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

1.在Fe（CN）_6 ~+、对苯醌、二氯酚靛酚、2-甲基-5，6-二氯酚靛酚等化合物存在下，用膜电极安培法成功地测定了工业葡萄糖杆菌、荧光假单胞菌和醋酸杆菌的完整细胞对D-葡萄糖、烟酸和乙醇的氧化反应<3->。6-二甲氧基苯醌作为电子受体对实验结果的分析表明，完整细胞的行为类似于氧化还原酶，其动力学遵循Michaelis-Menten型方程。催化常数和米氏常数表征单个细胞的催化活性。2悬浮在缓冲溶液中的醋酸杆菌全细胞作为均相催化剂，在2-甲基-5，6-二氢呋喃存在下产生乙醇氧化的电化学催化电流 关于我们 乙氧苯醌（Q_0）;乙酰曲霉催化Q_0氧化乙醇，并且由乙酰曲霉催化产生的还原形式的Q_0被电化学氧化以再生Q_0。用极谱动力学电流理论分析催化电流，计算出细菌细胞与Q_0之间的双分子速率常数。3固定化完整的醋酸杆菌细胞的碳糊电极在Q_0存在下对乙醇的氧化产生催化电流。对催化电流的分析表明，细菌细胞的催化活性是由于存在于细胞质膜上的乙醇脱氢酶。作为乙醇传感器的电极的性能进行了比较与纯化的酶修饰的电极。醇脱氢酶缺陷型菌株和含有醇脱氢酶基因的突变体也被检测为代替醋酸曲霉的生物催化剂。类似地，荧光假单胞菌和嗜酸副球菌分别作为构建烟酸和硝酸盐的基于细胞的生物传感器的生物催化剂。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