Isolation Of A Benzoate Sodium Degrading Bacterium And Cloning Of Gentisate 1, 2-Dioxygenase Gene And Heterologous Expression

Microcosm experiments were set up in order to study the dynamic changes of the bacterial communities in marine sediments supplemented with different model PAH compounds pyrene and sodium benzoate. High-throughput sequencing technology Illumina Miseq targeted the 16 S r RNA gene V3-V4 variable region was used to analysis the shift in the composition of the bacterial communities due to the addition of pyrene and sodium benzoate. Either pyrene or sodium benzoate could dramatically influence the bacterial community structures in marine sediments. Lower diversity and abundance were observed compared with the control group. Marinobacter and Pseudidiomarina became dominant community in the in experimental group treated with sodium benzoate. Balneola and Aequorivita were greatly enriched in the experimental group with the addition of pyrene. Our study will help to reveal the functional bacteria capable of degrading pyrene and sodium benzoate respectively. This result may also provide a theoretical basis for the bioremediation of PAH compounds in marine environment.Bacillus sp. LKG-A is a gram-positive benzoate degrader bacterium isolated from marine sediment, which degrades benzoate via the gentisate pathway to central metabolites. The god A gene, encoding the gentisate1, 2-dioxygenase responsible for catalysing the ring fission of gentisate with the formation of maleylpyruvate was cloned by touch-down PCR and thermal asymmetric interlaced PCR amplification. The open reading frame consists of 1143 bp encoding a protein with 379 amino acids.The deduced amino acid sequence of god A with a theoretical Mr of 42.9 k Da displays no more than 59% sequence identity with those previous pubulished gentisate1, 2-dioxygenase. The sequence of the gdo A gene product shared similarity with few known polypeptides, including gentisate 1, 2-dioxygenase from Paenibacillus sp. Ny Z101(49%) or gentisate 1, 2-dioxygenase from Escherichia coli O157: H7 str. Sakai(36%), gentisate 1, 2-dioxygenase from Bacillus halodurans C-125 share 50% identity. Homology modeling were employed to construct the three-dimensional structure of the gentisate 1, 2-dioxygenase protein from Bacillus sp. LKG-A. Analyzing three-dimensional structure of the protein subunits by homology modeling and docking result, we speculated His114, His116, His155 and ferrous ions were considered to constitute one catalytic core, and Trp91, Asp72, Asp189, Arg74 may play a certain role in the catalytic process. The probable biochemical mechanism of the involvement of the protein in aromatic hydrocarbon oxidation has also been investigated. It was cloned into p ET22 b vector then transformed into E. coli BL21(DE3) expressed by the induction with 1.0 m M of IPTG. The resulting product was purified to homogeneity and partially characterized. Studies showed that the dioxygenase had the largest activity at 30℃and p H 8.0. Ferrous ions could significantly improve its catalytic activity. The dioxygenase could not catalyze similar structure with gentisate, which was different from gentisate 1, 2-dioxygenase discovered earlier.The discoveries maight deepen our understanding of the gentisate metabolic pathways.