Screening, Isolation, Identification And Characteristics Of A New Nicotine Degrading Strain, Optimization Of Degradation Conditions And Elucidation Of The Catabolic Mechanism

Nicotine is highly toxic,biofilm-permeable,chemically stable,and an important precursor of tobacco-specific nitrosamines(TSNAs),which are not easily degraded in the environment and seriously pollute the environment.Due to its special water solubility,it can be miscible with water in any proportion,which is a great threat to human life and ecological environment.A new strain with high nicotine degradation activity was screened for the degradation of highly toxic and high-hazard nicotine,and the degradation characteristics were explored.The degradation process was optimized with the(response surface methodology(RSM).Through whole-genome sequencing(WGS),the nicotine degradatio-related gene cluster were analyzed and identified,and the nicotine catabolic pathway of ZUC-3 was initially constructed,and the key genes for degradation were functionally identified.The research aims to provide new microbial resources and theoretical basis for the research of biodegradation-related fields and degradation mechanisms of nicotine,the results of which showed that:A strain with high ability of nicotine degradation was obtained by isolation and purification from tobacco field soil,which was identified by morphological,physio-biochemical and 16 S rRNA gene analysis,and the characteristics of nicotine degradation under different conditions were investigated with dynamic process by HPLC analysis.It was identified as Stenotrophomonas sp.ZUC-3.The results of single-factor test showed that the suitable conditions of nicotine degradation by Stenotrophomonas sp.ZUC-3 was 30 ? with 10% inoculum at initial pH 7.0 and 180 r/min,in which the rate of nicotine degradation by Stenotrophomonas sp.ZUC-3 reached 91.53%.HPLC analysis illustrated the dynamic process and high efficiency of nicotine degradation by Stenotrophomonas sp.ZUC-3.In the optimization experiment of Box-Behnken Design-RSM of nicotine degradation by ZUC-3,an appropriate amount of peptone and NH4NO3 were added as nitrogen sources and supplemented a small amount of glucose and inorganic salts.Through Plackett-Burman(PB)Design,the most significant factors affecting the nicotine degradation were screened out,which were temperature,time and aerobic speed.Based on the results of PB Design,the steepest ascent experiment was used to approach the optimal region,and the climbing direction and step size were set by the results of PB Design.The center point of temperature,time and aerobic speed was 37 °C,32 h,and 150 r/min respectively,and the optimization of Box-Benhnken Design-RSM was designed by the center point.Therefore,the optimal degradation conditions of temperature,time and aerobic speed were determined,which were 30.13 °C,35.41 h,183.32 r/min respectively,in which the predicted value of nicotine degradation rate was 98.34%,and the actual value was 99.13%,and the fitting degree was 99.20 %.It proved that the rate of nicotine degradation was significantly higher.The analysis of whole-genome sequencing(WGS)of high-efficiency nicotine-degrading strain ZUC-3 resulted in a total of 90 Contigs in genome assembly and the genome sequence length was 6.08 Mb,while 74 tRNAs and 5519 coding sequences were obtained.The function of nicotine degradation-related genes of ZUC-3 were predicted to be located on Contig 3 by BLAST,and nicotine-degrading gene clusters of ZUC-3were mapped to construct the catabolic pathway.The relative expression levels of 9 degradation-related genes in the degradation pathway were determined by qRT-PCR,which showed that the expression of 9 degradation genes was all significantly up-regulated when nicotine was present.Molecular cloning and functional identification of the key degradation gene hspB of ZUC-3 were conducted,the hspB gene was obtained by PCR amplification,and pET-22 b was used as a vector to construct recombinant plasmid pET22b-hspB,which was transformed into E.coli BL 21(DE3)for heterologous expression.The protein HspB was detected by SDS-PAGE,and the enzymatic activity was measured.The recombinant plasmid pET22b-hspB was constructed and the protein HspB was successfully expressed.The molecular weight was 43.97 kDa.And the protein was determined to be expreesd in the form of inclusion bodies in cells.The enzymatic reaction showed that the HspB protein had a high activity of 6-hydroxy-3-succinoyl-pyridine monooxygenase.which confirmed the biological function and enzymatic activity of protein HspB.