Molecular characterization of laccase genes from the basidiomycete Coprinus cinereus and heterologous expression of the laccase Lcc1

A laccase from Coprinus cinereus is active at alkaline pH, an essential pro perty for some potential applications. We cloned and sequenced three laccas e genes (lcc1, lcc2, and lcc3) from the ink cap basidiomycete C. cinereus, The lcc1 gene contained 7 introns, while both lcc2 and Icc3 contained 13 in trons. The predicted mature proteins (Lcc1 to Lcc3) are 58 to 80% identical at the amino acid level. The predicted Led contains a 23-amino-acid C-term inal extension rich in arginine and lysine, suggesting that C-terminal proc essing may occur during its biosynthesis. We expressed the Led protein in A spergillus oryzae and purified it. The Lcc1 p rotein as expressed in A. ory zae has an apparent molecular mass of 66 kDa on sodium dodecyl sulfate-poly acrylamide gel electrophoresis and absorption maxima at 278 and 614 nm. Bas ed on the N-terminal protein sequence of the laccase, a 4-residue propeptid e was processed during the maturation of the enzyme. The dioxygen specifici ty of the laccase showed an apparent K-m of 21 +/- 2 mu M and a catalytic c onstant of 200 +/- 10 min(-1) for O-2 with 2,2'-azinobis(3-ethylbenzothiazo line-6-sulfonic acid) as the reducing substrate at pH 5.5. Led from A. oryz ae may be useful in industrial applications, This is the first report of a basidiomycete laccase whose biosynthesis involves both N-terminal and C-ter minal processing.