The Bioactivity Of Antibiotic Zwittermicin A And Establishment Of Detection Methods

Zwittermicin A?ZwA?is an antibiotic with broad-spectrum antimicrobial activity,produced by Bt bacteria metabolism.It is a small,water-soluble molecule with liner structure.ZwA can increase the insecticidal crystal protein's insecticidal activity by complexed with it.In this paper,ZwA was isolated and purified basing on its physicochemical properties.The detection methods of ZwA was established.And the synergistic effect of ZwA on Cry1Ca protein against plutella xylostella was studied.The results are as follows:The medium composition and culture conditions of Bt185 strain which is high yield ZwA were optimized.The optimum carbon source of Bt185 strain fermentation medium was glucose,the optimum nitrogen source was yeast powder,the optimum inorganic salt was sodium chloride,the optimum temperature is 30?and the optimum initial pH is 7.5.The potiomum condition to yield ZwA is the same excepet that its optimum initial pH is 8.0.The process of separation and purification of ZwA was constructed.The fermentation broth of Bt185 strain was first purified by LX-68M macroporous resin,eluted with deionized water,25%ethanol,50%ethanol,75%ethanol and 95%ethanol respectively.ZwA is concentrated in the elution phase of 50%ethanol.Then the ZwA samples were separated by the silica gel.,eluted with 20 mLethyl acetate:petroleum ether=2:1,50mL methanol:ethanol=2:1 and 20ml ethanol.ZwA is concentrated in the elution phase of methanol:ethanol=2:1.Strains of Streptomyces albus,Escherichia coli,Staphylococcus aureus,Bacillus subtilis,Bacillus cereus,Common variola,Corynebacterium glutamicum,Paratyphi B and Pseudomonas aeruginosa were used as test bacteria to test the antimicrobial spectrum of ZwA.The antibacterial activity of the fermentation broth showed that Bacillus thuringiensis Bt185 had different inhibitory effects on nine pathogenic bacteria,and its inhibitory effect on Erwinia herbicola was the strongest.The bioactivity of Cry1Ca protein and Cry1Ca protein complexed with ZwA against Plutella xylostella were determined.The synergism radio of ZwA to Cry1Ca was 1.7 fold.A chemiluminescence system of H₂SO₄-KMnO₄-CH₂O-ZwA was established.8mmol/L KMnO₄,1mol/L H₂SO₄ and 8%CH₂O were prepared with distilled water.Among them,KMnO₄ and H₂SO₄ were charged as an inverting,and CH₂O and sample were charged as carrier liquid,ampled by the auxiliary pump and mixed in the sixteen pass valves before flowing into the test cell.Finally,a chemiluminescence reaction occurred in the test cell.The flow rate of the main pump is 80r/min?4mL/min?,the auxiliary pump is 50r/min?2.5mL/min?,the main pump running time is 8s,the auxiliary pump running time is 4s.A new approach for the simultaneous determination of ZwA by ECL detection was developed.The optimal conditions for the determination were as follows:Ru?bpy?₃²⁺concentration 5?l/L,the buffer concentration was 60mmol/L,the buffer pH was 8.5.150?L buffer,100?L Ru?bpy?₃²⁺solution and 20?LZwA sample were added to the test cell,cyclic voltammetry,initial voltage 0.2V,high point 1.25V,low potential0.2V,scanning rate 0.1V/s,photomultiplier tube high voltage 800V.