased from 1010.24mg/L to 2130.12mg/L.The kinetics process and model of ES-2 for antimicrobial lipopeptide fermentationwere studied in this paper. The results showed that bacteria growth presents typical S curvemodel; the antimicrobial lipopeptide production and the mycelium growth are coupling.With the Logistic equation, the mycelium growth and the product synthesis kinetics processwere described; with the Luedeking-Piret equation, substrate consumption kineticsprocesses were described. The experimental result showed that three models all couldexplain well to the empirical datum.