联苯利用节杆菌YC-RL1中2,3-二羟基联苯-1,2-双加氧酶BphC功能验证

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

摘要:

【目的】通过节杆菌(Arthrobacter sp.) YC-RL1对多氯联苯降解过程中关键基因bphC的克隆与原核表达，鉴定其编码的2,3-二羟基联苯-1,2-双加氧酶BphC的酶活特性与功能。【方法】以菌株YC-RL1全基因组为模板进行PCR扩增获得bphC基因，将该基因转入Escherichia coli BL21(DE3)感受态细胞后进行原核表达；利用镍柱亲和层析法对BphC酶进行纯化并分别测定该酶在不同条件下对底物2,3-DHBP的催化特性，确定其最适反应pH、温度及不同金属离子对酶活特性的影响；进一步根据米氏方程对该酶的动力学参数进行测定与分析。【结果】通过PCR扩增获得了bphC基因，其大小为930 bp；对该基因进行原核表达，所得重组蛋白BphC携带有6个组氨酸标签，经纯化后体外仍具有活性，该酶作用于2,3-DHBP时的最适pH与温度分别为pH 7.4和30 °C，且在最适条件下，Fe2+、Cu2+及Cd2+等金属离子可明显促进其酶活作用，但多数金属离子对该酶有不同程度的抑制作用；该酶在与底物2,3-DHBP作用过程中，酶促动力学常数分别为Km：8.67 mmol/L，Vmax：27.32 μmol/s，kcat：15.55 s–1，kcat/Km：1.79 L/(mmol·s)，其催化效率同有关报道中同类酶的动力学特性比较均有所提高。【结论】菌株YC-RL中的bphC基因对于多氯联苯的生物降解具有至关重要的作用，其编码的BphC是重要的芳香环裂解酶，该酶对其底物具有较高的亲和性，可在体外环境中发挥高效的酶促作用，具有良好的应用价值。

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.