title: Cloning, expression, purification and characterization of fructose-1,6-bisphosphate aldolase from Anoxybacillus gonensis G2.
abstract: The fructose-1,6-bisphosphate aldolase gene from the thermophilic bacterium, Anoxybacillus gonensis G2, was cloned and sequenced. Nucleotide sequence analysis revealed an open reading frame coding for a 30.9 kDa protein of 286 amino acids. The amino acid sequence shared approximately 80-90% similarity to the Bacillus sp. class II aldolases. The motifs that are responsible for the binding of a divalent metal ion and catalytic activity completely conserved. The gene encoding aldolase was overexpressed under T7 promoter control in Escherichia coli and the recombinant protein purified by nickel affinity chromatography. Kinetic characterization of the enzyme was performed at 60 degrees C, and K(m) and V(max) were found to be 576 microM and 2.4 microM min(-1) mg protein(-1), respectively. Enzyme exhibits maximal activity at pH 8.5. The activity of enzyme was completely inhibited by EDTA.
authors.affiliation: Department of Chemistry; Department of Biology, Karadeniz Technical University, 61080 Trabzon, Turkey.
authors.forename: Nagihan Saglam
authors.initials: NS
authors.lastname: Ertunga
authors.forename: Ahmet
authors.initials: A
authors.lastname: Colak
authors.forename: Ali Osman
authors.initials: AO
authors.lastname: Belduz
authors.forename: Sabriye
authors.initials: S
authors.lastname: Canakci
authors.forename: Hakan
authors.initials: H
authors.lastname: Karaoglu
authors.forename: Cemal
authors.initials: C
authors.lastname: Sandalli
mesh_terms: D000595:Amino Acid Sequence; D001406:Bacillaceae / Q000201:enzymology*; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004492:Edetic Acid / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009532:Nickel / Q000737:chemistry; D011401:Promoter Regions, Genetic; D017386:Sequence Homology, Amino Acid
chemical_list: D009532:Nickel; D004492:Edetic Acid; D005634:Fructose-Bisphosphate Aldolase