High level production of the Magnaporthe grisea fructose 1,6-bisphosphate aldolase enzyme in Escherichia coli using a small volume bench-top fermentor.
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.
Labbé
Geneviève
G
Department of Chemistry, University of Waterloo, 200 University Ave. W, Waterloo, Ont., Canada N2L 3G1.
Bezaire
Jeremy
J
de Groot
Sarah
S
How
Christine
C
Rasmusson
Tim
T
Yaeck
Jason
J
Jervis
Eric
E
Dmitrienko
Gary I
GI
Guillemette
J Guy
JG
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
D005634:Fructose-Bisphosphate Aldolase; D015032:Zinc