Homologous expression of recombinant lignin peroxidase in Phanerochaete chrysosporium.
The glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter was used to drive expression of lip2, the gene encoding lignin peroxidase (LiP) isozyme H8, in primary metabolic cultures of Phanerochaete chrysosporium. The expression vector, pUGL, also contained the Schizophyllum commune ura1 gene as a selectable marker. pUGL was used to transform a P. chrysosporium Ura11 auxotroph to prototrophy. Ura+ transformants were screened for peroxidase activity in liquid cultures containing high-carbon and high-nitrogen medium. Recombinant LiP (rLiP) was secreted in active form by the transformants after 4 days of growth, whereas endogenous lip genes were not expressed under these conditions. Approximately 2 mg of homogeneous rLiP/liter was obtained after purification. The molecular mass, pI, and optical absorption spectrum of rLiPH8 were essentially identical to those of the wild-type LiPh8 (wt LiPH8), indicating that heme insertion, folding, and secretion functioned normally in the transformant. Steady-state and transient-state kinetic properties for the oxidation of veratryl alcohol between wtLiPH8 and rLiPH8 were also identical.
Sollewijn Gelpke
M D
MD
Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Portland, Oregon 97291-1000, USA.
Mayfield-Gambill
M
M
Lin Cereghino
G P
GP
Gold
M H
MH
D005822:Genetic Vectors; D007700:Kinetics; D010544:Peroxidases / Q000235:genetics* / Q000378:metabolism*; D020075:Phanerochaete / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D014170:Transformation, Genetic
D011994:Recombinant Proteins; D010544:Peroxidases; C042858:lignin peroxidase