title: Characteristics of a Recombinant 2,3-Dihydroxybiphenyl 1,2-Dioxygenase fromComamonassp. Expressed inEscherichia coli.
abstract: 2,3-Dihydroxybiphenyl 1,2-dioxygenase (2,3-DBDO) is an extradiol-type dioxygenase that involved in third step of biphenyl degradation pathway. The nucleotide sequence of thebphCgene fromComamonassp. SMN4, which encodes 2,3-DBDO with His-tag, was cloned into a plasmid pQE30 inE. coli. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified active 2,3-DBDO showed a single band around 33 kDa, corresponding the molecular mass of 2,3-DBDO subunit. Two fractions around 170 and 100 kDa were separated in gel filtration chromatography, but only former one (the fraction of 170 kDa) has 2,3-DBDO activity. The 2,3-DBDO was reported as the polymeric protein consisted of eight subunits. However, the fraction corresponding octameric protein of 2,3-DBDO was not found in the gel filtration chromatography. The 2,3-DBDO was exhibited the maximum activity at pH 9.0 and was stable at pH 8.0, relatively. The circular dichroism (CD) data showed that 2,3-DBDO had an α-helical folding structures at neutral pHs ranged from pH 4.5 to pH 9.0. However, this high stable folding structure was converted to unfolded structure in acidic region (pH 2.5) or in high pH (pH 12.0). The enzyme was thermally stable and active up to 40 °C. The conformational data by CD spectra were consistent with the stability of 2,3-DBDO by checking the activity. The binding affinity (Km) for 2,3-dihydroxybiphenyl, 3-metylcatechol, 4-methylcatechol and catechol was 11.7, 24 μM, 50 mM and 625 μM, respectively.
authors: Research Group of Gut Microbiome, Korea Food Research Institute, Baekhyun, Bundang, Seongnam, Gyeonggi 13539 Republic of Korea.
authors: Nari
authors: N
authors: Lee
authors: Research Group of Nutrition and Diet, Korea Food Research Institute, Baekhyun, Bundang, Seongnam, Gyeonggi 13539 Republic of Korea.
authors: Dae Yong
authors: DY
authors: Kwon
keywords: Binding affinity; Circular dichroism spectra; Comamonas; Dihydroxybiphenyl dioxygenase; bphC