期刊
EXTREMOPHILES
卷 9, 期 6, 页码 415-425出版社
SPRINGER JAPAN KK
DOI: 10.1007/s00792-005-0458-z
关键词
CueO; peptide mass fingerprinting; thermostability; tat system
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 similar to 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 Gln(23) to Lys(31). By signal peptide prediction, TTC1370 protein was assumed to be a secretory protein starting from Gln(23). 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 similar to 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.
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