| The paprent strain with nicotine degrading bacterium Bacillus amyloliquefaciens and Pseudomonas marginali was fused by the protoplast fusion technology, and applying the fusant to degradating nicotine of tobacco. Finally, the paper studied about the roughly separating of nicotine degrading enzyme from the generation of fusant. The following conclusions were made.1. Bacillus amyloliquefaciensT11 and Pseudomonas marginalis was used for parent strain.After using lysozyme to break the cell walls of parent strain,the Bacillus amyloliquefaciensT11 protoplast and heat-inactivated Pseudomonas marginalis protoplast were induced by 40%PEG-6000 in protoplast fusion experiments,then the nicotine degrading ability of fusant was measured.Through preliminary screening,176 fusion strains were obtained. The nicotine selective culture medium was used to rescreen the fusion strains,5 of them with higher nicotine degrading ability was acquired. After analyzing genetic inheritance stability of the 5 fusion strains and comparing the nicotine-degrading rate of parent strain,a better fusant A2 with higher nicotine degrading ability than parent strain was obtained.93% nicotine in culture medium was degraded in five days.2. A nicotine degrading enzyme with a molecular mass of about 35kDa was isolated and purified. The optimum pH of the nicotine degrading enzyme was 6.5-7.0, and the optimum temperature was 35?. By means of two-dimensional electrophoresis, mass spectrometry identification and detection of some metabolites of nicotine,the pathway of fusant degradating nicotine is pyridine pathway.3. The tobacco nicotine content of the tobacco leaf after spraying bacteria liquid of Bacillus amyloliquefaciensT11,Pseudomonas marginalis and fusant 3 times reduced and the degradation rate was 17.7%,18.57% and 25%.The nicotine content of tobacco leaves treated by Bacillus amyloliquefaciens decreased from 4.2% to 3.45%.In GFP tracer experiment,the strains will gradually die after spraying bacteria liquid. |
PDF Full Text Request