Laccase from the medicinal mushroom Agaricus blazei: production, purification and characterization

Abstract

The medicinal mushroom Agaricus blazei produced high amounts of laccase (up to 5,000 units l(-1)) in a complex, agitated liquid medium based on tomato juice, while only traces of the enzyme (<100 units l(-1)) were detected in synthetic glucose-based medium. Purification of the enzyme required three chromatographic steps, including anion and cation exchanging. A. blazei laccase was expressed as a single protein with a molecular mass of 66 kDa and an isoelectric point of 4.0. Spectroscopic analysis of the purified enzyme confirmed that it belongs to the "blue copper oxidases". The enzyme's pH optimum for 2,6-dimethoxyphenol (DMP) and syringaldazine was pH 5.5; but for 2,2'-azino-bis(3-ethylthiazoline-6-sulfonate) (ABTS) no distinct pH optimum was observed (highest activity at the lowest pH tested). Purified laccase was stable at 20 degrees C, pH 7.0 and pH 3.0, but rapidly lost its activity at 40 degrees C or pH 10. Sodium chloride strongly inhibited the enzyme activity, although the inhibition was completely reversible. The following kinetic constants were determined (K(m), k(cat)): 63 microM, 21 s(-1) for ABTS, 4 microM, 5 s(-1) for syringaldazine, 1,026 microM, 15 s(-1) for DMP and 4307 microM, 159 s(-1) for guaiacol. The results show that--in addition to the wood-colonizing white-rot fungi--the typical litter-decomposing basidiomycetes can also produce high titers of laccase in suitable liquid media.

Extracellular Laccase Produced by an Edible Basidiomycetous Mushroom,Grifola frondosa: Purification and Characterization

“…4, chloride ion also inhibited the activity of T. versicolor laccase, but the inhibitory effect was found to be much stronger on Lac 1. In addition, a similar inhibitory effect of chloride ion has been reported for other fungal laccases, including those from Agaricus blazei 45) and Polyporus versicolor. 46) It was suggested that the chloride ion blocks the electron pathway at the active site of the laccase.…”

Stimulation of laccases from Trametes pubescens: Use in dye decolorization and cotton bleaching

“…In contrast, the pH optimum for the other substrates was higher: a bell-shape pH profile with the maximum peak at 4.0-5.0-5.5 was observed for DMP, guaiacol and syringaldazine, respectively. These data are in accordance to most of the known laccases whose pH optimum is in an acidic range for ABTS and in a more alkaline interval (from pH 4 to 7) for phenol compounds (12).…”

“…Where indicated, the oxidation of syringaldazine at 525 nm (ε 525 = 65 mM -1 cm -1 ), guaiacol at 450 nm (ε 450 = 12.1 mM -1 cm -1 ) and DMP at 477 nm (ε 477 = 14.8 mM -1 cm -1 ) was followed (12). The activity was expressed in international units (1 U = 1 µmol of substrate oxidized per minute).…”

Enhanced production of thermostable laccases from a native strain of Pycnoporus sanguineus using central composite design

“…Our findings revealed that laccase activity enhancement also occurred in tomato juice medium without external inducers, as the activity achieved was higher (11 000 IU/L) than that produced in the optimized glucose-meat peptone medium and CuSO 4 . This medium served as a source of carbon and nitrogen for the growth of P. sanguineus and also might induce the enzyme production on account of its phenolic compounds as flavonoids and tannic acid (Carbajo et al, 2002;Ullrich et al, 2005).…”

“…As the carbon sources present in the medium play an important role in ligninolytic enzyme production (Eggert et al, 1996), different commercial products have been tested. Tomato-juice-based media were appropriate for cultivation of different fungal species under laboratory conditions and sufficient for laccase production (Ullrich et al, 2005;Michniewicz et al, 2006;Liers et al, 2007;Junghanns et al, 2008;Mueangtoom et al, 2010). Malt-extract and bran-flake media have been used to produce laccase specifically from Pycnoporus sanguineus (Trovaslet et al, 2007;Dantán-González et al, 2008).…”