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

1 .利用膜覆盖电极，采用电流法成功地测量了工业葡萄杆菌、荧光假单胞菌和乙酰杆菌的完整细胞在Fe(CN)_6^<3->、对苯二氮醌、二氯酚吲哚酚等化合物存在下对d -葡萄糖、烟酸和乙醇的氧化反应。实验结果分析表明，完整的细胞表现为氧化还原酶，其动力学遵循Michaelis-Menten型方程。催化常数和米切里斯常数表征了单个细胞的催化活性。据计算，细菌催化反应的催化常数可达每秒一百万次在2-甲基-5,6-dim . More乙氧基苯醌（Q_0）存在的情况下，悬浮在缓冲溶液中的乙酰乙杆菌（Acetobacter aceti）细胞作为均相催化剂产生电化学催化电流氧化乙醇；a.b hii用Q_0催化乙醇氧化，由a.b hii催化产生的还原物Q_0经电化学氧化再生Q_0。极谱动力学电流理论可用于分析催化电流，由此可以计算出细菌细胞与Q_0之间的双分子速率常数将完整的乙酰a.b i细胞固定在碳糊电极上，在Q_0存在的情况下产生乙醇氧化的催化电流。催化电流分析表明，细菌细胞的催化活性是由存在于细胞质膜上的醇脱氢酶引起的。将该电极作为乙醇传感器的性能与用纯化酶修饰的电极进行了比较。乙醇脱氢酶缺陷菌株和携带乙醇脱氢酶基因的突变体也被检测为替代乙酰a.b乙酰的生物催化剂。同样，荧光假单胞菌和反硝化副球菌分别作为构建基于细胞的烟酸和硝酸盐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