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 95°c. 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. copyright 2014 zoological society of pakistan.