The electrochemical properties of a laccase from Thermus thermophilus HB27 (Tth-laccase) were characterized. The gene encoding the laccase was cloned and overexpressed in Escherichia coli. One-step purification of the corresponding apo-enzyme was achieved by nickel-affinity chromatography. Copper was incorporated into the apo-laccase as the cofactor to yield the holo-enzyme. The temperature-dependent catalytic activity of the laccase was investigated by spectrophotometric as well as electrochemical methods. Specifically, the catalytic properties of the enzyme were characterized by employing a photometric assay based on the oxidation of the substrate 2,2-azino-bis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS). The electroactive substrate ABTS can be also monitored by cyclic voltammetry, thus allowing for determination of the enzymatic activity electrochemically. It was found that the recombinant laccase exhibited higher activity as the temperature increased up to 65 °C. Spectroscopic studies of Tth-laccase based on circular dichroism and fluorescence measurements are consistent with a thermally stable secondary structure of the protein.
A salicylate‐selective electrode based on calix[4]arene derivative was developed and its response characteristics were investigated. The optimum membrane composition was 1 % ionophore, 30 % PVC, 69 % DOS. The electrode exhibited a Nernstian slope of 58.8±0.5 mV/pSal in the range of 1.0×10−5–1.0×10−1 M with a detection limit of 4.3×10−6 M at pH 4.0, 20±1 °C. The potentiometric response of the electrode in the presence of different anions was investigated by the separate solution method. The lifetime was found at least 4 months, and its response time was 5–10 s. It was successfully used for the potentiometric determination of salicylate in pharmaceutical preparations.
Knowledge of the protonation constants of small dipeptide is important, interesting and necessary for complete understanding of the physiochemical behavior of dipeptide. In this study, the protonation constants of some aliphatic dipeptides (Gly-Gly, Gly-Val, Gly-Leu, Gly-Thr, Gly-Phe and Gly-Met) were studied in water and ethanol-water mixtures [20% ethanol-80% water, 40% ethanol-60% water, 60% ethanol-40% water, (v/v)] at 25 +/- 0.1 degrees C under nitrogen atmosphere and ionic strength at 0.10 mol dm(-3) by potentiometry. The constants of the systems were calculated by using BEST computer program, and distribution species diagrams were produced using the SPE computer program. The protonation constants were influenced by changes in solvent composition, and their variations were discussed in terms of solvent and structural properties. The concentration distribution of the various species in ethanol-water mixtures was evaluated.
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