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

Ertunga N. S., Colak A., Belduz A. O., Canakci S., Karaoglu H., Sandalli C.

JOURNAL OF BIOCHEMISTRY, cilt.141, sa.6, ss.817-825, 2007 (SCI-Expanded) identifier identifier identifier

  • Yayın Türü: Makale / Tam Makale
  • Cilt numarası: 141 Sayı: 6
  • Basım Tarihi: 2007
  • Doi Numarası: 10.1093/jb/mvm085
  • Dergi Adı: JOURNAL OF BIOCHEMISTRY
  • Derginin Tarandığı İndeksler: Science Citation Index Expanded (SCI-EXPANDED), Scopus
  • Sayfa Sayıları: ss.817-825
  • Anahtar Kelimeler: Anoxybacillus gonensis, cloning, fructose-1,6-bisphosphate aldolase, sequencing, thermophilic, CLASS-II ALDOLASE, MOLECULAR-CLONING, NUCLEOTIDE-SEQUENCE, STRUCTURAL-ANALYSIS, FDA GENE, MECHANISM, BINDING, IDENTIFICATION, REPLACEMENT, ZINC
  • Karadeniz Teknik Üniversitesi Adresli: Evet

Özet

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 30.9kDa protein of 286 amino acids. The amino acid sequence shared similar to 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 mu M and 2.4 mu M min(-1) mg protein(-1), respectively. Enzyme exhibits maximal activity at pH 8.5. The activity of enzyme was completely inhibited by EDTA.