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http://dx.doi.org/10.7841/ksbbj.2012.27.4.257

Gene Cloning and Enzymatic Properties of Thermostable Laccase from Thermus thermophilus HJ6  

Lee, So-Young (Department of Biomaterial Control (BK21 program), Dong-Eui University)
Jung, Young-Hoon (Department of Biotechnology & Bioengineering, Dong-Eui University)
Seo, Min-Ho (Department of Biotechnology & Bioengineering, Dong-Eui University)
Jeon, Sung-Jong (Department of Biomaterial Control (BK21 program), Dong-Eui University)
Publication Information
KSBB Journal / v.27, no.4, 2012 , pp. 257-262 More about this Journal
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 $90^{\circ}C$ The half-life of heat inactivation was about 120 min at $90^{\circ}C$ 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 $K_m$ value being for 4-hydroxyindole, highest $k_{cat}$ value for syringaldazine, and highest $k_{cat}/K_m$ for guaiacol.
Keywords
Laccase; Thermus thermophilus; thermostability; copper;
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