Expression, purification and functional characterization of a recombinant 2,3-dihydroxybiphenyl-1,2-dioxygenase from Rhodococcus rhodochrous.
A 2,3-dihydroxybiphenyl (2,3-DHBP) dioxygenase gene from a Rhodococcus sp. strain, named RrbphCI and involved in the degradation of polychlorinated biphenyls (PCBs), was synthesized. RrbphCI was expressed in Escherichia coli and its encoded enzyme was purified. SDS-PAGE analysis indicated that the size of the protein encoded by RrbphCI was about 32 kDa. The activity of the 2,3-DHBP dioxygenase was 82.8 U/mg when the substrate was 2,3-DHBP, with optimum pH 8.0 at 30°C, and optimum temperature was 40°C at pH 8.0. The RrbphCI gene was transformed into Pseudomonas putida strain EG11, to determine the ability of the enzyme to degrade 2,3-DHBP. The wild type EG11 degraded 61.86% of supplied 2,3-DHBP and the transformed EG11 (hosting the RrbphCI gene) utilized 52.68% after 2 min of treatment at 30°C. The overexpressed and purified enzyme was able to degrade 2,3-DHBP. The 2,3-DHBP dioxygenase is a key enzyme in the PCB degradation pathway. RrbphCI and its encoded 2,3-DHBP dioxygenase may have transgenic applications in bioremediation of PCBs.
Shanghai Key Laboratory of Agricultural Genetics and Breeding, Biotechnological Research Institute, Shanghai Academy of Agricultural Sciences, 2901 Beidi Rd, 201106 Shanghai, China.
Fei
F
Xiong
Jian-Jun
JJ
Shuai
Ri-He
RH
Peng
Yong-Sheng
YS
Tian
Wei
W
Zhao
Quan-Hong
QH
Yao
Ai-Sheng
AS
Xiong
D001673:Biodegradation, Environmental / Q000187:drug effects; D001713:Biphenyl Compounds / Q000378:metabolism; D002396:Catechols / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D049308:Dioxygenases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007477:Ions; D008670:Metals / Q000494:pharmacology; D030781:Organisms, Genetically Modified; D011994:Recombinant Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D012240:Rhodococcus / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D013696:Temperature; D013997:Time Factors
D001713:Biphenyl Compounds; D002396:Catechols; D004791:Enzyme Inhibitors; D007477:Ions; D008670:Metals; D011994:Recombinant Proteins; C454813:2,3-dihydroxybiphenyl; D049308:Dioxygenases; C074272:2,3-dihydroxybiphenyl oxygenase