Molecular characterization of laccase genes from the basidiomycete Coprinus cinereus and heterologous expression of the laccase lcc1

Abstract

A laccase from Coprinus cinereus is active at alkaline pH, an essential property for some potential applications. We cloned and sequenced three laccase genes (lcc1, lcc2, and lcc3) from the ink cap basidiomycete C. cinereus. The lcc1 gene contained 7 introns, while both lcc2 and lcc3 contained 13 introns. The predicted mature proteins (Lcc1 to Lcc3) are 58 to 80% identical at the amino acid level. The predicted Lcc1 contains a 23-amino-acid C-terminal extension rich in arginine and lysine, suggesting that C-terminal processing may occur during its biosynthesis. We expressed the Lcc1 protein in Aspergillus oryzae and purified it. The Lcc1 protein as expressed in A. oryzae has an apparent molecular mass of 66 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and absorption maxima at 278 and 614 nm. Based on the N-terminal protein sequence of the laccase, a 4-residue propeptide was processed during the maturation of the enzyme. The dioxygen specificity of the laccase showed an apparent K(m) of 21 +/- 2 microM and a catalytic constant of 200 +/- 10 min(-1) for O(2) with 2, 2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) as the reducing substrate at pH 5.5. Lcc1 from A. oryzae may be useful in industrial applications. This is the first report of a basidiomycete laccase whose biosynthesis involves both N-terminal and C-terminal processing.

FIG. 1

Construction of expression vector pDSY67 for C. cinereus lcc1.

FIG. 2

Partial alignment of the deduced amino acid sequence of the C. cinereus Lcc1 laccase and other known laccase amino acid sequences. The sequences were aligned with the Clustal algorithm (DNASTAR, Madison, Wis.). The numbers refer to the amino acid sequence. The region in boldface is the peptide that may be removed during the biosynthesis of Lcc1. C. cin lcc1, lcc2, and lcc3, C. cinereus Lcc1, Lcc2, and Lcc3, respectively; T. vil lcc1 and lcc2, T. villosa Lcc1 (GenBank accession no. L49376) and Lcc2 (L49377), respectively; T. ver. lcc1, T. versicolor Lcc1 (X84683); C. hir and P. rad, C. hirsutus (M60560; J05562) and P. radiata (X52134), respectively; P. cin and PM1, P. cinnabarinus (AF025481) and the basidiomycete PM1 (Z12156) laccases, respectively; A. bis, N. cra, and M. ther, A. bisporus Lcc1 (L10664), N. crassa laccase (M18333; M18334), and M. thermophila Lcc1 (T10922), respectively; R. sol lcc1 and lcc4, R. solani Lcc1 (Z54275) and Lcc4 (Z54277), respectively.

FIG. 3

Alignment of Lcc1, Lcc2, and Lcc3 amino acid sequences with other known laccase amino acid sequences at the four putative copper binding regions. The sequences were aligned with the Clustal algorithm. Conserved histidines and cysteines are boxed. The numbers refer to the amino acid sequence. The dashes are gaps in the alignment.