Molecular cloning and expression of the pyranose 2-oxidase cDNA from Trametes ochracea MB49 in Escherichia coli.

Abstract

A cDNA of a structural gene encoding pyranose 2-oxidase (P2O) from Trametes ochracea strain MB49 was cloned into Escherichia coli strain BL21(DE3) on a multicopy plasmid under the control of the trc promoter. Synthesis of P2O was studied in batch cultures in LB or M9-based mineral medium at 28 degrees C. While there was a low specific activity of P2O in LB medium, the enzyme was synthesised constitutively in mineral medium and represented 3% of the cell soluble protein (0.3 U mg(-1)). The effect of isopropyl beta-D-thiogalactoside on the expression of P2O was studied in mineral medium at 25 and 28 degrees C. The synthesis of P2O at 28 degrees C corresponded to 39% of the cell soluble protein but the major portion of P2O (93%) was in the form of non-active inclusion bodies (activity of P2O equalled 0.19 U mg(-1)). At 25 degrees C, the amount of P2O represented 14% of the cell soluble protein and the activity of P2O was 1.1 U mg(-1). The soluble enzyme represented 70% of the total amount of P2O.