Cloning, characterization and expression of a novel laccase gene Pclac2 from Phytophthora capsici
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 f...
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Sociedade Brasileira de Microbiologia
2014-01-01
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doaj-9ea892c706644e968944f37ce67604282020-11-24T22:51:17ZengSociedade Brasileira de MicrobiologiaBrazilian Journal of Microbiology1678-44052014-01-01451351358S1517-83822014000100050Cloning, characterization and expression of a novel laccase gene Pclac2 from Phytophthora capsiciBao Zhen Feng0Peiqian Li1Yuncheng UniversityYuncheng UniversityLaccases 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.http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822014000100050&lng=en&tlng=enPhytophthora capsicilaccaseexpressionpurificationactivity |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Bao Zhen Feng Peiqian Li |
spellingShingle |
Bao Zhen Feng Peiqian Li Cloning, characterization and expression of a novel laccase gene Pclac2 from Phytophthora capsici Brazilian Journal of Microbiology Phytophthora capsici laccase expression purification activity |
author_facet |
Bao Zhen Feng Peiqian Li |
author_sort |
Bao Zhen Feng |
title |
Cloning, characterization and expression of a novel laccase gene Pclac2 from Phytophthora capsici |
title_short |
Cloning, characterization and expression of a novel laccase gene Pclac2 from Phytophthora capsici |
title_full |
Cloning, characterization and expression of a novel laccase gene Pclac2 from Phytophthora capsici |
title_fullStr |
Cloning, characterization and expression of a novel laccase gene Pclac2 from Phytophthora capsici |
title_full_unstemmed |
Cloning, characterization and expression of a novel laccase gene Pclac2 from Phytophthora capsici |
title_sort |
cloning, characterization and expression of a novel laccase gene pclac2 from phytophthora capsici |
publisher |
Sociedade Brasileira de Microbiologia |
series |
Brazilian Journal of Microbiology |
issn |
1678-4405 |
publishDate |
2014-01-01 |
description |
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. |
topic |
Phytophthora capsici laccase expression purification activity |
url |
http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822014000100050&lng=en&tlng=en |
work_keys_str_mv |
AT baozhenfeng cloningcharacterizationandexpressionofanovellaccasegenepclac2fromphytophthoracapsici AT peiqianli cloningcharacterizationandexpressionofanovellaccasegenepclac2fromphytophthoracapsici |
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1725670515800014848 |