Blue laccase from Galerina sp.: Properties and potential for Kraft lignin demethylation

We purified a laccase isoenzyme, Lac1 from Galerina sp. HC1 using a combination of anion exchange- and hydrophobic interaction chromatography. Lac1 has a molecular mass of 64 kDa, an isoelectric point of 4, and 3.35 copper atoms/enzyme molecule. The enzyme has features typical of fungal blue laccases. The sequences of two internal peptides were highly similar to reported laccase sequences from other fungi such as Trametes sp. Lac1 exhibited optimal activity on substrate 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) at pH 3 and 60 °C. It had high stability at ≤20 °C and at high pH. Lac1 exhibited high substrate affinity and the highest catalytic efficiency reported among laccases for ABTS, and among the lowest for syringaldazine. The most potent inhibitors of Lac1 were sodium azide, sodium cyanide, disulfide reducing agents, and metal ions in the order Li+ > Sn2+ > Hg2+. The laccase efficiently catalyzed demethylation of eucalyptus hard wood Kraft lignin.