Characterization of 2,3-dihydroxybiphenyl-1,2-dioxygenase BphC from biphenyl degrader Arthrobacter sp. YC-RL1

Abstract:

[Objective] This study focused on gene cloning, expression and functional characterization of 2,3-dihydroxybiphenyl-1,2-dioxygenase BphC encoded by bphC gene from Arthrobacter sp. strain YC-RL1 in the biodegradation process of polychlorinated biphenyls (PCBs). [Methods] Cloning bphC gene using the whole genome of YC-RL1 as a template and then transformed into Escherichia coli BL21(DE3) for prokaryotic expression. The recombinant enzyme BphC was purified through Ni2+ column based on affinity chromatography and its activity was measured in different ranges of pH and temperatures using 2,3-dihydroxybiphenyl as a substrate. We also assayed effects of different metal-ion on the enzyme and further detected the kinetic parameters according to the Michaelis equation. [Results] The bphC gene, size of 930 bp, was cloned by PCR and expressed in E. coli BL21(DE3). The 6-His tagged recombinant BphC was then purified and the optimum pH and temperature were pH 7.4 and 30 °C in vitro, respectively. Effects of metal ions on BphC differed from each other as Fe2+, Cu2+ and Cd2+ promoted the activity while others inhibited in varying degrees. Kinetic parameters of BphC acting on 2,3-DHBP were measured as below: Km: 8.67 mmol/L, Vmax: 27.32 μmol/s, kcat: 15.55 s–1, kcat/Km: 1.79 L/(mmol·s), respectively, of which the catalytic efficiency was much higher than some other homogeneous enzymes. [Conclusion] The gene bphC of strain YC-RL1 is vital in the process of PCBs biodegradation and the encoded enzyme BphC was revealed to be a very important aromatic ring lyase, which had high affinity to its substrates and could efficiently degrade them in vitro. This study showed a very good applicable value of bphC gene from Arthrobacter sp. strain YC-RL1.