High level production of the Magnaporthe grisea fructose 1,6-bisphosphate aldolase enzyme in Escherichia coli using a small volume bench-top fermentor
| Autor: | Sarah de Groot, Jeremy Bezaire, Jason Yaeck, Christine How, Tim Rasmusson, Eric Jervis, J. Guy Guillemette, Gary I. Dmitrienko, Geneviève Labbé |
|---|---|
| Rok vydání: | 2007 |
| Předmět: |
Fructose 1
6-bisphosphate Molecular Sequence Data Gene Expression medicine.disease_cause chemistry.chemical_compound Bioreactors Fructose-Bisphosphate Aldolase Escherichia coli medicine Magnaporthe grisea Amino Acid Sequence Cloning Molecular Protein Structure Quaternary chemistry.chemical_classification Binding Sites biology Aldolase A Active site Fructose Hydrogen-Ion Concentration biology.organism_classification Molecular Weight Kinetics Magnaporthe Zinc Enzyme chemistry Biochemistry Fermentation biology.protein Dimerization Sequence Alignment Biotechnology |
| Zdroj: | Protein Expression and Purification. 51:110-119 |
| ISSN: | 1046-5928 |
| DOI: | 10.1016/j.pep.2006.06.020 |
| Popis: | The Class II fructose 1,6-bisphosphate aldolase from the Rice Blast causative agent Magnaporthe grisea was subcloned in the Escherichia coli vector pT7-7. The enzyme was overexpressed using fed-batch fermentation in a small bench-top reactor. A total of 275 g of cells and 1.3 g of highly purified enzyme with a specific activity of 70 U/mg were obtained from a 1.5L culture. The purified enzyme is a homodimer of 39.6 kDa subunits with a zinc ion at the active site. Kinetic characterization indicates that the enzyme has a K(m) of 51 microM, a k(cat) of 46 s(-1), and a pH optimum of 7.8 for fructose 1,6-bisphosphate cleavage. The fermentation system procedure reported exemplifies the potential of using a lab-scale bioreactor for the large scale production of recombinant enzymes. |
| Databáze: | OpenAIRE |
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