Efficient secretion of a true laccase by the acidophilic green microalga Chlamydomonas pitschmannii switched to alkaline pH

Laccases are extracellular multicopper oxidases that react with a broad range of aromatic substrates and are used for many biotechnological applications. Recently, microalgal laccases were discovered and started to be characterized in some Chlorophyceae (Tetracystis aeria and a few other species). A volumetric activity of maximum 27 U L-1 could be reached with Chlamydomonas moewusii, which is largely insufficient to compete with fungi and heterologous expression systems. Here, the acidophilic green microalga Chlamydomonas pitschmannii was shown to be a new promising source of laccase competing with fungi in terms of secretion yields. By modulating culture pH and CO2 supply, laccase volumetric activity in the supernatant could be increased from 12 to 459 U L-1. Laccase-containing supernatants were found to oxidize many phenolic and non-phenolic substrates with an efficiency that was pH-dependent. 1D SDS-PAGE was achieved and lighted up that C. pitschmannii laccase is a glycosylated protein. Functional measurements pointed out the participation of glycosylation in functional enzymatic regulation and integrity. Two bands of about 50 and 45 kD were detected that presumably corresponded to laccase. In agreement with previous studies in T. aeria, we hypothesize that C. pitschmannii laccase is a heterodimer, a feature that denotes with most known laccases, essentially monomeric. Altogether, those results demonstrate that efforts of discovery and characterization of microalgal laccases must be continued with regards to the amazing potential they offer for biotechnological applications.