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

作　　者：乔铖 任磊[1] 樊双虎[1] 贾阳[1] 赵百锁[1] 闫艳春[1] 

机构地区：[1]中国农业科学院研究生院,北京100081

出　　处：《微生物学通报》2017年第7期1639-1648,共10页Microbiology China

基　　金：国家自然科学基金项目(No.31170119;31540067);中国农业科学院基本科研业务基金项目(No.0042012003;0042014011)~~

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bph C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Bph C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bph C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Bph C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,Fe^(2+)、Cu^(2+)及Cd^(2+)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K_m:8.67 mmol/L,V_(max):27.32μmol/s,k_(cat):15.55 s^(–1),k_(cat)/K_m: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。[Objectivel 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 genorne 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 ℃ 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： Kin： 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 ofbphC gene from Arthrobacter sp. strain YC-RLI.

关 键 词：多氯联苯 生物降解 2 3-二羟基联苯-1 2-双加氧酶 酶促动力学 

分 类 号：Q55[生物学—生物化学] Q78