Construction Of Metagenomic Library From PCBs-contaminated Soil And The New BphC Genes Cloning And Expression

Polychlorinated biphenyls is noted as an chamieal organic matter with toxicy.It can affect the healthy of organism by enriched in the humen's body.It can be absorbed by organism's skin lung and stomach, when it is flowed to liver organize of muscle and fattiness by blood. The toxic effect to humans is can't be reversed.In this study, the metagenomic DNA were isolated indirectly from PCBs-contaminated soil, size of 23Kb.After the agarose gels,further purification of the DNA fragments on 9-23 Kb andwas used to constract the metagenomic library.The metagenomic library was constructed, which contain 8000 clones with an average DNA insert size of 4 kb, the library had an average size of inserted fragments of 45%. The metagenomic library represented about 14.4 Mb.Functional screening was employed to search,the library contained positive cloning.the new gene named pBphCA.The analysis indieated that this pBphCA contained an ORF of 2,3-dihydroxybiphenyl dioxygenase,the fragment of size was 939bp.ORF coding and Catechol 2,3-dioxygenase from Ralstonia eutropha JMP134 with 59% homology by Blast software.ORF was named bphCA, the registered number was GU479462. A DNA sequence containing the DNA region encoding the protease gene was amplified by PCR and cloned into E.coli expression vector pET28a.Enzyme of the active was preliminarily characterized. The recombinant BphCA was stable at temperature 0-30℃and pH8-9,low temperature at 15℃and pH8-9 has more than 60% of the activity.The optimal temperature and pH of BphCA were 35℃and pH 8.5. The activity of BphCA was enhanced by Mn2+ and inhibited by Al3+,Cu2+ and little influence by Co2+,K+,Fe2+. The Km toward 3-methylcatechol was 22.69μM, Show that the substrate and enzyme had the largest affinity, comparing Vmax/Km, with the result that 3-methylcatechol as BphCA the optimal substrate. Innovation of this paper is:obtained a novel 2,3-dihydroxybiphenyl dioxygenase gene and its cloning and characterization of enzyme activity;the first time obtain the cold adaptation 2,3 - dihydroxybiphenyl dioxygenase.