title: Electrochemical properties and temperature dependence of a recombinant laccase from Thermus thermophilus.
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.
authors.affiliation: Department of Chemistry, University of Kentucky, Lexington, KY 40506-0055, USA.
authors.forename: Xin
authors.initials: X
authors.lastname: Liu
authors.forename: Megan
authors.initials: M
authors.lastname: Gillespie
authors.forename: Ayca Demirel
authors.initials: AD
authors.lastname: Ozel
authors.forename: Emre
authors.initials: E
authors.lastname: Dikici
authors.forename: Sylvia
authors.initials: S
authors.lastname: Daunert
authors.forename: Leonidas G
authors.initials: LG
authors.lastname: Bachas
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D042845:Laccase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D042845:Laccase