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

Cited 17 time in wos
Cited 19 time in scopus
Title
Expression, refolding, and characterization of a small laccase from Thermus thermophilus HJ6
Other Titles
Thermus thermophilus HJ6 유래의 small laccase의 발현, refolding과 특성
Authors
Kim, Han-Woo
Jeon, Sung-Jong
Park, Hyun
이소영
Keywords
LaccaseRefoldingThermostabilityThermus thermophilus
Issue Date
2015
Citation
Kim, Han-Woo, et al. 2015. "Expression, refolding, and characterization of a small laccase from Thermus thermophilus HJ6". PROTEIN EXPRESSION AND PURIFICATION, 114: 37-43.
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 6 M 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 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 C. The optimum pH for the oxidation of 2,20-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 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.
URI
https://repository.kopri.re.kr/handle/201206/7354
DOI
http://dx.doi.org/10.1016/j.pep.2015.06.004
Type
Article
Indexed
SCI
Appears in Collections  
2014-2016, Antarctic Organisms: Cold-Adaptation Mechanism and Its Application (14-16) / Park; Hyun (PE14070; PE15070; PE16070)
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