Purification and Characterization of a Thermo- and pH-Stable Laccase From the Litter-Decomposing Fungus Gymnopus luxurians and Laccase Mediator Systems for Dye Decolorization

Abstract

An extracellular laccase (GLL) was purified from fermentation broth of the litter-decomposing fungus <i>Gymnopus luxurians</i> by four chromatography steps, which resulted in a high specific activity of 118.82 U/mg, purification fold of 41.22, and recovery rate of 42.05%. It is a monomeric protein with a molecular weight of 64 kDa and N-terminal amino acid sequence of AIGPV TDLHI, suggesting that GLL is a typical fungal laccase. GLL demonstrated an optimum temperature range of 55°C-65°C and an optimum pH 2.2 toward 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). It displayed considerably high thermostability and pH stability with about 63% activity retained after 24 h at 50°C, and 86% activity retained after 24 h at pH 2.2, respectively. GLL was significantly enhanced in the presence of K⁺, Na⁺, and Mg²⁺ ions. It demonstrated <i>K</i> _<i>m</i> of 539 μM and <i>k</i> _<i>cat</i> /<i>K</i> _<i>m</i> of 140 mM^-1⋅s^-1 toward ABTS at pH 2.2 and 37°C. Acetosyringone (AS) and syringaldehyde (SA) were the optimal mediators of GLL (0.4 U/ml) for dye decolorization with decolorization rates of about 60%-90% toward 11 of the 14 synthetic dyes. The optimum reaction conditions were determined to be mediator concentration of 0.1 mM, temperature range of 25°C -60°C, and pH 4.0. The purified laccase was the first laccase isolated from genus <i>Gymnopus</i> with high thermostability, pH stability, and effective decolorization toward dyes, suggesting that it has potentials for textile and environmental applications.