Gene Cloning and Characterization of a Type II Pullulanase Hydrolase from a Hyperthermophilic Archaeon Pyrobaculum calidifontis

The genome search of the hyperthermophilic archaeon Pyrobaculum calidifontis revealed the presence of an open reading frame Pcal_1616 encoding for a type II pullulanase hydrolase. Pcal_1616 composed of 1006 amino acid residues with a molecular mass of 111 kDa including a 17-residue signal peptide. The amino acid sequence analysis revealed the presence of five conserved regions that are characteristic of GH57 family hydrolases. Pcal_1616 gene was cloned and expressed in Escherichia coli. The recombinant enzyme exhibited the highest activity at 95C. The optimum pH of the enzyme activity was 5.5. However Pcal-1616 exhibited more than 80% activity over a broad pH range (4.0-8.0). The metal ions including Ca2+ did not show a significant effect on the enzyme activity.