Cloning, sequencing, and expression of the gene encoding extracellular α-amylase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme

Abstract

The gene encoding the hyperthermophilic extracellular α-amylase from Pyrococcus furiosus was cloned by activity screening in Escherichia coli. The gene encoded a single 460-residue polypeptide chain. The polypeptide contained a 26-residue signal peptide, indicating that this Pyrococcus α- amylase was an extracellular enzyme. Unlike the P. furiosus intracellular α- amylase, this extracellular enzyme showed 45 to 56% similarity and 20 to 35% identity to other amylolytic enzymes of the α-amylase family and contained the four consensus regions characteristic of that enzyme family. The recombinant protein was a homodimer with a molecular weight of 100,000, as estimated by gel filtration. Both the dimer and monomer retained starch- degrading activity after extensive denaturation and migration on sodium dodecyl sulfate-polyacrylamide gels. The P. furiosus αamylase was a liquefying enzyme with a specific activity of 3,900 U mg-1 at 98°C. It was optimally active at 100°C and pH 5.5 to 6.0 and did not require Ca2+ for activity or thermostability. With a half-life of 13 hat 95°C, the P. furiosus enzyme was significantly more thermostable than the commercially available Bacillus licheniformis α-amylase (Taka-therm).