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Braz. j. microbiol ; Braz. j. microbiol;45(1): 351-358, 2014. ilus
Article de Anglais | LILACS | ID: lil-709455

RÉSUMÉ

Laccases are blue copper oxidases (E.C. 1.10.3.2) that catalyze the one-electron oxidation of phenolics, aromatic amines, and other electron-rich substrates with the concomitant reduction of O2 to H2O. A novel laccase gene pclac2 and its corresponding full-length cDNA were cloned and characterized from Phytophthora capsici for the first time. The 1683 bp full-length cDNA of pclac2 encoded a mature laccase protein containing 560 amino acids preceded by a signal peptide of 23 amino acids. The deduced protein sequence of PCLAC2 showed high similarity with other known fungal laccases and contained four copper-binding conserved domains of typical laccase protein. In order to achieve a high level secretion and full activity expression of PCLAC2, expression vector pPIC9K with the Pichia pastoris expression system was used. The recombinant PCLAC2 protein was purified and showed on SDS-PAGE as a single band with an apparent molecular weight ca. 68 kDa. The high activity of purified PCLAC2, 84 U/mL, at the seventh day induced with methanol, was observed with 2,2'-azino-di-(3-ethylbenzothialozin-6-sulfonic acid) (ABTS) as substrate. The optimum pH and temperature for ABTS were 4.0 and 30 ºC, respectively . The reported data add a new piece to the knowledge about P. Capsici laccase multigene family and shed light on potential function about biotechnological and industrial applications of the individual laccase isoforms in oomycetes.


Sujet(s)
Laccase/génétique , Laccase/métabolisme , Phytophthora/enzymologie , Clonage moléculaire , Séquence conservée , Stabilité enzymatique , Expression des gènes , Concentration en ions d'hydrogène , Laccase/composition chimique , Laccase/isolement et purification , Masse moléculaire , Cadres ouverts de lecture , Structure tertiaire des protéines , Phytophthora/génétique , Pichia/génétique , Signaux de triage des protéines/génétique , Protéines recombinantes/composition chimique , Protéines recombinantes/génétique , Protéines recombinantes/isolement et purification , Protéines recombinantes/métabolisme , Similitude de séquences d'acides aminés , Température
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