Investigation Of Optimization Of Fermentation Conditions Of An Endophytic Lysobacter Enzymogenes R-2-1 With A Broad Spectrum Of Antibacterial Activities And Its Secondary Metabolites

Plant pest threat to agricultural production seriously,the use of chemical pesticides can ensure crop yield,but because of its long-term unreasonable use,resulte in serious environmental pollution problems affecting human health.Because of the efficient,easily degradable,environmentally friendly features,biopesticides are being closely watched.In recent years,the rapid progress of global bio-pesticide industry,biopesticides has replaced more than 20%of chemical pesticides,of which the microbial pesticides are account for 90%.An endophytic strain R-2-1,L.enzymogenes,was firstly isolated from traditional Chinese medicine Artemisia annua Linn.and its fermentation extract was found to have a broad spectrum of antimicrobial activities.The present work focused on optimization of its fermentation conditions using orthogonal experiment and response surface analysis(RSA).The former was used to optimize the LB medium.The best medium included the following components:corn flour 20 g·L-1,peanut meal 20 g·L-1,bean pulp 20 g·L-1,yeast extract 2.0 g·L-1,tryptone 4.0 g·L-1,(NH4)2SO4 6.0 g·L-1?K2HPO4 6.0 g·L-1,riboflavin 3.0 g·L-1,MgSO4·7H2O 4.8 g·L-1,MnSO4-H2O 0.020 g·L-1,FeSO4·7H2O 0.010 g·L-1,CaCl2·2H2O 0.12 g·L-1,NaCl 2.4 g·L-1,thiamine 1.2 g·L-1,Vitamin B60.010 g·L-1,biotin 0.10 g·L-1,and the number of living microbe of strain R-2-1 reached up to(6.94±0.02)×108 cfu·mL-1 under the optimal condition after 24.0 h.The fermentation of L.enzymogenes was optimized by using RSA with statistical software Design Expert 7.00.Under the optimum conditions(fermentation time 87.2 h,initial pH 7.60,broth volume 204 mL,inoculum volume 7.90 mL),the yield reached up to 0.518±0.050 g·200 mL-1.The further condition development for fermentation indicated that the yield had been raised to 0.523 ± 0.023 g·200 mL-1 at 210 rpm and 30.0?.The results could provide a reference to scale-up production of bactericide from L.enzymogenes R-2-1.Research combined the widespread purification methods:silica gel column chromatography,Sephadex LH20 gel column chromatography,C18 reverse phase column chromatography and HPLC.As a result,11 compounds are obtained.Compound 1(2,5-2 ketone piperazine dipeptide).Other isolated compounds were including vaseline(2),glycerine(3),hypoxanthine(4),phenylalanine(5),uracil(6),pyrimidine diol(7),adenine(8),9H-purine-6,8-diol(9),(3-(1H-indol-3-yl)methyl-6-(4-hy droxy-1-oxo-1,2,3,4-tetrahy drophthalazin-6-yl)piperazine-2,5-dione(10),rhamnose(11).Research detected extracellular enzyme of Lysobacter enzymogenes R-2-1.The upper fermented liquid by centrifugation as crude enzyme fluid,and casein and glucan as substrates.As a result of ultra-violet analysis,R-2-1 strain could secrete protease and ?-1,3-glucanse with enzyme-activity unit 1.55 ?g/min and 80.0 ?g/min,respectively.