title: Cloning, characterization and expression of a novel laccase gene Pclac2 from Phytophthora capsici.
abstract: 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 °C, 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.
authors.affiliation: Department of Life Sciences Yuncheng University Yuncheng China.
authors.forename: Bao Zhen
authors.initials: BZ
authors.lastname: Feng
authors.affiliation: Department of Life Sciences Yuncheng University Yuncheng China.
authors.forename: Peiqian
authors.initials: P
authors.lastname: Li
mesh_terms: D003001:Cloning, Molecular; D017124:Conserved Sequence; D004795:Enzyme Stability; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D042845:Laccase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D016366:Open Reading Frames; D010838:Phytophthora / Q000201:enzymology* / Q000235:genetics; D010843:Pichia / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013696:Temperature
chemical_list: D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D042845:Laccase
keywords: Phytophthora capsici; activity; expression; laccase; purification