A hyperthermophilic laccase from Thermus thermophilus HB27.
A copper-inducible laccase activity was detected in Thermus thermophilus HB27. The enzyme was partially purified and separated by SDS-PAGE. After staining, a gel slice containing a approximately 53-kDa protein was excised and treated with trypsin, and the in-gel digests were analyzed by mass spectrometry. By mass fingerprinting, the peptides were found to share identity with the TTC1370 protein of the thermophile, which was tentatively annotated as a laccase in the whole genome analysis, albeit experimental evidence was lacking. The assigned mass nearest to the N-terminal sequence was that from Gln23 to Lys31. By signal peptide prediction, TTC1370 protein was assumed to be a secretory protein starting from Gln23. The DNA encoding the mature protein was then cloned and expressed in Escherichia coli. The recombinant enzyme, expressed as an apoprotein, was dialyzed against copper-containing buffer to yield a holoprotein. The holoprotein was purified to homogeneity, which displayed a blue color typical of laccases and oxidized canonical laccase substrates such as guaiacol and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate). The enzyme was most notable for its striking thermophilicity; the optimal reaction temperature was approximately 92 degrees C and the half-life of thermal inactivation at 80 degrees C was >14 h, ranking it as the most thermophilic laccase reported thus far.
Miyazaki
Kentaro
K
Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan. miyazaki-kentaro@aist.go.jp
D000595:Amino Acid Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D019327:Copper Sulfate / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000378:metabolism; D016678:Genome; D005973:Glutamine / Q000737:chemistry; D006358:Hot Temperature; D006835:Hydrazones / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D042845:Laccase / Q000737:chemistry*; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010100:Oxygen / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013053:Spectrophotometry; D013379:Substrate Specificity; D013696:Temperature; D016963:Thermus thermophilus / Q000201:enzymology*; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology; D014466:Ultraviolet Rays
D006835:Hydrazones; D010455:Peptides; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D005973:Glutamine; C009370:syringaldazine; D004247:DNA; D042845:Laccase; D014357:Trypsin; D008239:Lysine; D019327:Copper Sulfate; D010100:Oxygen