Chemotaxis Of Aromatic Contaminants Degrader YC-RL1 And Functional Study On The Degradation Gene Of BphC

We isolated the bacterium from activated sludge that was historically exposed into aromatic compounds,and identified it as Arthrobacter sp.strain,named YC-RL1.A large number of pre-studies had shown a wide spectrum of substrates of YC-RL1 for biodegradation such as phenol and polychlorinated biphenyls.Microbial chemotactic system enables them to move towards or away from certain chemotant.Strain YC-RL1,which could efficiently degrade p-nitrophenol,was used in swarm plate assay,soil chemotaxis assay and capillary chemotaxis assay with p-nitrophenol as the sole substrate,respectively.In the meanwhile,a test of in situ soil assay was carried out in order to assess the application performance of YC-RL1.HPLC test revealed the ability of YC-RL1 to degrade over 99%of p-nitrophenol within 72 h.A series of chemotaxis assays indicated that strain YC-RL1 had obvious chemotaxis toward p-nitrophenol.Specifically in the capillary assay,YC-RL1 was positively chemotactic to p-nitrophenol within a certain range of concentrations?5⁸00 mg/L?,but was gradually inhibited while the concentration was higher than 800 mg/L.Furthermore,test of in situ soil assay showed a higher degradation efficiency in unsterilized soil than in sterilized soil where the degradation rate could reach up to 95%within 14 days.Our data revealed an excellent adaptability of YC-RL1 to the environment and claimed the potential application in environmental remediation of pollutants.This study further focused on gene cloning,expression and functional characterization of2,3-dihydroxybiphenyl-1,2-dioxygenase encoded by bph C from YC-RL1 in the biodegradation process of PCBs.We cloned bphC gene using the whole genome of strain YC-RL1 as a template and then transformed into E.coli BL21?DE3?competent cell for prokaryotic expression;The recombinant enzyme BphC was purified based on affinity chromatography and its activity was measured in different ranges of pH and temperatures using 2,3-dihydroxybiphenyl as substrate;We also assayed effects of different metal-ion on the enzyme and further detected the kinetic parameters according to the Michaelis equation.Results showed that bphC gene,size of 930 bp,was successfully cloned by PCR and then transformed into E.coli BL21?DE3?;The 6-His tagged recombinant BphC was then purified and the optimum pH and temperature were pH 7.4 and 30°C in vitro,respectively;Effects of metal ions on BphC differed from each other as Fe²⁺,Cu²⁺and Cd²⁺promoted the activity while others inhibited;Kinetic parameters of BphC acting on 2,3-DHBP were also measured,which showed a catalytic efficiency much higher than some other homogeneous enzymes.In this study,amino acid sequence analysis and 3D structure prediction and molecular docking studies were performed.The results indicated that the protein had good affinity and enzymatic hydrolysis to 2,3-DHBP.In conclusion,YC-RL not only well adapted in aromatic environment,but also played a crucial role in the biodegradation of pollutants.This study showed a very good applicable value and potential of Arthrobacter sp.strain YC-RL1.