Molecular cloning and expression of the pyranose 2-oxidase cDNA from Trametes ochracea MB49 in Escherichia coli.
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.
Vecerek
B
B
Laboratory of Enzyme Technology, Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
Maresová
H
H
Kocanová
M
M
Kyslík
P
P
D002237:Carbohydrate Dehydrogenases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000201:enzymology; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D002479:Inclusion Bodies / Q000201:enzymology; D008969:Molecular Sequence Data; D020072:Polyporales / Q000201:enzymology* / Q000235:genetics; D011401:Promoter Regions, Genetic; D012331:RNA, Fungal / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013696:Temperature
D018076:DNA, Complementary; D012331:RNA, Fungal; D011994:Recombinant Proteins; D002237:Carbohydrate Dehydrogenases; C022489:pyranose oxidase