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

The gene encoding the Pyrococcus furiosus hyperthermophilic amylopullu lanase (APU) was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a single 827-residue polypeptide with a 26-residue s ignal peptide. The protein sequence had very low homology (17 to 21% i dentity) with other APUs and enzymes of the alpha-amylase family. In p articular, none of the consensus regions present in the cy-amylase fam ily could be identified. P. furiosus APU showed similarity to three pr oteins, including the P. furiosus intracellular cw-amylase and Dictyog lomus thermophilum alpha-amylase A. The mature protein had a molecular weight of 89,000. The recombinant P. furiosus APU remained folded aft er denaturation at temperatures of less than or equal to 70 degrees C and showed an apparent molecular weight of 50,000 in sodium dodecyl su lfate-polyacrylamide gel electrophoresis. Denaturating temperatures of above 100 degrees C were required for complete unfolding. The enzyme was extremely thermostable, with an optimal activity at 105 degrees C and pH 5.5. Ca2+ increased the enzyme activity, thermostability, and s ubstrate affinity. The enzyme was highly resistant to chemical denatur ing reagents, and its activity increased up to twofold in the presence of surfactants.