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

Otto, Benjamin, Schlosser, Dietmar
Planta 2014 v.240 no.6 pp. 1225-1236
Chlorophycota, anion exchange, anthraquinones, biotransformation, decolorization, dyes, electrophoresis, enzyme activity, fungi, gels, iron, laccase, molecular weight, monophenol monooxygenase, oxidation, pH, polypeptides, protein subunits, size exclusion chromatography, substrate specificity, tyrosine
MAIN CONCLUSION : 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 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 Fe²⁺. 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 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.