Production and characterization of recombinant Phanerochaete chrysosporium cellobiose dehydrogenase in the methylotrophic yeast Pichia pastoris.
The hemoflavoenzyme cellobiose dehydrogenase (CDH) from the white-rot fungus Phanerochaete chrysosporium has been heterologously expressed in the methylotrophic yeast Pichia pastoris. After 4 days of cultivation in the induction 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. Analysis with SDS-PAGE and Coomassie Brilliant Blue (CBB) staining revealed a major protein band with an approximate molecular mass of 100 kDa, which was identified as rCDH by Western blotting. The absorption spectrum of rCDH shows that the protein contains one flavin and one heme cofactor per protein molecule, as does wtCDH. The kinetic parameters for rCDH using cellobiose, ubiquinone, and cytochrome c, as well as the cellulose-binding properties of rCDH were nearly identical to those of wtCDH. From these results, we conclude that the rCDH produced by Pichia pastoris retains the catalytic and cellulose-binding properties of the wild-type enzyme, and that the Pichia expression system is well suited for high-level production of rCDH.
Yoshida
M
M
Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan.
Ohira
T
T
Igarashi
K
K
Nagasawa
H
H
Aida
K
K
Hallberg
B M
BM
Divne
C
C
Nishino
T
T
Samejima
M
M
D002237:Carbohydrate Dehydrogenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism; D003001:Cloning, Molecular; D005822:Genetic Vectors; D007700:Kinetics; D020075:Phanerochaete / Q000201:enzymology* / Q000235:genetics; D010843:Pichia / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic
D011994:Recombinant Proteins; D002482:Cellulose; D002237:Carbohydrate Dehydrogenases; C019859:cellobiose-quinone oxidoreductase