Determination of valence and coordination of iron in oxidic compounds by means of the x-ray fluorescence emission spectrum

Abstract

A novel laccase gene Lac1 and its cDNA were cloned from a white-rot fungus Cerrena sp. and characterized. The 1554-bp cDNA of Lac1 encoded a mature protein with 497 amino acids, preceded by a signal peptide of 20 amino acids. An unconventional intron splice site and incomplete splicing variants of Lac1 were observed. Lac1 was heterologously expressed in the yeast host Pichia pastoris, and a maximal laccase activity of 6.3 U mL−1 in the fermentation broth was achieved after fermentation for 9 days. The recombinant protein rLac1 was purified, and its enzymatic properties and functional characteristics were investigated. When ABTS was used as the substrate, the enzyme was most active at pH 3.5 and 55 °C, and stable at pH 4–10 and 20–60 °C. The Km and kcat values of rLac1 toward ABTS were 28.9 μM and 332.4 s−1, respectively. Furthermore, rLac1 was tolerant to common metal ions up to 100 mM concentration and capable of decolorizing structurally different dyes in the absence of a redox mediator. Hence, Lac1 may be useful for industrial applications, such as dye decolorization and bioremediation.