Cloning of Lentinula edodes lemnp2, a manganese peroxidase that is secreted abundantly in sawdust medium

Manganese peroxidase (MnP), which is one of the lignin-degrading enzymes from white-rot fungi, possesses oxidative activity against phenolic compounds, making it a useful enzyme for bioremediation. A novel MnP-encoding gene (lemnp2) was isolated from Lentinula edodes. The deduced amino acid sequence showed approximately 48.8% homology to LeMnP1. The cDNA clone was approximately 1.4 kbp whereas the genomic sequence was 1.9 kbp, and comparison of the two indicated that lemnp2 contains 13 introns. The upstream region of lemnp2 contains putative CAAT, TATA, and metal response elements. Additionally, LeMnP2 contains conserved motifs that are observed in fungal MnPs, including 10 cysteines, a Mn-binding site, and Ca2+-binding sites. The lemnp2 transcript was identified in mycelium cultivated on sawdust medium, and the protein was secreted into the medium. MnP activity was purified from the sawdust medium as one peak during purification. Western blot analysis confirmed that LeMnP2, but not LeMnP1, was secreted into the sawdust medium. These results collectively demonstrate that LeMnP2 is the major MnP secreted into sawdust medium.