High-level expression of a fungal pyranose oxidase in high cell-density fed-batch cultivations of Escherichia coli using lactose as inducer.
Expression of a recombinant pyranose oxidase (P2O) from the basidiomycete Trametes ochracea has been increased 10-fold in shaking flask cultures of Escherichia coli BL21(DE3) harboring plasmid pSE33 by optimizing the composition of the culture medium using an experimental design approach. Inexpensive lactose was used as a medium component and inducer of expression of the P2O gene, which is under the control of a trc promoter. The expression system was studied in detail in batch and fed-batch cultivations with the aim to improve the expression level of active recombinant protein and to minimize the formation of inclusion bodies. In batch cultivations, the highest specific P2O activity of 0.9 U (mg of soluble protein)(-1) was measured in oxygen-limited cultures grown at 25 degrees C. The highest overall volumetric productivity of 33 mg of active P2O per liter and hour (corresponding to 345U (L h)(-1)) has been determined in a high-density fed-batch process with a feed-forward exponential feeding strategy. During the fed-batch process, lactose was added intermittently to the culture. A final biomass concentration of 33 g L(-1) (based on cell dry weight) was obtained. Compared to shaking flask cultures in not optimized culture media, the overall volumetric P2O productivity has been improved by a factor of 110 using the fed-batch strategy and the optimized culture medium. Recombinant P2O was expressed in the cytoplasm with 9% of the total soluble protein being active P2O. In terms of physical and enzyme kinetic properties, the purified recombinant P2O was found to be similar to the previously published data of P2O isolated from its original host.
Kotik
Michael
M
Laboratory of Enzyme Technology, Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídenská 1083, 142 20 Prague 4. kotik@biomed.cas.cz
Kocanová
Marcela
M
Maresová
Helena
H
Kyslík
Pavel
P
D001487:Basidiomycota / Q000201:enzymology; D002237:Carbohydrate Dehydrogenases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D005786:Gene Expression Regulation; D007700:Kinetics; D007785:Lactose / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D013379:Substrate Specificity
D005656:Fungal Proteins; D011994:Recombinant Proteins; D002237:Carbohydrate Dehydrogenases; C022489:pyranose oxidase; D007785:Lactose