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.

Expression, purification, and characterization of a novel laccase from Setosphaeria turcica in Eschericha coli

“…In this study, the StLAC4 gene was successfully expressed in E. coli for the first time and the maximum activity of the purified laccase reached 127.78 U · mg −1 at a lower production cost. The results showed the optimum pH for Stlac4 is 4.0, which is similar to most of the studied fungal laccases , but the optimum temperature is 60 °C, which is significantly higher than most . If this advantage can be enhanced , it may improve the potential of Stlac4 in industrial applications.…”

“…Laccases (EC 1.10.3.2) are a group of multicopper‐containing oxidases (MCOs) which can oxidize a range of aromatic compounds such as phenols and aromatic amines . Laccases are wide‐spread in nature and over 220 species of laccases have been isolated from many sources,including plants, fungi, bacteria, and insects . Fungal laccases are the most extensively studied multicopper oxidases, and while primarily considered to be involved in lignin degradation , recent studies have shown that fungal laccases participate in fruiting body formation , pigment biosynthesis , pathogenesis, and stress defense .…”

Purification and Characterization of a White Laccase with Pronounced Dye Decolorizing Ability and HIV-1 Reverse Transcriptase Inhibitory Activity from Lepista nuda

“…However, most of the laccases studied so far originated from fungal species and the laccase activity has been demonstrated in Basidiomycetes, Ascomycetes and Deuteromycetes [4,5]. In fungi, laccases have more diverse physiological functions such as sporulation, pigment production, fruit body formation and plant pathogenesis [6,7,8].…”

The L-type Ca2+ Channel Blocker Nifedipine Inhibits Mycelial Growth, Sporulation, and Virulence of Phytophthora capsici

“…Recent studies of the biological function of fungal laccases suggest that this enzyme plays an important role in fungal morphogenesis and fungal virulence ( Li et al, 2013 ). In P. capsici , pectate lyase and laccase activities are important for successful infection during plant–pathogen interactions ( Feng and Li, 2014 ; Fu et al, 2015 ). Laccases, which served as blue copper oxidases, catalyze the one-electron oxidation (e.g., aromatic amines and phenolics) and other electron-rich substrates; there also has a reduction of O 2 to H 2 O concomitantly.…”