Immobilization Of Extradiol Dioxygenase BphC And Its Application In Enhancing Biodegradation Of Biphenyl

The aromatic compounds are widely used in industry, wheras they have been accumulated in the environment causing serious environmental problems due to the recalcitrant characteristics. Microbial degradation has been proven to be an efficenct and the main approach for aromatics treatment. The critical step is to cleave the benzene ring, mainly conducted by extridol dioxygenase. In this dissertation, the complete genome of strain Dyella sp. LA-4was obtained, and a unique bph gene cluster involved in biphenyl metabolism was identified using bioinformatic analysis. Then, magnetic nanoparticles Fe3O4and mesoporous silica SBA-15were utilized to immobilize the2,3-dihydroxybiphenyl1,2-dioxygenase (BphC), which catalyzed the rate-limiting step in the biphenyl metabolism pathway. Furthermore, the immobilized enzymes were used to enhance the biphenyl biodegradation process of Dyella sp. LA-4. This study could give a new insight into the practitical application of BphC in aromatics treatment process.Firstly, the draft genome sequence of stain LA-4was obtained by Illumina sequencing. The unique continuous biphenyl metabolic cluster bphAlA2(orfl)A3A4BCX0was found in a small contig. Then, the three-dimensional structure of the rate-limiting enzyme BphC was predicted through homology modeling for further analysis.Magnetic Fe3O4was firstly used to immobilize BphC. The optimum conditions for immobilization were:Fe3O4was treated with ultrasonic for10-30min, and incubated with BphC for30min at pH9.0. Under these conditions, the loading amount of BphC could reach40.7mg/g and the immobilized enzyme retained42.3%of its initial activity. Storage stability and thermostability were also enhanced by immobilization. Meanwhile, the optimum pH of immobilized BphC rised from8.0to9.0.In order to further improve the catalytic efficiency and stability of immobilized BphC, mesoporous silicas SBA-15was used as an alternative matrix. Protein titration curves and surface electrostatics analysis predicted that the optimum pH for immobilization was8.0, and this result was validated experimentally. The successfully immobilization of BphC was confirmed by Fourier transform infrared spectroscopy (FTIR) and N2adsorption/desorption isotherms. Under the experimental conditions, the maximum loading amount of the immobilized enzyme could reach124.58mg/g. The BphC-SBA-15could be stored at4℃for 5d, while the free enzyme was deactive quickly within12h. The result of circular dichroism (CD) supposed that the stability and activity of the immobilized BphC was altered by the changes of secondary structure.Ultimately, both of the immobilized enzymes were used to enhance the biphenyl biodegradation process of Dyella sp. LA-4. By adding the immobilized enzyme BphC-Fe3O4, the product HOPDA was accumulated faster than the system which only contained Dyella sp. LA-4. However, BphC-SBA-15did not show any enhanced capacity for biphenyl degradation. These results indicated the feasibility of using immobilized BphC to enhance biphenyl biodegradation, while the enhance capacity was dependent on the immobilization matrix.