Studies On Breeding And Fermentation Condition Optimization Of The Producing Strain GX-29 And Antibiotic's Separation

GX-29 is one strain of Streptomyces fulvissimus which was screened from soil by our Lab.In previous researched,it was found that strain GX-29 can produce an antibiotic which could kill Brine Shrimp and silkworm intensely and strongly inhibit Magnaporthe grisea,Bacillussubtilis,Aspergillus ornatus and Rhodotorula glulinis.By using Bacillussubtilis as an indicative strain in primary screening and Brine Shrimp in second screening assay,its spores were treated by some physical factors to improve in mutative breeding.The optimizing and separate-purifying experiments were carried on at the same time.1.The initial strain 0^#(with half-leathal time of 26.4 min)was treated by means of ultraviolet radiation,⁶⁰Co radiation and microwave radiation respectively.Every time,a strain with higher activity acqucired from one step was selected as the starting strain of the next step.High-yield mutant Strain 101^# (with half-leathal time of 22.6 min)was got by ultraviolet radiation;strain 202^# (with half-leathal time of 21.3 min)was obtained by ⁶⁰Co;strain 310^#(with half-leathal time of 19.2 min)was gained by microwave radiation.Compared with the strain 0^#,the time of half-leathal of 310^# was 19.2 min,shorten 7.2 minutes.Ultraviolet radiation which contirbuted most to the improvement of the production among the three methods,was 52.78%,and was an effective method for the mutation and breeding of the antibiotic producing strain GX-29; microwave radiation was the second one;and the ⁶⁰Co was the last one,which was only 18.06%.2.The fermentation conditions of strain 310^# were optimized by the single-factor and the Orthogonal design experiments.The study has shown that the optimum fermentation medium were composed as folows(%):soluble starch 1.6,glucose 0.3,yeast powder 0.4,K₂HPO₄ 0.1,NaCl 0.1,MgSO₄·7H₂O 0.1, FeSO₄ 0.001.The optimum culture conditions were as follows:the culture time of seed 48h,the seed volume 8%,the initial pH 7.4,liquid volume 70mL/ 250mL,the temperature of the shaking table 28℃,rotating speed 150r/min, the fermentation time 4d.Under the optimum conditions,the half-leathal time of the strain 310^# decreased to 13.9 min,compared to that of original conditions.3.Before the procedure of purification,some properties of antibiotic were identified through a series of experiments.At last,it was found that the antibiotic's molecular weight is no more than 3000Da and it has high polarity which could dissovle in water.The result also shown that it has a better insecticidal stability,including temperature,anti-ultraviolet,in pH6.0-10.0. 4.The compound of antibiotic was isolated from the fermentation broth by ion exchange chromatography combined with gel filtration chromatography.The ion exchange resin 732^# could work better in conditions:solution's pH 6.0-7.0, absorbing at flow 2.0 ml/min,0.2%NaCl solution is chosen as eluant,eluating at flow 0.5 ml/min.The eluant with high bioactivity was collected and concentrated to get the crystal of the antibiotic.5.Elucidated through wavelength scanning and MS,the molecular weight of antibiotic was identified as about 1081,and the detective wavelength is 290nm.