Liucija Marcinkeviciene

Regulation of a Novel Acidithiobacillus caldus Gene Cluster Involved in Metabolism of Reduced Inorganic Sulfur Compounds

ABSTRACT Acidithiobacillus caldus has been proposed to play a role in the oxidation of reduced inorganic sulfur compounds (RISCs) produced in industrial biomining of sulfidic minerals. Here, we describe the regulation of a new cluster containing the gene encoding tetrathionate hydrolase (tetH), a key enzyme in the RISC metabolism of this bacterium. The cluster contains five cotranscribed genes, ISac1, rsrR, rsrS, tetH, and doxD, coding for a transposase, a two-component response regulator (RsrR and RsrS), tetrathionate hydrolase, and DoxD, respectively. As shown by quantitative PCR, rsrR, tetH, and doxD are upregulated to different degrees in the presence of tetrathionate. Western blot analysis also indicates upregulation of TetH in the presence of tetrathionate, thiosulfate, and pyrite. The tetH cluster is predicted to have two promoters, both of which are functional in Escherichia coli and one of which was mapped by primer extension. A pyrrolo-quinoline quinone binding domain in TetH was predicted by bioinformatic analysis, and the presence of an o-quinone moiety was experimentally verified, suggesting a mechanism for tetrathionate oxidation.

Reagentless biosensors based on co-entrapment of a soluble redox polymer and an enzyme within an electrochemically deposited polymer film

A novel biosensor architecture, which is based on the combination of a manual and a non-manual deposition technique for sensor components on the electrode surface is reported. A water-soluble Os-poly(vinyl-imidazole) redox hydrogel is deposited on a graphite electrode by drop-coating (i.e. manually) followed by the electrochemically-induced deposition of an enzyme-containing non-conducting polymer film. The local polymer deposition is initiated by electrochemical generation of H(3)O(+) exclusively at the electrode surface causing a pH-shift to be established in the diffusion zone around the electrode (i.e. non-manually). This pH-shift leads to the protonation of a dissolved polyanionic polymer which in consequence changes significantly its solubility and is hence precipitating on the electrode surface. In the presence of a suitable enzyme, such as quinohemoprotein alcohol dehydrogenase (QH-ADH), the polymer precipitation leads to an entrapment of the redox enzyme within the polymer film. Simultaneously, the water-soluble Os-poly(vinyl-imidazole) redox hydrogel, which is slowly dissolving from the electrode surface after addition of the electrolyte, is co-entrapped within the precipitating polymer layer. This provides the pre-requisite for an efficient electron-transfer pathway from the redox enzyme via the polymer-bound redox centres to the electrode surface. The sensor preparation protocol has been optimised aiming on a high mediator concentration in the polymer film and an effective electron transfer.

An oxygen-independent ethanol sensor based on quinohemoprotein alcohol dehydrogenase covalently bound to a functionalized polypyrrole film

In the present work the characteristics of a phenazine methosulphate mediated alcohol biosensor based on a newly isolated quinohemoprotein alcohol dehydrogenase are described. The enzyme was covalently linked at a functionalized polypyrrole film which had been electrochemically deposited on the surface of a platinum-black electrode. The biosensor architecture developed was characterized with regard to sensitivity, selectivity, and long-term operational stability. Owing to the inherent properties of the new enzyme the related biosensors are oxygen-independent and exhibit improved selectivity to ethanol in contrast to alcohol biosensors based on alcohol oxidase or on cationic nicotinamide adenine dinucleotide dependent alcohol dehydrogenase.