Abstract
The gene encoding Thermus thermophilus HJ6 laccase (Tt-laccase) was cloned, sequenced, and comprised of 1,389 nucleotides encoding a protein (462 amino acids) with a predicted molecular mass of 51,049 Da. The deduced amino acid sequence of Tt-laccase showed 99.7% and 44.3% identities to the Thermus thermophilus HB27 laccase and Synechococcus sp. RS9917 laccase, respectively. Tt-laccase gene was expressed as a fusion protein with six histidine residues in E. coli Rosetta-gami (DE3) cells, and the recombinant protein was purified to homogeneity. UV-Vis spectrum analysis revealed that the enzyme has copper atoms, a type I Cu(II) and a type III binuclear Cu(II). The optimum pH for the oxidation of guaiacol was 5.0 and the optimum temperature was <TEX>$90^{\circ}C$</TEX> The half-life of heat inactivation was about 120 min at <TEX>$90^{\circ}C$</TEX> The enzyme reaction was inhibited by sodium azide, L-cystein, EDTA, dithiothreitol, tropolone, and kojic acid. The enzyme oxidized various known laccase substrates, its lowest <TEX>$K_m$</TEX> value being for 4-hydroxyindole, highest <TEX>$k_{cat}$</TEX> value for syringaldazine, and highest <TEX>$k_{cat}/K_m$</TEX> for guaiacol.
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