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 man ganese peroxidase (MnP) and laccase but do not express lignin peroxidase (L iP). To facilitate structure/function studies of MnP from D. squalens, we h eterologously expressed the enzyme in the well-studied basidiomycete, Phane rochaete chrysosporium. The glyceraldehyde-3-phosphate-dehydrogenase (gpd) promoter of P. chrysosporium was fused to the coding region of the mnp2 gen e of D. squalens, 5 bp upstream of the translation start site, and placed i n a vector containing the ural gene as a selectable marker. Purified recomb inant protein (rDsMnP) was similar in kinetic and spectral characteristics to both the wild-type MnPs from D. squalens and P. chrysosporium (PcMnP). T he N-terminal amino acid sequence of the rDsMnP was determined and was iden tical to the predicted sequence. Cleavage of the propeptide followed a cons erved amino acid motif (A-A-P-S/T) in both rDsMnP and PcMnP. However, the p rotein from D. squalens was considerably more thermostable than its P. chry sosporium homolog with half-lives 15- to 40-fold longer at 55 degreesC. As previously demonstrated for PcMnP, addition of exogenous Mn-II and Cd-II co nferred additional thermal stability to rDsMnP, However, unlike PcMnP, Zn-I I also confers some additional thermal stability to rDsMnP at 55 degreesC. Some differences in the metal-specific effects on thermal stability of rDsM nP at 65 degreesC were noted. (C) 2001 Academic Press.