First laccase in green algae: purification and characterization of an extracellular phenol oxidase from Tetracystis aeria

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 a parts per thousand currency sign2.5, 7.0, and 6.5; K (m) values of 28.8, 40.5, and 1,830 A mu M; respectively), but not l-tyrosine or Fe2+. ABTS is by far the most efficient substrate. Two polypeptides, A (similar to 110 kDa) and B (71 kDa), were co-purified by the applied procedure, both being highly N-glycosylated (a parts per thousand yen similar to 53 and a parts per thousand yen27 %, respectively). As suggested by various gel electrophoretic analyses, the native enzyme (apparent molecular mass of similar to 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 B (2) . 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.