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

An open reading frame of the Thermus thermophilus HJ6 hypothetical laccase, which composed of 729 bases, was cloned and expressed as a fusion protein with six histidine residues in Escherichia coli SoluBL21(TM) cells. The resulting insoluble bodies were separated from cellular debris by centrifugation and solubilized with 6 M guanidine HCI. The solubilized protein was refolded by a simple on-column refolding procedure using Ni-chelation affinity chromatography and then the refolded protein was purified by gel filtration chromatography. It showed a single band with a molecular mass of 27 kDa in SDS-PAGE. The results from UV-visible absorption and electron paramagnetic resonance (EPR) analysis suggested that the enzyme had the typical copper sites, type-1, 2, and 3 Cu(II) of laccase. The purified enzyme exhibited the laccase activity with the optimal catalytic temperature at 75 degrees C. The optimum pH for the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and syringaldazine was 4.5 and 6.0, respectively. The recombinant protein showed high thermostability, and the half-life of heat inactivation was about 50 min at 85 degrees C. The enzyme oxidized various known laccase substrates, its lowest Km value being for syringaldazine, highest k(cat) value for guaiacol, and highest k(cat)/K-m for 2,6-dimethoxy-phenol. The enzyme reaction was strongly inhibited by the metal chelators and the thiol compounds. (C) 2015 Elsevier Inc. All rights reserved.