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KMID : 0380020120270040257
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Korean Journal of Biotechnology and Bioengineering
2012 Volume.27 No. 4 p.257 ~ p.262
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Gene Cloning and Enzymatic Properties of Thermostable Laccase from Thermus thermophilus HJ6
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Lee So-Young
Jung Young-Hoon
Seo Min-Ho
Jeon Sung-Jong
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Abstract
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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℃ The half-life of heat inactivation was about 120 min at 90℃ 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 Km value being for 4-hydroxyindole, highest kcat value for syringaldazine, and highest kcat/Km for guaiacol.
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KEYWORD
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Laccase, Thermus thermophilus, thermostability, copper
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