A green algal phenol oxidase was firstly purified, confirmed to be a laccase, and a hetero-oligomeric quaternary structure is suggested. The operation of a laccase-mediator system is firstly described in algae. Laccases (EC 1.10.3.2) catalyze the oxidation of a multitude of aromatic substrates. They are well known in higher plants and fungi, while their presence in green algae appears uncertain. Extracellular laccase-like enzyme activity has previously been described in culture supernatants of the green soil alga Tetracystis aeria [Otto et al. in Arch Microbiol 192:759-768, (2010)]. As reported herein, the T. aeria enzyme was purified 120-fold by employing a combination of anion exchange and size exclusion chromatography. The purified enzyme was confirmed to be a laccase according to its substrate specificity. It oxidizes 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), syringaldazine, and 2,6-dimethoxyphenol (pH optima of pH ≤ 2.5, 7.0, and 6.5; K m values of 28.8, 40.5, and 1,830 µM; respectively), but not L-tyrosine or Fe(2+). ABTS is by far the most efficient substrate. Two polypeptides, A (~110 kDa) and B (71 kDa), were co-purified by the applied procedure, both being highly N-glycosylated (≥~53 and ≥ 27 %, respectively). As suggested by various gel electrophoretic analyses, the native enzyme (apparent molecular mass of ~220 kDa) most probably is a hetero-oligomer with the composition AB 2 , wherein A is the catalytic subunit and B forms a disulfide-linked homo-dimer B2. The decolorization of anthraquinone (Acid Blue 62 and Remazol Brilliant Blue R) and diazo dyes (Reactive Black 5) was studied in the presence of redox-mediating compounds (ABTS and syringaldehyde), demonstrating the operation of the laccase-mediator system in algae for the first time. Thus, laccases from green algae may participate in the biotransformation of a wide spectrum of natural and xenobiotic compounds.

In order to explore the abundance and potential environmental functions of green algal laccases, we screened various algae for extracellular laccase-like activities, characterized basic features of these activities in selected species and exemplarily studied the transformation of environmental pollutants and complex natural compounds by the laccase of Tetracystis aeria. Oxidation of the classical laccase substrate ABTS was found to be widespread in chlorophycean algae. The oxidation activity detected in members of the 'Scenedesmus' clade was caused by an unknown thermostable low-molecular-mass compound. In contrast, species of the Moewusinia, including Chlamydomonas moewusii and T. aeria, excreted putative 'true' laccases. Phenolic substrates were oxidized by these enzymes optimally at neutral to alkaline pH. The Tetracystis laccase efficiently transformed bisphenol A, 17α-ethinylestradiol, nonylphenol and triclosan in the presence of ABTS as redox mediator, while anthracene, veratrylalcohol and adlerol were not attacked. Lignosulfonate and humic acid underwent slight (de)polymerization reactions in the presence of the laccase and mediator(s), probably involving the oxidation of phenolic constituents. Possible natural functions of the enzymes, such as the synthesis of complex polymers or detoxification processes, may assist the survival of the algae in adverse environments. In contaminated surface waters, laccase-producing green algae might contribute to the environmental breakdown of phenolic pollutants.

Laccases (EC 1.10.3.2) are versatile multi-copper oxidases so far found in higher plants, fungi, insects, prokaryotes and lichens. In the present study, the production of an extracellular laccase-like enzyme by the coccoid green soil alga Tetracystis aeria was investigated and the enzyme was partly characterized, thereby providing the first description of a laccase-like enzyme in soil algae. Enzyme production in algae cultures was considerably increased by addition of the fungal laccase inducer copper sulphate. Maximal enzyme production was observed during the stationary growth phase. Peroxidase or tyrosinase activity was not detected. The native enzyme exhibits an apparent molecular mass of about 212 kDa as observed with size exclusion chromatography and about 210-260 kDa as estimated by zymograms. The enzyme efficiently oxidizes 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), 2,6-dimethoxyphenol (2,6-DMP), syringaldazine (SGZ) and the anthraquinone dye Acid Blue 62, while guaiacol and Remazol Brilliant Blue R are only poorly oxidized. The apparent kinetic parameters obtained for ABTS, 2,6-DMP and SGZ oxidation are within the range reported for fungal laccases. Oxidation of the phenolic substrate 2,6-DMP displays a remarkably high pH optimum (pH 8.0-8.5), which is interesting with respect to potential biotechnological applications.