Biochemical characterization of a recombinant pullulanase from Thermococcus kodakarensis KOD1

Lett Appl Microbiol. 2013 Oct;57(4):336-43. doi: 10.1111/lam.12118. Epub 2013 Jul 8.


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 Escherichia coli and purified to homogeneity. Pul-Tk can hydrolyse both pullulan and soluble starch. The purified enzyme was optimal at pH 5·5-6·0 and 100°C and exhibited good stability over a broad pH range (4-8). The Vmax and Km values were 118·39 ± 1·76 μmol mg(-1 ) min(-1) and 0·37 ± 0·02 mg ml(-1) for pullulan and 53·19 ± 11·66 μmol mg(-1 ) min(-1) and 0·36 ± 0·05 mg ml(-1) for starch. All these favourable enzymatic properties make it valuable in various industries.

Significance and impact of the study: Pullulanases have a great potential in industrial applications including the starch industry, the production of maltose syrups and high-purity glucose and fructose. In this study, a pullulanase from hyperthermophilic archaeon Thermococcus kodakarensis KOD1 was successfully expressed in Escherichia coli and the recombinant enzyme can be purified and characterized. The high activity, broad pH range and stability implicate it as a potential enzyme for industrial applications.

Keywords: GH13 family; Industrial application; Pullulanase; Thermococcus kodakarensis KOD1.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Archaeal Proteins / chemistry*
  • Archaeal Proteins / genetics
  • Archaeal Proteins / isolation & purification
  • Archaeal Proteins / metabolism
  • Enzyme Stability
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Glucans / metabolism
  • Glycoside Hydrolases / chemistry*
  • Glycoside Hydrolases / genetics
  • Glycoside Hydrolases / isolation & purification
  • Glycoside Hydrolases / metabolism
  • Kinetics
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Starch / metabolism
  • Thermococcus / enzymology*
  • Thermococcus / genetics


  • Archaeal Proteins
  • Glucans
  • Recombinant Proteins
  • pullulan
  • Starch
  • Glycoside Hydrolases
  • pullulanase