Cloning, expression, purification and characterization of fructose-1,6-bisphosphate aldolase from Anoxybacillus gonensis G2

J Biochem. 2007 Jun;141(6):817-25. doi: 10.1093/jb/mvm085. Epub 2007 Mar 29.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacillaceae / enzymology*
  • Base Sequence
  • Chromatography, Affinity
  • Cloning, Molecular
  • Edetic Acid / chemistry
  • Escherichia coli / metabolism
  • Fructose-Bisphosphate Aldolase / chemistry*
  • Fructose-Bisphosphate Aldolase / genetics*
  • Fructose-Bisphosphate Aldolase / isolation & purification
  • Hydrogen-Ion Concentration
  • Kinetics
  • Molecular Sequence Data
  • Nickel / chemistry
  • Promoter Regions, Genetic
  • Sequence Homology, Amino Acid

Substances

  • Nickel
  • Edetic Acid
  • Fructose-Bisphosphate Aldolase