title: Expression, refolding, and characterization of a small laccase from Thermus thermophilus HJ6.
abstract: 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™ cells. The resulting insoluble bodies were separated from cellular debris by centrifugation and solubilized with 6M guanidine HCl. 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 27kDa 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°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 50min at 85°C. The enzyme oxidized various known laccase substrates, its lowest Km value being for syringaldazine, highest kcat value for guaiacol, and highest kcat/Km for 2,6-dimethoxy-phenol. The enzyme reaction was strongly inhibited by the metal chelators and the thiol compounds.
authors.affiliation: Division of Life Sciences, Korea Polar Research Institute (KOPRI), Korea University of Science and Technology, 26, Songdomirae-ro, Incheon 406-840, Republic of Korea; Department of Polar Sciences, Korea University of Science and Technology, 26, Songdomirae-ro, Incheon 406-840, Republic of Korea.
authors.forename: Han-Woo
authors.initials: HW
authors.lastname: Kim
authors.affiliation: Department of Smart-Biohealth, Dong-Eui University, Busan 614-714, Republic of Korea.
authors.forename: So-Yeong
authors.initials: SY
authors.lastname: Lee
authors.affiliation: Division of Life Sciences, Korea Polar Research Institute (KOPRI), Korea University of Science and Technology, 26, Songdomirae-ro, Incheon 406-840, Republic of Korea; Department of Polar Sciences, Korea University of Science and Technology, 26, Songdomirae-ro, Incheon 406-840, Republic of Korea.
authors.forename: Hyun
authors.initials: H
authors.lastname: Park
authors.affiliation: Department of Smart-Biohealth, Dong-Eui University, Busan 614-714, Republic of Korea; Department of Biotechnology & Bioengineering, Dong-Eui University, Busan 614-714, Republic of Korea. Electronic address: jeon.sj@deu.ac.kr.
authors.forename: Sung-Jong
authors.initials: SJ
authors.lastname: Jeon
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D042845:Laccase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D042845:Laccase
keywords: Laccase; Refolding; Thermostability; Thermus thermophilus