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

Protein Expr Purif. 2015 Oct:114:37-43. doi: 10.1016/j.pep.2015.06.004. Epub 2015 Jun 11.

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.

Keywords: Laccase; Refolding; Thermostability; Thermus thermophilus.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / isolation & purification
  • Bacterial Proteins / metabolism*
  • Enzyme Stability
  • Escherichia coli / genetics
  • Laccase / chemistry*
  • Laccase / genetics
  • Laccase / isolation & purification
  • Laccase / metabolism*
  • Molecular Sequence Data
  • Protein Refolding
  • Recombinant Proteins / chemistry*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism*
  • Sequence Alignment
  • Thermus thermophilus / enzymology*
  • Thermus thermophilus / genetics

Substances

  • Bacterial Proteins
  • Recombinant Proteins
  • Laccase