Cloning and heterologous expression of a laccase isozyme gene from Coriolopsis polyzona MUCL 38443

Laccases (EC 1.10.3.2) are enzymes which catalyze oxidation of phenolic and non-phenolic lignin-related compounds coupled with reduction of oxygen to water. Laccases belongs to a protein superfamily which includes multicopper oxidases (MCO). Laccases are highly interested enzymes as a result of their beneficial applications in detoxification of industial effluents from the paper, pulp and textile industries and bioremediation of the herbicides and pesticides along with their applications in beverage industry and medical diagnosis. Three different partial cDNA sequences, Cplcc1, Cplcc2 and Cplcc3, which belongs to laccase isozymes from white-rot fungus Coriolopsis polyzona MUCL 38443 strain were previously identified by our research group. These partial sequences showed around 80% similarity via with laccase-encoding sequences, which were previously submitted to National Center for Biotechnology Information (NCBI) database, via BLAST analysis. Subsequently, first, full-lenght Cplcc1 cDNA and, next, full-length Cplcc2 cDNA were isolated and characterized in our group. In the present study, characterization of full-length cDNA belongs to Cplcc3 partial cDNA sequences, cloning of the full-length gene into yeast expression vector pPICZB and heterologous expression of the protein were aimed. Initially, cDNA ends belong to partial Cplcc3 cDNA were characterized. With this characterization, it was clarified that this partial cDNA belongs to Cplcc2 gene of Coriolopsis polyzona. After that, full-length Cplcc2 open-reading frame was cloned into the yeast shuttle expression vector pPICZB with its own secretion signal sequence. Cplcc2 was heterologously expressed in methylotrophic yeast Pichia pastoris. CpLcc2 expression and secretion in yeast transformants were, first-screened in the expression-inducing media. CpLcc2-expressing transformants were, then screened in methanol-containing broth media in order to select best enzmye-producing transformant after applying laccase assay to the daily-collected samples for 3 days. Moreover, expression conditions of CpLcc2 was optimized to achieve production of laccase enzymes at highest level. In order to do so, cultivation conditions, methanol concentration, copper concentration, L-Alanine concentration and growth temperature were optimized. Optimum conditions for CpLcc2 expression were determined as 1.5% methanol, 0.8 mM CuSO4, 0.4% L-alanine at 28°C with 250 rpm shaking and 7 days of incubation. Optimal expression conditions were found to be similar to the recombinant CpLcc1 laccase previously isolated from Coriolopsis polyzona MUCL 38443. Maximum CpLcc2 laccase activity was found as 1159.041 U/L, which was higher than the recombinant Cplcc1 (800U/l). Molecular weight of Cplcc2 was determined according to SDS-PAGE analysis as 72.4 kDa, which is higher than hypothetical molecular weight of enzyme due to glycosylation in the expression host. Most probably, this glycosylation was stated as the reason for higher activity of CpLcc2 than CpLcc1. Molecular weight of recombinant Cplcc2 was found consistent with laccase from Coriolopsis polyzona WR710-1. Level of glycosylation was found more than most fungal laccases.