Study On The Characterization Of The Nitroreductase From Psychrobacter Sp.ANT206 And Its Metabolism Pathways Of Nitro-aromatic Compounds

Nitro-aromatic compounds（NACs）with the characteristics of structure stability and degradation refractory are significant raw ingredients and intermediate products in chemical industries.These contaminants could not only pollute the environment,but also affect the growth and propagation of animals,plants and microorganisms,threaten lives and health of human beings.In comparison with other degradation methods,the biological degradation method has been widely concerned by researchers as a result of the advantages of low cost and simple reaction conditions.Nevertheless,in the practical biodegradation of the industrial wastewater containing NACs,the growth and metabolism of those normal microbes are easily inhabited and make it difficult to achieve the efficient biodegradation of pollutants.Therefore,a psychrophilic strain with efficient NACs degradation capability under low temperature was isolated from Antarctic environment,the nitroreductase gene（psntr）from the strain was cloned and the enzymatic characteristics,relationship between structures and low temperature catalytic properties of Ps NTR were analyzed.Furthermore,the in-frame deletion mutant strain was constructed for the exploration of biodegradation mechanism of NACs by the strain under low temperature.And the strain was immobilized using the synthesized magnetic nanoparticles to improve its reusability.This research provided theoretical basis and technical supports for the efficient biodegradation of NACs under low temperature.In this paper,psychrophile ANT206 that could utilize nitrobenzene as sole carbon and nitrogen source was isolated from Antarctic sea-ice,which belonged to Moraxellaceae Psychrobacter after identification.The novel psntr gene was cloned from Psychrobacter sp.ANT206 using the designed primers and bioinformatics analyzed.Subsequently,psntr was heterologously expressed into Escherichia coli BL21 using cold-shock plasmid p Cold I.The purified recombinant Ps NTR（r Ps NTR）with good salinity tolerance and thermolability exhibited its highest activity at 25℃and still maintained46.90%of the activity at 0°C,which could be identified to be cold-adapted enzyme.Furthermore,Ps NTR showed good substrate binding affinity for nitrobenzene and p-nitrophenol.The results of Gas Chromatography-Mass Spectrometer（Gc-MS）indicated that those NACs could be converted to their corresponding amine derivatives.Arg49,Ser50,Arg52,Val254,Tyr255 of Ps NTR were identified to be key amino acids in the biodegradation of NACs,according to the results of molecular docking,chemical modification and site-directed mutagenesis.Homology modelling and molecular dynamic simulation were utilized for the analysis of the cold-adaption mechanism of Ps NTR.Ps NTR with longer loops and reduced number of hydrogen bonds,displayed better flexibility under low temperature and could reduce its stability by increasing protein flexibility.All of these characters made Ps NTR showed better catalytic efficiency under low temperature.The role of cold-adapted Ps NTR of psychrophile Psychrobacter sp.ANT206 strain played in the biodegradation of NACs and its mechanism were further analyzed.The in-frame deletion mutant of psntr（Δpsntr-ANT206）with excellent genetic stability and kanamycin tolerance stability was successfully constructed using allelic replacement method.The comparative results of the morphologies and growth curves of the wild-type strain（strain ANT206）andΔpsntr-ANT206 under identical culture conditions indicated that the in-frame deletion of psntr gene could not affect the growth of the strain and Ps NTR from ANT206 played an irreplaceable role in the bio-reduction of NACs using the wild-type strain.The metabolites of ANT206 were further analyzed using integrated GC-MS,and the content of peptides was the highest.In addition,the analysis of the differences of metabolites of wild-type strain andΔpsntr-ANT206 cultured at 16℃at different time periods revealed that ANT206 was able to convert NACs into their corresponding amine derivatives through the production of hydroxylamino intermediates.Cell immobilization is an important method for the improvement of the pollutants’biodegradation capability and reusability of strains.The low-cost dopamine（DA）functionalized magnetic nanoparticles were synthesized using co-precipitation method,and characterized using various methods.Fe₃O₄@Si O₂@DA nanoparticles were utilized for the immobilization of the wild-type strain Psychrobacter sp.ANT206,and the magnetic nanoparticles with excellent biocompatibility could be absorbed on the surface of ANT206 strain,due to the small size effect.The immobilization conditions were further optimized.The highest immobilization yield of the immobilized cells was 90.67%under the optimized immobilization conditions of 10℃,p H 7.5,2 h and cell/support 1.2 mg/mg,and activity recovery of the immobilized cells was identified to be 89.41%.The further analysis of the influence of different factors on the immobilized cells revealed that Psychrobacter sp.ANT206 immobilized using Fe₃O₄@Si O₂@DA owned better salinity stability and reusability,which could maintain more than 52%of its initial activity after10 cycles.Additionally,in comparison with the free ANT206 strains,the immobilized ANT206 strain exhibited better biodegradation capability of nitrobenzene and p-nitrophenol,which could degrade those compounds completely in 36 and 42 h,respectively.These results could provide a theoretical basis of the practical industrial application of the efficient biodegradation of NACs by immobilized strains under low temperature.