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Journal of Biochemistry Advance Access published online on March 29, 2007
Journal of Biochemistry, doi:10.1093/jb/mvm085
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© 2007 The Japanese Biochemical Society

Cloning, Expression, Purification and Characterization of Fructose-1,6-bisphosphate Aldolase from Anoxybacillus gonensis G2

Nagihan Saglam Ertunga1, Ahmet Colak1,*, Ali Osman Belduz2, Sabriye Canakci2, Hakan Karaoglu2 and Cemal Sandalli3

1Department of Chemistry, Karadeniz Technical University, 61080 Trabzon, Turkey
2Department of Biology, Karadeniz Technical University, 61080 Trabzon, Turkey
3Department of Biology, Rize University, 53100 Rize, Turkey

* Corresponding author. Ahmet Colak, Karadeniz Technical University, Faculty of Arts and Sciences, Department of Chemistry, 61080 Trabzon, Turkey, Tel.: +90-462-3772489; Fax: +90-462-3773661, E-mail: acolak{at}ktu.edu.tr

Received February 14, 2007; Accepted March 19, 2007


  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 °C, and Km and Vmax were found to be 576 µM and 2.4 µM min-1 mg protein-1, respectively. Enzyme exhibits maximal activity at pH 8.5. The activity of enzyme was completely inhibited by EDTA.

Key Words: Anoxybacillus gonensis, Cloning, Fructose-1,6-bisphosphate Aldolase, Sequencing, Thermophilic


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