Production and characterization of recombinant Phanerochaete chrysosporiumcellobiose dehydrogenase in the methylotrophic yeast Pichia pastoris

The hemoflavoenzyme cellobiose dehydrogenase (CDH) from the white-rot fungu s Phanerochaete chrysosporium has been heterologously expressed in the meth ylotrophic yeast Pichia pastoris. After 4 days of cultivation in the induct ion medium, the expression level reached 1800 U/L (79 mg/L) of CDH activity , which is considerably higher than that obtained previously for wild-type CDH (wtCDH) and recombinant CDH (rCDH) produced by P. chrysosporium. Analys is with SDS-PAGE and Coomassie Brilliant Blue (CBB) staining revealed a maj or protein band with an approximate molecular mass of 100 kDa, which was id entified as rCDH by Western blotting. The absorption spectrum of rCDH shows that the protein contains one flavin and one heme cofactor per protein mol ecule, as does wtCDH. The kinetic parameters for rCDH using cellobiose, ubi quinone, and cytochrome c, as well as the cellulose-binding properties of r CDH were nearly identical to those of wtCDH. From these results, we conclud e that the rCDH produced by Pichia pastoris retains the catalytic and cellu lose-binding properties of the wild-type enzyme, and that the Pichia expres sion system is well suited for high-level production of rCDH.