HOMOLOGOUS EXPRESSION OF RECOMBINANT MANGANESE PEROXIDASE IN PHANEROCHAETE-CHRYSOSPORIUM

The promoter region of the glyceraldehyde-3-phosphate dehydrogenase ge ne (gpd) was used to drive expression of mnp1, the gene encoding Mn pe roxidase isozyme 1, in primary metabolic cultures of Phanerochaete chr ysosporium. A 1,100-bp fragment of the P. chrysosporium gpd promoter r egion was fused upstream of the mnp1 gene to construct plasmid pAGM1, which contained the Schizophyllum commune ade5 gene as a selectable ma rker. pAGM1 was used to transform a P. chrysosporium ade1 auxotroph to prototrophy. Ade(+) transformants were screened for peroxidase activi ty on a solid medium containing high carbon and high nitrogen (2% gluc ose and 24 mM NH4 tartrate) and o-anisidine as the peroxidase substrat e. Several transformants that expressed high peroxidase activities wer e purified and analyzed further in liquid cultures. Recombinant Mn per oxidase (rMnP) was expressed and secreted by transformant cultures on day 2 under primary metabolic growth conditions (high carbon and high nitrogen), whereas endogenous wild-type mnp genes were not expressed u nder these conditions. Expression of rMnP was not influenced by the le vel of Mn in the culture medium, as previously observed for the wild-t ype Mn peroxidase (wtMnP). The amount of active rMnP expressed and sec reted in this system was comparable to the amount of enzyme expressed by the wild-type strain under ligninolytic conditions. rMnP was purifi ed to homogeneity by using DEAE-Sepharose chromatography, Blue Agarose chromatography, and Mono Q column chromatography. The M(r), and absor ption spectrum of rMnP were essentially identical to the M(r) and abso rption spectrum of wtMnP, indicating that heme insertion, folding, and secretion were normal. The steady-state kinetic values for the oxidat ion of Mn(II) and 2,6-dimethoxyphenol by rMnP and wtMnP also were very Similar. This system is suitable for generating site-directed mutants of Mn peroxidase.