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Journal of Biochemistry Advance Access originally published online on March 29, 2007
Journal of Biochemistry 2007 141(6):817-825; 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; 2Department of Biology, Karadeniz Technical University, 61080 Trabzon; and 3Department of Biology, Rize University, 53100 Rize, Turkey

*To whom correspondence should be addressed. Tel: +90-462-3772489, Fax: +90-462-3773661, E-mail: acolak{at}ktu.edu.tr

Received February 14, 2007; Accepted February 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 ~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 576µM and 2.4µM 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

Abbreviations: FBP, fructose-1,6-bisphosphate; IPTG, isopropyl ß-D-thiogalactopyranoside


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