title: High level production of the Magnaporthe grisea fructose 1,6-bisphosphate aldolase enzyme in Escherichia coli using a small volume bench-top fermentor.
abstract: 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.
authors.affiliation: Department of Chemistry, University of Waterloo, 200 University Ave. W, Waterloo, Ont., Canada N2L 3G1.
authors.forename: Geneviève
authors.initials: G
authors.lastname: Labbé
authors.forename: Jeremy
authors.initials: J
authors.lastname: Bezaire
authors.forename: Sarah
authors.initials: S
authors.lastname: de Groot
authors.forename: Christine
authors.initials: C
authors.lastname: How
authors.forename: Tim
authors.initials: T
authors.lastname: Rasmusson
authors.forename: Jason
authors.initials: J
authors.lastname: Yaeck
authors.forename: Eric
authors.initials: E
authors.lastname: Jervis
authors.forename: Gary I
authors.initials: GI
authors.lastname: Dmitrienko
authors.forename: J Guy
authors.initials: JG
authors.lastname: Guillemette
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D019149:Bioreactors; D003001:Cloning, Molecular / Q000379:methods*; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology; D005285:Fermentation; D005634:Fructose-Bisphosphate Aldolase / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D020082:Magnaporthe / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020836:Protein Structure, Quaternary; D016415:Sequence Alignment; D015032:Zinc / Q000032:analysis
chemical_list: D005634:Fructose-Bisphosphate Aldolase; D015032:Zinc