Cloning, sequence and expression in Escherichia coli of the gene encoding phosphofructokinase from Bacillus macquariensis.
A chromosomal DNA fragment containing the Bacillus macquariensis (Bm) ATP-dependent phosphofructokinase-encoding gene (pfk) was cloned from a subgenomic library in pUC19 using a PCR-derived probe. The region containing pfk, including flanking sequences, was sequenced and the deduced amino acid sequence (aa) was found to be homologous to other PFK, but it contained two single-aa changes conserved in a range of other organisms from pro- and eukaryotic origins. Enzymatic studies with PFK purified from overproducing Escherichia coli (Ec) host cells showed that the Bm enzyme is similar to B. stearothermophilus (Bs) PFK in many respects and that it is relatively cold stable.
Rouwendal
G J
GJ
Department of Bioconversion, Agrotechnological Research Institute (ATO-DLO), Wageningen, The Netherlands. g.j.a.rouwendal@ato.dlo.nl
Zwiers
L H
LH
Wolbert
E J
EJ
Springer
J
J
Mooibroek
H
H
Huizing
H J
HJ
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology / Q000235:genetics*; D001482:Base Composition; D001483:Base Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010732:Phosphofructokinase-1 / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D011993:Recombinant Fusion Proteins; D010732:Phosphofructokinase-1