Cloning, expression and characterization of xylose isomerase from thermophilic Geobacillus caldoxylosilyticus TK4 strain

Abstract

Aim: To clone the gene of Xylose isomerase (XyI) from thermophilic Geobacillus cal-doxylosilyticus TK4 strain and to express and characterize this enzyme. Methods: XyI gene of Geobacillus caldoxylolyticus was cloned into pET28a(+) vector and expressed in Escherichia coli. The recombinant protein was purified by using nickel affinity chromatography and characterization of the purified enzyme was done. Results: The recombinant enzyme had 1326 bp and optimum pH and temperature of the XyI were found to be 6.5 and 80°C, respectively. At 4.0-9.0 pH range and 4°C, after 15 days incubation, the enzyme was quite stable. The enzyme retained nearly 80% of its original activity after 3 hours incubation at 60°C and 70°C. In the presence of glucose as substrate, Km and Vmax values of the XyI were determined as 20.58 mM and 0.67 U/ mg protein, respectively. Conclusion: A XyI gene from Geobacillus caldoxylosilyticus TK4 was cloned and expressed in E. coli. The XyI shared common characteristics with known XyIs in terms of conserved amino acid residues, electrophoretic behaviour and kinetic parameters. The recombinant XyI may be used in industrial applications need high temperatures and low pHs. © TurkJBiochem.com.