| Chlorothalonil(2,4,5,6-tetrachloroisophthalonitrile,TPN)is a kind of systemic broad-spectrum chlorinated benzene nitrile fungicide.It was one of the largest used fungicides in the world.At present,the production amount of chlorothalonil reaches 8000 tons in China.Chlorothalonil behaves stably in natural environments,owing to its toxic propertity,cumulative toxicity;it would cause pollution on the farmland.Therefore,it causes serious harmness to the ecological environment,human health and food safety.Thus,remediation of chlorothalonil contamination has attracted widespread attention.(1)In order to effectively deal with serious pollution problems caused by the wide use of chlorothalonil,Novel TPN hydrolytic dehalogenase(Chd)from Pseudomonas sp.CTN-3 had been identified in our previous study.To stable expression of chlorothalonil hydrolytic dehalogenase,this study will use the method of homologous recombination for gene knock out.The successful realization of Chd gene in Bacillus subtilis WB800 exocytosis of stably expression solved the problem of expression unstable work.(2)Bacillus subtilis can be in normal growth under 30? to 37?,but grow better under 37? in temperature test.In comparison the two recombinant strains,Bacillus subtilis WB800-Chd,and the previously constructed WB800(pP43Chd),WB800(pP43Chd)showed highest enzyme activity reached 16.54 U/L,whereas WB800-Chd reached 10.63 U/L in flask cultrue.It reduced by 55%,but the WB800-Chd exhibited better stably.(3)The batched submerged fermentation was used to increase the production ofyield chlorothalonil hydrolytic dehalogenase.Submerged fermentation of Chd enzymeproduced by strains WB800-Chd and WB800(pP43Chd),and strains WB800(pP43Chd)showed that glucose levels were decreased from 50 g/L to 1.987 g/L,96%glucose was utilized as carbon source during the fermentation period of 20 h.At the same time enzyme activity reached a maximu of 22.30 U/L,with the stability of 94%to 45%from the first generation to the sixth generation.In comparision,submerged fermentation period of WB800-Chd was prolonged to 27 h.Glucose concentration was reduced from 50 g/L to 0.038 g/L,showed that the carbon source was consumptedby 99.8%.Enzyme activity was up to 13.00 U/L,and the stability was from 98%of the first generation to 95%of the sixth generation.(4)Three kinds of storage methods were adopted for enzyme:4? preservation,ultrafiltration and freeze drying preservation method.In comparison,ultrafiltration method retained the highest enzyme activity.(5)Chlorothalonil residues caused not only environmental pollution but also biomass bioconversion inhibition.The relief inhibition of Chd on biomass bioconversion was studied,therefore its degradation is imminent.Chlorothalonil degradation assay was performed to testify the degradation ability of chlorothalonil on biological conversion by the Chd.Then using two different ways of solid state fermentation and enzymolysis,by wheat straw processed biological conversionto degradation of chlorothalonil in biomass pesticide residues.we can saw that gradually increasing the concentration of chlorothalonil generated inhibition to wheat straw bioconversion more and more seriously.However,the concentration of chlorothalonil was gradually reduced due to the degradation of Chd.When adding 120 ?g/mLof chlorothalonil generated biggest inhibition reached 45%,but under the same conditions to add Chd generated bioconversion inhibition only reached 12%.The results showed that Chd has great potentials in removing chlorothalonil residues in environmental pollution and relieving its inhibition on biomass bioconversion. |
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