title: First laccase in green algae: purification and characterization of an extracellular phenol oxidase from Tetracystis aeria.
abstract: 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.
authors.affiliation: Institute of Biology, General and Applied Botany, Leipzig University, 04103, Leipzig, Germany, benjamin.otto@t-online.de.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Otto
authors.forename: Dietmar
authors.initials: D
authors.lastname: Schlosser
mesh_terms: D001673:Biodegradation, Environmental; D000460:Chlorophyta / Q000201:enzymology*; D003116:Color; D004396:Coloring Agents / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005110:Extracellular Space / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D042845:Laccase / Q000378:metabolism*; D014442:Monophenol Monooxygenase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D010084:Oxidation-Reduction; D013379:Substrate Specificity
chemical_list: D004396:Coloring Agents; D042845:Laccase; D014442:Monophenol Monooxygenase