Electrochemical properties and temperature dependence of a recombinant laccase from Thermus thermophilus
Open Access
- 14 November 2010
- journal article
- research article
- Published by Springer Science and Business Media LLC in Analytical and Bioanalytical Chemistry
- Vol. 399 (1), 361-366
- https://doi.org/10.1007/s00216-010-4345-9
Abstract
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.This publication has 26 references indexed in Scilit:
- Kinetic studies of laccase enzyme of Coriolus versicolor MTCC 138 in an inexpensive culture mediumBiochemical Engineering Journal, 2009
- Nanostructured carbon electrodes for laccase-catalyzed oxygen reduction without added mediatorsElectrochimica Acta, 2008
- Stabilization studies of Fomes sclerodermeus laccasesBioresource Technology, 2008
- Crystal structures of E. coli laccase CueO at different copper concentrationsBiochemical and Biophysical Research Communications, 2007
- Production, purification and partial characterisation of a novel laccase from the white-rot fungus Panus tigrinus CBS 577.79Antonie van Leeuwenhoek, 2006
- How to study proteins by circular dichroismBiochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2005
- The Structure of Rigidoporus lignosus Laccase Containing a Full Complement of Copper Ions, Reveals an Asymmetrical Arrangement for the T3 Copper PairJournal of Molecular Biology, 2004
- Crystal structure of the type-2 Cu depleted laccase from Coprinus dnereus at 2.2 Å resolutionNature Structural & Molecular Biology, 1998
- Multicopper Oxidases and OxygenasesChemical Reviews, 1996
- The structure and function of fungal laccasesMicrobiology, 1994