Molecular Cloning And Expression Of AgnD And AgnH Genes And Functional Analysis Of AgnH Gene Involved In Nicotine-Degradation Pathways In Agrobacteriumtumefaciens SCUEC1 Strain

Nicotine?1-methy-l-2-[3-pyridyl-pyrrolidine]?is an alkaloid found in Solanaceae,which is a toxic heterocyclic compound that is harmful to humans,animals and the environment.Degradation of nicotine by microorganisms is a highly efficient,cost–effective and environmentally friendly method because that its chemical structure is so stable and it is hard to be degraded naturally.Before this,the laboratory has previously isolated and screened a strain which has a high ability to degrade nicotine from the soil of tobacco plantation in Xiangyang city,Hubei Province,Which was identified as Agrobacterium tumefaciens strain,and named SCUEC1.In this study,the two genes agnD and agnH involved in nicotine degradation metabolic pathway in Agrobacterium tumefaciens SCUEC1 strain were cloned and expressed,the biological function and enzymatic characteristics of gene agnH were analyzed.The main experimental results of this paper are as follows:?1?The genes agnD and agnH were cloned by PCR with the total DNA of the Agrobacterium tumefaciens SCUEC1 strain as a template.The size of the genes agnD and agnH were respectively 1176 bp and 585 bp in length by sequencing.After amino acid sequence analysis,the AgnD protein that the agnD gene encoded was predicted to be a hydrophilic non-secreted protein which has the function with 6-hydroxy-3-succinylpyridine oxidase.The AgnH protein that the agnH gene encoded was predicted to be a hydrophobic non–secreted protein which has the function with maleic acid cis-trans isomerase.?2?The genes agnD and agnH were ligated to the plasmid pET28a?+?respectively,and the recombinant plasmids pET28a?+?-agnD and pET28a?+?-agnH were transformed into E.coli BL21?DE3?strain in order to be expressed heterologously.The effects of different concentration of IPTG,induction temperature and induction time on the expression of the recombinant protein AgnD and AgnH were studied.The results of SDS-PAGE showed that:the molecular weight of AgnD and AgnH protein were 44.0kDa and 28.0 kDa respectively,which was consistent with the predicted results.The suitable conditions for AgnD and AgnH protein to be expressed were that the induction temperature and time were 25°C and 25 h respectively,and the concentration of IPTG was 0.4 mmol/L.The solubility of the protein AgnD and AgnH were analyzed,and protein AgnH was eluted.The results of SDS-PAGE showed that the protein AgnD and AgnH were mostly distributed in the precipitate;it was found that a higher concentration of the purer AgnH protein was obtained with 250 mmol/L imidazole eluent.?3?Maleic acid and fumaric acid have specific absorption peaks at 210 nm.The retention time of maleic acid standard was 3.98 min by HPLC?high performance liquid chromatography?,and the retention time of fumaric acid standard was 5.46 min.The functional analysis of AgnH protein was carried out with maleic acid as the reaction substrate by HPLC.The results showed that when the enzymatic reaction was for 0 min,the peak area of maleic acid was 9048.85 mAU·s,and the peak area of fumaric acid was0 mAU·s,and when the enzymatic reaction was carried out for 30 min,the maleic acid peak area has decreased to 3256.05 mAU·s,on the other hand,the peak area of fumaric acid has increased to 4771.82 mAU·s.Under the catalysis of AgnH protein,the peak area of maleic acid decreased and the area of fumaric acid peak increased gradually with the increase of reaction time,it indicated that AgnH protein has the same activity as maleic acid cis-trans isomerase.?4?The effects of different temperatures,pH and metal ions on the biological activity of AgnH protein with maleic acid as the reaction substrate,and the reaction products were detected by HPLC.The results showed that the suitable temperature reaction of the AgnH protein was at 37°C;pH 8.0 is more suitable for the enzymatic reaction of AgnH protein.The enzyme activity of AgnH protein has been promoted by Mg²⁺,and it has been inhibited by Ca²⁺,Zn²⁺,Fe²⁺,Li⁺,Mn²⁺,Cu²⁺and Ni²⁺,among which,Cu²⁺has a worse inhibitory effect.The thermal stability of AgnH protein has been researched,the results showed that the enzyme activity of AgnH protein had no significant effect in the water bath at 0°C for 120 min,the relative enzyme activity decreased to 24.79%and 8.53%respectively at 55°C and 65°C for 120 min.