Expression of the pyranose 2-oxidase from Trametes pubescens in Escherichia coli and characterization of the recombinant enzyme
| Autor: | Marcela Hradská, Marie-Helene Saniez, Nathalie Libessart, Branislav Večerek, Helena Marešová, Stanislav Bečka, Pavel Kyslík |
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| Rok vydání: | 2005 |
| Předmět: |
Protein Folding
Trametes pubescens Gene Expression Bioengineering Biology medicine.disease_cause Applied Microbiology and Biotechnology law.invention Fungal Proteins Species Specificity law medicine Escherichia coli chemistry.chemical_classification Inclusion Bodies Fungal protein Basidiomycota Pyranose oxidase General Medicine biology.organism_classification Molecular biology Kinetics Enzyme chemistry Pyranose Biochemistry Recombinant DNA Carbohydrate Dehydrogenases Heterologous expression Biotechnology |
| Zdroj: | Journal of biotechnology. 120(4) |
| ISSN: | 0168-1656 |
| Popis: | cDNA-encoding pyranose 2-oxidase (P2O) from Trametes pubescens was sequenced and cloned into Escherichia coli strain BL21/DE3 on a multicopy plasmid under the control of trc promoter. The synthesis of P2O was studied in a batch culture in M9-based mineral medium: the enzyme was synthesized constitutively at 28 degrees C in amount corresponding to 8% of the cell soluble protein (0.6 Umg(-1)). Only small portion of P2O (11%) was in the form of non-active inclusion bodies. Purified recombinant enzyme has similar physico-chemical and kinetic parameters with other P2Os. When compared to the expression of p2o of Trametes ochracea, a ratio of the mature enzyme to inclusion bodies found in the same E. coli host at 28 degrees C is as much as nine times higher. The finding makes the enzyme from T. pubescens preferable for the large-scale production by recombinant bacteria. The difference in amino acid sequences of the P2O from T. ochracea and T. pubescens may explain the favourable trait of the latter enzyme regarding protein folding. |
| Databáze: | OpenAIRE |
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