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.

Production, purification and characterization of laccase from Pleurotus ostreatus grown on tomato pomace

“…This chromatographic matrix was also useful for purification of other fungal laccases such as Coriolus versicolor and Ganoderma lucidium (data not shown). To authors' knowledge, this is the first report about one-step purification of laccase by using affinity chromatography containing either urea, acetamide or ethanolamine as affinity ligands since the literature contains several reports on laccase purification based on multi-step procedures by using ion-exchange, gel filtration chromatography and affinity chromatography on dye and Concanavalin A (Liu et al 2011;Wang and Ng 2006). However, the results presented in this purification table are in agreement with the data reported in the literature.…”

“…However, these results were obtained with different substrates for enzyme assays compared with the present work. This enzyme has been isolated from several sources by using different chromatographic techniques such as immobilized metal affinity chromatography (IMAC), affinity chromatography on Concavalin A and dye affinity chromagraphy (Rosconi et al 2005;Freixo et al 2008b;Liu et al 2011;Wang and Ng 2006). However, most of these reports involve the use of several additional purification steps such as heat treatment, ion-exchange and gel filtration chromatography which are both tedious and time-consuming (Rosconi et al 2005;Liu et al 2011;Wang and Ng 2006) For instance, laccase from Ganoderma lucidum was purified by using a multi-step isolation scheme, which involves ion exchange and gel filtration chromatography as well as affinity chromatography on dye and Concanavalin A with a final recovery of laccase activity of about 20.0% (Wang and Ng 2006).…”

Preparation of Biodegradable PLGA-Nanoparticles Used for pH-Sensitive Intracellular Delivery of an Anti-inflammatory Bacterial Toxin to Macrophages

“…Purification of the His-Tagged SubAB Oligo histidinetagged (6 × His-tagged) recombinant SubAB (His-tagged SubAB) and catalytically inactive mutant His-tagged SubA S272A B were purified by Ni-NTA agarose (Qiagen, Hilden, Germany) chromatography as previously reported. 26) Surface Modification of the PLGA NPs The PLGA NPs (100 µL) dispersion was centrifuged (12000 × g, 5 min) and the supernatant was removed. The precipitate was re-dispersed in 100 µL of a solution containing 7.7 mM NHS and 7.7 mM WSC in 20 mM phosphate buffer (pH 8.2).…”

Expression, refolding, and characterization of a small laccase from Thermus thermophilus HJ6

“…The physiological function of 64 plant and fungal laccases has been exhaustively studied and shown 65 to be related to the synthesis and degradation of lignin, respec-66 tively [16]. Among the increasing number of bacterial laccases 67 reported, several with distinctive functions have been described, 68 including roles in morphogenesis and sporulation processes, 69 pigment production, and resistance to copper and phenolic 70 compounds [12,[17][18][19] biosensing and biofuel cells [20][21][22]. 77 One of the general prerequisites for an enzyme to be applicable 78 in industrial processes is thermostability or thermotolerance [23].…”