厌氧氨氧化细菌铜氧化酶异源表达及酶学分析

Heterologous Expression and Enzymatic Analysis of Copper Oxidase of Anammox Bacteria

厌氧氨氧化细菌是在地球生物氮循环过程中具有重要意义的一类微生物,利用其脱氮性能发展的厌氧氨氧化工艺是目前最具前景的污水生物脱氮技术,但是厌氧氨氧化细菌对环境变化敏感限制了实际工程的应用,因此有必要研究参与厌氧氨氧化细菌应激机制的功能蛋白。铜氧化酶是细菌中参与细胞应激反应的一类蛋白,能够帮助细菌抵抗多种因素(如过氧化物、金属离子等)对细胞的侵害。该研究克隆了来自厌氧氨氧化细菌的铜氧化酶基因,并将其转入大肠杆菌BL21(DE3)中过量表达,培养温度30℃,异丙基-β-D-硫代半乳糖苷终浓度0.2 mmol/L为最佳诱导条件。对以包涵体形式存在的铜氧化酶进行纯化,经10%变性聚丙烯酰胺凝胶电泳分析,得知目的蛋白分子质量约为35 kD。优化了铜氧化酶的复性条件,获得了具有漆酶活性的目的蛋白,针对漆酶底物2,2'-联氮双(3-乙基苯并噻唑啉-6-磺酸)二铵盐的最适pH值是3,最适温度是35℃,在最适反应条件下,K_(m)为1538.91μmol/L,V_(max)为17.76μmol/(L·min),k_(cat)为1.09 s^(-1)。该研究为该类铜氧化酶在厌氧氨氧化细菌细胞应激过程中可能发挥的功能提供了新线索。

Anammox bacteria are of great significance in the biological nitrogen cycle of the earth.The anammox process developed by the unique performance of anammox bacteria is currently the most promising sewage treatment.However,sensitivity to changes in the surrounding environment is one of the limiting factors in the engineering application of anammox bacteria.Therefore,it is necessary to study the functional proteins that may participate in the stress process of anammox bacteria.Copper oxidase is a type of protein involved in cell stress response in bacteria,which can help bacteria resist the damage of cells by various factors(such as peroxides,metal ions,etc.).The copper oxidase gene was cloned from anammox bacteria and overexpressed in E.coli BL21(DE3).The best induction conditions is 30℃,the final concentration of IPTG is 0.2 mmol/L.In this study,the copper oxidase was purified from inclusion bodies and about 35 kD protein was detected on 10%denaturing polyacrylamide gel.A large number of target proteins with laccase activity were obtained by optimizing the renaturation conditions.The copper oxidase showed optimal laccase activity to ABTS at pH 3 and 35℃.Under the optimal reaction conditions,K_(m) is 1538.91μmol/L,V_(max) is 17.76μmol/(L·min),and k_(cat) is 1.09 s^(-1).This study provides new clues for the possible function of the copper oxidase in stress process of anammox bacteria cells.