CLONING AND EXPRESSION OF A NOVEL LACCASE GENE FROM PHYTOPHTHORA CAPSICI
B.Z. Feng, P.Q. Li
Phytophthora capsici is an aggressive plant pathogen that affects solanaceous and cucurbitaceous hosts. From this fungus, the novel laccase gene pclac1 was recovered and its corresponding full-length cDNA was cloned. The 1716 bp full-length cDNA of pclac1 encoded a mature laccase protein containing 571 amino acids. The deduced protein sequence showed high similarity with other known fungal laccases and contained four copper-binding conserved domains typical of laccase protein. Expression pattern of pclac1 in the host plant was evaluated, showing that expression levels increased until the 4th day post inoculation (dpi). Heterologous expression of PCLAC1 was achieved using the expression vector pPIC9K with the Pichia pastoris expression system. The purified recombinant PCLAC1 protein migrated as a single band in SDS-PAGE, with an apparent molecular weight of ca. 68 kDa. A high activity of purified PCLAC1 (88.46 U/ml), induced with methanol with 2,2’-azino-di-(3-ethylbenzothialozin-6-sulfonic acid) (ABTS) as substrate, was observed at the 7th dpi. The reported data add new knowledge on P. capsici laccase multigene family and shed light on the potential function of individual laccase isoforms of oomycetes for biotechnological and industrial applications.