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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 188.8.131.52) 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.