Cloning, sequencing, and expression of the gene encoding amylopullulanase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme

Abstract

The gene encoding the Pyrococcus furiosus hyperthermophilic amylopullulanase (APU) was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a single 827-residue polypeptide with a 26-residue signal peptide. The protein sequence had very low homology (17 to 21% identity) with other APUs and enzymes of the α-amylase family. In particular, none of the consensus regions present in the α-amylase family could be identified. P. furiosus APU showed similarity to three proteins, including the P. furiosus intracellular α-amylase and Dictyoglomus thermophilum α-amylase A. The mature protein had a molecular weight of 89,000. The re-combinant P. furiosus APU remained folded after denaturation at temperatares of ≤70°C and showed an apparent molecular weight of 50,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Denaturating temperatures of above 100°C were required for complete unfolding. The enzyme was extremely thermo stable, with an optimal activity at 105°C and pH 5.5. Ca2+ increased the enzyme activity, thermostability, and substrate affinity. The enzyme was highly resistant to chemical denaturing reagents, and its activity increased up to twofold in the presence of surfactants.