title: Expression, purification, and characterization of a 2,3-dihydroxybiphenyl-1,2-dioxygenase from Bacillus sp. JF8 in Escherichia coli.
abstract: 2,3-Dihydroxybiphenyl-1,2-dioxygenase plays an important role in the degradation of polychlorinated biphenyls. The gene (BsbphCI) encoding a 2,3-DHBP dioxygenase from Bacillus sp. JF8 is 960 bp. We synthesized a 960 bp BsbphCI gene encoding a 2,3-DHBP dioxygenase derived from Bacillus sp. JF8 and expressed it in Escherichiacoli. The recombinant protein was about 36 kDa, confirmed by SDS-PAGE. The concentration of the purified protein was about 1.8 mg/mL. With 2,3-DHBP as a substrate, the optimal temperature for enzyme activity at pH 8.5 was 50 °C. The optimal pH for the 2,3-DHBP dioxygenase was 8.5. The enzyme retained 33% activity after heating at 60 °C for 60 min. We found that Cu(2+), K(+), Zn(2+), Mg(2+), Ni(2+), Co(2+), and Cd(2+) activated the enzyme. However, Ca(2+), Fe(2+), Li(+), and Cr(3+) inhibited it. Enzyme activity was reduced by exposure to H(2)O(2), SDS, and KI. The results of HPLC indicated that the transgenic E. coli strain with the BsbphCI gene degraded 2,3-DHBP more quickly than the wild type strain.
authors.affiliation: State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Horticulture, Nanjing Agricultural University, 1 Weigang, Nanjing 210095, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Bian
authors.forename: Jian-Jun
authors.initials: JJ
authors.lastname: Shuai
authors.forename: Fei
authors.initials: F
authors.lastname: Xiong
authors.forename: Ri-He
authors.initials: RH
authors.lastname: Peng
authors.forename: Quan-Hong
authors.initials: QH
authors.lastname: Yao
authors.forename: Ai-Sheng
authors.initials: AS
authors.lastname: Xiong
mesh_terms: D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D049308:Dioxygenases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005813:Genes, Synthetic; D059748:Proteolysis; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D011994:Recombinant Proteins; D049308:Dioxygenases; C074272:2,3-dihydroxybiphenyl oxygenase