Biochemical characterization of a recombinant pullulanase from Thermococcus kodakarensis KOD1.

In this report, a glycoside hydrolase 13 family pullulanase gene (Tk0977) was cloned from a thermophilic anaerobic archaeon Thermococcus kodakarensis KOD1 (Pul-Tk). Pul-Tk encodes a protein of 765 amino acids including a putative 22-residue signal peptide. The protein has four consensus motives and a catalytic triad of glycoside hydrolase 13 family in the deduced amino acid sequence. The recombinant enzyme was expressed in Escherichiacoli and purified to homogeneity. Pul-Tk can hydrolyse both pullulan and soluble starch. The purified enzyme was optimal at pH55-60 and 100 degrees C and exhibited good stability over a broad pH range (4-8). The V-max and K-m values were 11839176molmg(-1)min(-1) and 037 +/- 002mgml(-1) for pullulan and 5319 +/- 1166molmg(-1)min(-1) and 036 +/- 005mgml(-1) for starch. All these favourable enzymatic properties make it valuable in various industries.