Molecular Cloning And Expression Of AgnB And AgnE Genes And Functional Analysis Of AgnE Involved In Nicotine-degradation Pathways In Agrobacterium Tumefaciens Strain SCUEC1

Nicotine,the main natural alkaloid in the tobacco plants,is harmful to the human health and the environment.The removal of nicotine from the environment via chemical and physical processes is relatively time-consuming,inefficient and can also cause secondary pollution.Therefore,the highly efficient and environmentally friendly strategy to fight pollution is the use and manipulation of the detoxification abilities of microorganisms.A strain with nicotine degradation ability was previously isolated from tobacco planting soil in Xiangyang city of Hubei province.It was identified as Agrobacterium tumefaciens,named SCUEC1.In this study,two genes agnB and agnE from Agrobacterium tumefaciens strain SCUEC1 and involved in nicotine degradation were cloned and expressed,and the function of agnE was analyzed.The main experimental results of this paper are as follows:(1)The agnB and agnE were cloned by PCR using the total DNA of the Agrobacterium tumefaciens SCUEC1 strain as a template.The size of the agnB and the agnE were 1314 bp and 1029 bp,respectively.Predicted with on-line software,the molecular weight of agnB encode AgnB and agnE encoded AgnE were 48.8 k Da and 42.0 k Da,respectively.AgnB and AgnE were hydrophilic and non-secretory proteins.(2)The agnB and the agnE were ligated to the plasmid p ET28a(+),respectively,and the recombinant plasmid was transformed into E.coli BL21(DE3)to obtain a recombinant plasmid transformed strain.The plasmid was extracted and verified by digestion and sequencing.The recombinant plasmids p ET28a(+)-agnB and p ET28a(+)-agnE were induced to express with different concentrations of IPTG,induction temperature and induction time to obtain optimal expression conditions.The results showed that the recombinant plasmids p ET28a(+)-agnB and p ET28a(+)-agnE had a highly expression under induction temperature of 30? and induction time of 20 h.IPTG concentration of 0.2-1.0 mmol/L has no obvious effect on expression of p ET28a(+)-agnB and p ET28a(+)-agnE.The SDS-PAGE was used to detect the target protein in the whole broth,supernatant and precipitate.The results showed that AgnB and AgnE were mostly distributed in the precipitate.The AgnE was purified and extracted by using different concentrations of imidazole eluent and nickel column.It was found that pure AgnE was obtained when nickel column was eluted with 250 mmol/L imidazole eluent.(3)According to the laboratory's previous work,it was assumed that the AgnE has the function of 2,5-dihydroxypyridine dioxygenase.The function analysis of AgnE was performed by using 2,5-dihydroxypyridine as a substrate.The enzyme activity of AgnE with 2,5-dihydroxypyridine as reaction substrate was determined spectrophotometrically by monitoring the decrease of 2,5-dihydroxy pyridine at 320 nm.The absorbance of the enzyme reaction at 0,5,10 and 20 min was 0.6170,0.2273,0.0907,0.0667,respectively.The AgnE was shown to have 2,5-dihydroxypyridine dioxygenase activity.AgnE has higher 2,5-dihydroxy pyridine dioxygenase activity at p H 8.0 and temperature 20 ?.The effect of different metal ions on the biological activity of AgnE indicates that Ni2+ and Cu2+ have a certain inhibitory effect on the biological activity of AgnE,while Ca2+ and Fe2+ have a certain role in promoting the biological activity of AgnE,and Fe2+ has a higher promoting effect on the biological activity of AgnE.(4)The gene encoding the AgnE was randomly mutated and three mutants of AgnE were obtained,namely AgnE33-46,AgnE90-199 and AgnE260.The glutamic acid at position 33 and the glutamine at position 46 in the mutant AgnE33-46 were mutated to aspartic acid and arginine,respectively;the isoleucine at position 90 and the glutamine at position 199 in the mutant AgnE90-199 were mutated to leucine and proline,respectively;the tryptophan at position 260 in the mutant AgnE260 was mutated to terminator.The biological activity of non-mutated AgnE was set to 100%,the relative biological activity of AgnE33-46 was 12.60%,and the relative biological activity of AgnE260 was 4.50%.The relative biological activity of AgnE90-199 was 110.30%.The results showed that either glutamic acid at position 33 or glutamine at position 46 is an essential amino acid for the biological activity of the AgnE.