Heterologous Expression of a Thermostable Manganese Peroxidase from Dichomitus squalens in Phanerochaete chrysosporium

Dichomitus squalens belongs to a group of white-rot fungi which express manganese peroxidase (MnP) and laccase but do not express lignin peroxidase (LiP). To facilitate structure/function studies of MnP from D. squalens, we heterologously expressed the enzyme in the well-studied basidiomycete, Phanerochaete chrysosporium. The glyceraldehyde-3-phosphate-dehydrogenase (gpd) promoter of P. chrysosporium was fused to the coding region of the mnp2 gene of D. squalens, 5 bp upstream of the translation start site, and placed in a vector containing the ura1 gene as a selectable marker. Purified recombinant protein (rDsMnP) was similar in kinetic and spectral characteristics to both the wild-type MnPs from D. squalens and P. chrysosporium (PcMnP). The N-terminal amino acid sequence of the rDsMnP was determined and was identical to the predicted sequence. Cleavage of the propeptide followed a conserved amino acid motif (A-A-P-S/T) in both rDsMnP and PcMnP. However, the protein from D. squalens was considerably more thermostable than its P. chrysosporium homolog with half-lives 15- to 40-fold longer at 55°C. As previously demonstrated for PcMnP, addition of exogenous MnII and CdII conferred additional thermal stability to rDsMnP. However, unlike PcMnP, ZnII also confers some additional thermal stability to rDsMnP at 55°C. Some differences in the metal-specific effects on thermal stability of rDsMnP at 65°C were noted.