title: Biochemical characterization of a recombinant pullulanase from Thermococcus kodakarensis KOD1.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: College of Plant Sciences, Jilin University, Changchun, China.
authors.forename: T
authors.initials: T
authors.lastname: Han
authors.forename: F
authors.initials: F
authors.lastname: Zeng
authors.forename: Z
authors.initials: Z
authors.lastname: Li
authors.forename: L
authors.initials: L
authors.lastname: Liu
authors.forename: M
authors.initials: M
authors.lastname: Wei
authors.forename: Q
authors.initials: Q
authors.lastname: Guan
authors.forename: X
authors.initials: X
authors.lastname: Liang
authors.forename: Z
authors.initials: Z
authors.lastname: Peng
authors.forename: M
authors.initials: M
authors.lastname: Liu
authors.forename: J
authors.initials: J
authors.lastname: Qin
authors.forename: S
authors.initials: S
authors.lastname: Zhang
authors.forename: B
authors.initials: B
authors.lastname: Jia
mesh_terms: D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005936:Glucans / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013213:Starch / Q000378:metabolism; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics
chemical_list: D019843:Archaeal Proteins; D005936:Glucans; D011994:Recombinant Proteins; C009109:pullulan; D013213:Starch; D006026:Glycoside Hydrolases; C100181:pullulanase
keywords: GH13 family; Industrial application; Pullulanase; Thermococcus kodakarensis KOD1