Construction and characterization of a novel whole cell biosensor with electrocatalytic function

1在Fe(CN)_6、对苯二酚、二氯苯酚、2-甲基-5，6-二甲氧基苯醌等化合物存在下，用膜覆盖电极成功地测定了工业葡萄糖杆菌、荧光假单胞菌和乙酸乙酸杆菌完整细胞对D-葡萄糖、烟酸和乙醇的氧化反应。结果表明，完整细胞的氧化反应类似氧化还原酶，其动力学符合Michaelis-Menten方程。催化常数和米氏常数表征了单个电池的催化活性。2.在2-甲基-5，6-二胺…存在下，悬浮在缓冲溶液中的醋酸杆菌全细胞作为均相催化剂，对乙醇的氧化产生电化学催化电流更多的乙氧基苯醌(Q_0)；乙酸乙酯催化乙醇与Q_0的氧化，乙酸乙酯催化生成的还原形式的Q_0被电化学氧化生成Q_0。利用极谱动力学电流理论对催化电流进行了分析，计算出细菌细胞与Q0之间的双分子速率常数。3将完整的醋酸菌细胞固定在碳糊电极上，在Q0存在下产生催化电流。对催化电流的分析表明，细菌细胞的催化活性是由于细胞质膜中存在乙醇脱氢酶所致。将该电极作为乙醇传感器的性能与纯酶修饰的电极进行了比较。一株酒精脱氢酶缺陷菌株和携带酒精脱氢酶基因的突变株也被检测为替代醋酸曲霉的生物催化剂。类似地，荧光假单胞菌和反硝化副单胞菌分别作为构建烟酸和硝酸盐生物传感器的生物催化剂。较少

1 The oxidation reactions of D-glucose, nicotinic acid and ethanol by intact cells of Gluconobacter industrius, Pseudomonas fluorescens, and Acetobacter aceti, respectively, are successfully measured by an amperometric method using a membrane-covered electrode in the presence of such compounds as Fe(CN)_6^<3->, p-bsnzoquinone, dichlorophenolindophenol, or 2-methyl-5,6-dimethoxy benzoquinone as an electron acceptor Analysis of the experimental results reveals that the intact cells behave like oxidoreductases whose kinetics follows a Michaelis-Menten type equation. The catalytic constant and the Michaelis constant characterize the catalytic activity of a single cell. The catalytic constant is calculated to be as large as a million per second for the bacterial catalytic reactions.2 Whole cells of Acetobacter aceti suspended in a buffer solution function as homogeneous catalysts to produce an electrochemical catalytic current for the oxidation of ethanol in the presence of 2-methyl-5,6-dim … More ethoxy benzoquinone(Q_0) ; A.aceti catalyzes the oxidation of ethanol with Q_0, and the reduced form of Q_0 produced by the A.aceti catalysis is electrochemically oxidized to regenerate Q_0. The theory of polarographic kinetic current is available for analyzing the catalytic current, from which the bimolecular rate constant between the bacterial cell and Q_0 can be calculated.3 Carbon paste electrodes on which intact cells of A.aceti are immobilized produce a catalytic current for the oxidation of ethanol in the presence of Q_0. Analysis of the catalytic current reveals that the catalytic activity of the bacterial cell is due to alcohol dehydrogenase existing in the cytoplasmic membranes. Performance of the electrode as an ethanol sensor is compared with that of an electrode modified with purified enzyme. An alcohol dehydrogenase deficient strain and the mutant harboring alcohol dehydrogenase gene are also examined as biocatalyst in place of A.aceti. Similarly, Pseudomonas fluorescens and Paracoccus denitrificans serves as biocatalysts for constructing cell based biosensors for nicotinic acid and nitrate, respectively. Less