中文摘要：

已知源于枯草芽孢杆菌内生孢子的Cot A蛋白具有漆酶和胆红素氧化酶活性。然而,其分离纯化极为困难。本研究对表达与纯化的重组Cot A蛋白的胆红素氧化酶特性及氧化还原功能进行鉴定。基因转染及筛选获得了表达Cot A的P.pastoris菌株;继而,表达的重组Cot A蛋白经DEAESepharose FF及Sephadex G-75层析分离与纯化,产物得率为25%,纯化产物的酶比活性为4 U/mg。经SDS-PAGE和MALDI-TOF MS分析显示,其分子质量为65 k D。纯化的Cot A蛋白能够催化胆红素氧化,生成胆绿素,且催化反应速率受反应溶液中溶解氧含量的影响,提示纯化的重组CotA具有胆红素氧化酶活性。酶反应进一步证明,Cot A的胆红素氧化酶反应最适p H值为p H 8.0,最适温度为60℃。该酶在90℃条件下的半衰期为7 h,提示Cot A胆红素氧化酶具有高度的热稳定性。Cot A修饰的摄谱仪石墨电极可直接电催化分子氧（O2）还原,具有很好的电流响应。我们的结果表明,重组的Cot A蛋白具有耐高温胆红素氧化酶活性。更重要的是,我们的结果还提示重组的Cot A蛋白在酶生物燃料电池阴极的制备上具有较好的应用潜能。

英文摘要：

Cot A protein from B.subtilis endospore has been shown to function as laccase and bilirubin oxidase.This natural protein,however,is very difficult to be isolated from the endospore protein complex.In this study,we described the enzymatic kinetics and redox function of thermostable recombinant Cot A as bilirubin oxidase,which was obtained by recombinant DNA technology.The P.pastoris transformant expressing recombinant Cot A protein was obtained by transformation and screening.The recombinent protein was isolated and purified by the combination of DEAE-Sepharose FF chromatography with Sephadex G-75 chromatography with a yield of 25%.The specific activity of the purified protein was 4 U/mg.SDS-PAGE and MALDI-TOF MS analyses revealed that the molecular weight of the protein was 65 k D.The purified Cot A could catalyze the oxidation of bilirubin to biliverdin,whose reaction rate was affected by the concentration of oxygen dissolved in the reaction solution,indicating that the recombinant Cot A possesses bilirubin oxidase activity.Furthermore,enzymatic reaction showed that the optimal p H and temperature for the reactions catalyzed by Cot A/bilirubin oxidase were 8.0 and 60℃,respectively.In addition,the half-life of the Cot A/bilirubin oxidase was about 7hours at 90℃,indicating that recombinant Cot A was a highly thermostable bilirubin oxidase.The direct bioelectrocatalytic reduction of O2 was achieved on Cot A modified spectrographic graphite electrode displayed a good current response.These data suggest that the recombinant Cot A has catalytic activity of thermostable bilirubin oxidase.More importantly,these data also indicate potential application of recombinant Cot A in preparation of cathode in enzymatic biofuel cells.