Homologous expression of recombinant lignin peroxidase in Phanerochaete chrysosporium

The glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter was used to dri ve expression of lip2, the gene encoding lignin peroxidase (LiP) isozyme H8 , in primary metabolic cultures of Phanerochaete chrysosporium. The express ion vector, pUGL, also contained the Schizophyllum commune ura1 gene as a s electable marker. pUGL was used to transform a P. chrysosporinm Ura11 auxot roph to prototrophy, Ura(+) transformants were screened for peroxidase acti vity in liquid cultures containing high-carbon and high-nitrogen medium. Re combinant LiP (rLiP) was secreted in active form by the transformants after 4 days of growth, whereas endogenous lip genes were not expressed under th ese conditions. Approximately 2 mg of homogeneous rLiP/liter was obtained a fter purification. The molecular mass, pI, and optical absorption spectrum of rLiPH8 were essentially identical to those of the wild-type LiPh8 (wt Li PH8), indicating that heme insertion, folding, and secretion functioned nor mally in the transformant. Steady-state and transient-state kinetic propert ies for the oxidation of veratryl alcohol between wtLiPH8 and rLiPH8 were a lso identical.