Rapid purification of an active recombinant His-tagged 2,3-dihydroxybiphenyl 1,2-dioxygenase from Pseudomonas putida OU83.
2,3-Dihydroxybiphenyl 1,2-dioxygenase (2,3-DBPD) is an extradiol-type dioxygenase that catalyzes the aromatic ring fission of 2,3-dihydroxybiphenyl, the third step in the biphenyl degradation pathway. The nucleotide sequence of the Pseudomonas putida OU83 gene bphC, which encodes 2,3-DBPD, was cloned into a plasmid pQE31. The His-tagged 2,3-DBPD produced by a recombinant Escherichia coli strain, SG13009(pREP4)(pAKC1), and purified with a Ni-nitrilotriacetic acid resin affinity column using the His-bind Qiagen system. The His-tagged 2,3-DBPD construction, carrying a single 6 x His tail on the N-terminal of the polypeptide, was active. SDS-PAGE analysis of the purified active 2,3-DBPD gave a single band of 34 kDa; this is in agreement with the size of the bphC coding region. The K(m) for 2,3-dihydroxybiphenyl was 14.5 +/- 2 microM. The enzyme activity was enhanced by ferrous ion but inhibited by ferric ion. The enzyme activity was inhibited by thiol-blocking reagents and heavy metals HgCl2, CuSO4, NiSO4, and CdCl2. The yield was much higher and the time required to purify recombinant 2,3-DBPD from clone pAKCl was faster than by the conventional chromatography procedures.
Khan
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Microbiology Division, Food and Drug Administration, Jefferson, AR 72079, USA.
Nawaz
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MS
Cerniglia
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CE
D049308:Dioxygenases; D005296:Ferrous Compounds / Q000494:pharmacology; D010105:Oxygenases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D016958:Pseudomonas putida / Q000201:enzymology*; D011994:Recombinant Proteins / Q000096:biosynthesis; D013379:Substrate Specificity
D005296:Ferrous Compounds; D011994:Recombinant Proteins; D010105:Oxygenases; D049308:Dioxygenases; C074272:2,3-dihydroxybiphenyl oxygenase