CLONING, SEQUENCING, AND EXPRESSION OF THE GENE ENCODING EXTRACELLULAR ALPHA-AMYLASE FROM PYROCOCCUS-FURIOSUS AND BIOCHEMICAL-CHARACTERIZATION OF THE RECOMBINANT ENZYME

The gene encoding the hyperthermophilic extracellular alpha-amylase fr om Pyrococcus furious was cloned by activity screening in Escherichia coli. The gene encoded a single 460-residue polypeptide chain. The pol ypeptide contained a 26-residue signal peptide, indicating that this P yrococcus alpha-amylase was an extracellular enzyme. Unlike the P. fur iosus intracellular alpha-amylase, this extracellular enzyme showed 45 to 56% similarity and 20 to 35% identity to other amylolytic enzymes of the alpha-amylase family and contained the four consensus regions c haracteristic of that enzyme family. The recombinant protein was a hom odimer with a molecular weight of 100,000, as estimated by gel filtrat ion. Both the dimer and monomer retained starch-degrading activity aft er extensive denaturation and migration on sodium dodecyl sulfate-poly acrylamide gels. The P. furiosus alpha-amylase was a liquefying enzyme with a specific activity of 3,900 U mg(-1) at 98 degrees C. It was op timally active at 100 degrees C and pH 5.5 to 6.0 and did not require Ca2+ for activity or thermostability. With a half-life of 13 h at 98 d egrees C, the P. furiosus enzyme was significantly more thermostable t han the commercially available Bacillus licheniformis alpha-amylase (T aka-therm).